ALBERT

Description:    Antitumor agents are used in therapy against many forms of human cancer. One of these is mitomycin-C (MMC). As with many agents, it can interact with biological molecules and can induce genetic hazards in non-tumor cells. One of the possible approaches to protect DNA from this damage is to supply antioxidants that can remove free radicals produced by antitumor agents. Lipoic acid (LA) is known as one of the most powerful antioxidants. The aim of this study was to investigate antigenotoxic effects of LA against MMC induced chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronucleus (MN) formation in human lymphocytes. Lymphocytes were treated with 0.2 μg MMC/heparinized mL for 48 h. Three different concentrations (0.5, 1, 2 μg/mL) of LA were used together with MMC in three different applications; 1 h pre-treatment, simultaneous treatment and 1 h post-treatment. A negative, a positive and a solvent control were also included. In all the cultures treated with MMC + LA, the frequency of abnormal cells and CA/cell significantly decreased compared to MMC. Statistically significant reduction was also observed in SCE/cell and MN frequencies in all treatments. These results demonstrated anticlastogenic and antimutagenic effects of LA against MMC induced genotoxicity. LA showed the most efficient effect during 1 h pretreatment. On the other hand, MMC + LA treatments induced significant reduction in mitotic index than that of MMC treatment alone. These results are encouraging that LA can be a possible chemopreventive agent in tumorigenesis in both cancer patients and in health care persons handling anti-cancer drugs. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9504-8 Authors Fatma Unal, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Gokce Taner, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Deniz Yuzbasioglu, Department of Biology, Science Faculty, Gazi University, Ankara, Turkey Serkan Yilmaz, Department of Midwifery, Faculty of Health Sciences, Ankara University, Ankara, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps. Content Type Journal Article Category Brief Report Pages 1-10 DOI 10.1007/s10616-012-9509-3 Authors Iria Maria Gomez-Touriño, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Ana Senra, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Francisco Garcia, CIMUS (Department of Physiology), School of Medicine, University of Santiago de Compostela, Avd. Barcelona, 15782 Santiago de Compostela, Spain Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The present study aims to investigate the heptonephro-protective effect of grape seeds proanthocyanidin extract (GSPE) against the risks induced by gibberellic acid (GA3) in male rats. The results recorded that GA3 caused a significant increase in total lipids, total cholesterol, triglycerides and LDL-C levels in serum, concomitant with a significant decrease in serum HDL-C. A significant increase in serum AST, ALT, urea and creatinine, while, a significant decrease in total protein content in serum was observed in rats given GA3. Hepatic and renal lipid peroxidation product (MDA) was significantly increased, meanwhile, total antioxidant capacity (TAC), glutathione, and catalase levels were significantly decreased. In addition, there was a negative change in liver structure including dilatation in the central veins with degeneration of endothelium cells and cellular injury around the veins as well as in the kidney structure such as lesion in both glomeruli and tubules, detachment of the Malpighian corpuscles from the Bowman’s capsule’s epithelium, shrinkage in the glomerular capillary network. However, almost all of these adverse effects seemed to be ameliorated by oral administration of GSPE with GA3 to rats for 2 month indicating the protective effect of grape seeds GSPE on GA3 induced oxidative stress in rats. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9506-6 Authors Hanaa A. Hassan, Faculty of Science, Zoology Department, Mansoura University, Mansoura, Egypt Maisaa M. Al-Rawi, Biology Department, Practical Science College, Umm Al-Qura University, Mecca, Saudi Arabia Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9499-1 Authors Wenjing Zhang, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Yan Lan, The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China Qilai Huang, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Zichun Hua, The State Key Laboratory of Quality Research in Chinese Medicine, Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa Macau, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue ( p  〈 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl 2 as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl 2 domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation. Content Type Journal Article Category Original Research Pages 1-12 DOI 10.1007/s10616-012-9470-1 Authors Hanaa A. Hassan, Physiology Division, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt Hani S. Hafez, Zoology Department, Faculty of Science, Siez, Suez Canal University, Ismailia, Egypt Mona S. Goda, Genetic Department, Hospital Medicine, Mansoura University, Mansoura, Egypt Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg −1 and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”. Content Type Journal Article Category Method in Cell Science Pages 1-16 DOI 10.1007/s10616-012-9491-9 Authors P. Jayesh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Seena Jose, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Rosamma Philip, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India I. S. Bright Singh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 682016 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Polyethylenimine (PEI) has been used widely in transient gene expression studies of mammalian cells. We performed transient gene expression in suspension Chinese hamster ovary cells using a one-step transfection procedure in which DNA and PEI were simultaneously added to a cell culture in suspension without prior PEI/DNA complex incubation. To further understand the effect of PEI/DNA formation on the transfection and expression of exogenous gene in shaking state, we investigated the diameter and overcharge of the PEI/DNA complex. The results showed that the diameter of the complex was smaller with more positive charge when the PEI/DNA ratio was higher. Moreover, DNA more easily penetrated cells and nuclei at higher PEI concentrations. The highest transcription level, transfection efficiency, and GFP expression were obtained when the PEI/DNA ratio was 5:1. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9483-9 Authors Qiuling Xie, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Guo Xinyong, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Chen Xianjin, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Wang Yayu, College of Life Science and Technology, Jinan University, Guangzhou, 510632 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Pdx - 1 and Irs - 1 , genes highly associated with diabetes onset, were knocked down in mouse embryonic stem (ES) cells in order to develop cell line models for diabetes. ES cells with different gene knockdown levels were induced to differentiate to the stage of insulin production. Among the cell lines that differentiated, we identified two in which the levels of expression of both genes were 20–40 % of that of control cells. These cell lines showed appreciable deficiencies in three characteristic malfunctions associated with diabetes, namely, insulin production, insulin reception signaling, and glucose-stimulated insulin secretion. These dysfunctions were consistent with results reported elsewhere from in vivo and in vitro studies. Both cell lines did not show any abnormal morphology such as size, shape, color, and surface roughness. No abnormal expression profiles for 17 genes relevant to diabetes were observed. Therefore, these cell lines fulfilled the criteria for a validated cell model for diabetes. The model cell lines developed here are promising biomaterials for cell-based screening tests of new medicines that may be effective in treating diabetes. Content Type Journal Article Category Original Research Pages 1-14 DOI 10.1007/s10616-012-9466-x Authors Mikako Saito, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Aya Hayakawa, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Nobuya Inagaki, Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507 Japan Hideaki Matsuoka, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Open reading frame 17 ( Bm17 ) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9451-4 Authors Hongxing Shen, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Yang Zhou, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Wen Zhang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Bin Nin, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Hua Wang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Xiaochun Wang, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Shihe Shao, School of Medical Science and Laborarory Medicine, Jiangsu University, Zhenjiang, 212013 People’s Republic of China Huiqing Chen, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Zhongjian Guo, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Xiaoyong Liu, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Qin Yao, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Keping Chen, Institute of Life Sciences, Jiangsu University, 301# xuefu road, Zhenjiang, 212013 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H 2 O 2 levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H 2 O 2 level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC 50 ) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC 50 by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H 2 O 2 levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening. Content Type Journal Article Category Original Research Pages 1-7 DOI 10.1007/s10616-011-9423-0 Authors Wenwei Mao, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Xinlin Chen, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Tian Yang, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Yu Yin, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Mei Ge, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Minyu Luo, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Daijie Chen, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Xiuping Qian, Lab of Microbial and Biochemical Pharmaceutical, School of Pharmacy, Shanghai Jiaotong University, 800 Dong Chuan Road, Minhang District, Shanghai, 200240 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    A short half-life and low levels of growth factors in an injured microenvironment necessitates the sustainable delivery of growth factors and stem cells to augment the regeneration of injured tissues. Our aim was to investigate the ability of VEGF 165 expressing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro. We isolated, cultured and identified rabbit BMMSCs, then electroporated the BMMSCs with VEGF 165 -pCMV6-AC-GFP plasmid. G418 was used to select transfected cells and the efficiency was up to 70%. The groups were then divided as follows: Group A was electroporated with pCMV6-AC-GFP plasmid + HGF + EGF and Group B was electroporated with VEGF 165 -pCMV6-AC-GFP plasmid +HGF + EGF. After 14 days, BMMSCs were induced into short spindle and polygonal cells. Alpha-fetoprotein (AFP) was positive and albumin (ALB) was negative in Group A, while both AFP and ALB were positive in group B on day 10. AFP and ALB in both groups were positive on day 20, but the quantity of AFP in group B decreased with prolonged time and was about 43.5% less than group A. The quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day 10 and 20. Our results demonstrated that VEGF 165 -pCMV6-AC-GFP plasmid modified BMMSCs still had the ability to differentiate into hepatocytes. The VEGF 165 gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9439-0 Authors Yan Tan, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China En-hua Xiao, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Li-zhi Xiao, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China You-hong Yuan, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Cong Ma, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Quan-liang Shang, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Du-jun Bian, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Yan-hui Li, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Zhu Chen, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Qian Chang, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, 410011 Hunan Province, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis , is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed. Content Type Journal Article Category Original Research Pages 581-598 DOI 10.1007/s10616-011-9364-7 Authors Shaoqin Ge, Hebei University Health Science Center, 071000 Baoding, People’s Republic of China Suixin Wang, Langfang Health Vocational College, 065000 Langfang, People’s Republic of China Xianjiang Kang, College of Life Science, Hebei University, 071000 Baoding, People’s Republic of China Fei Duan, Hebei University Health Science Center, 071000 Baoding, People’s Republic of China Yan Wang, Hebei University Health Science Center, 071000 Baoding, People’s Republic of China Wenyan Li, Hebei University Health Science Center, 071000 Baoding, People’s Republic of China Mingshen Guo, College of Life Science, Hebei University, 071000 Baoding, People’s Republic of China Shumei Mu, College of Life Science, Hebei University, 071000 Baoding, People’s Republic of China Yuhua Zhang, Hebei University Health Science Center, 071000 Baoding, People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 63 Journal Issue Volume 63, Number 6

Description:    Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitative PCR (RT-qPCR) and differentiation into adipocytes, osteocytes and chondrocytes precursors was analyzed by cytochemical staining. This study showed that amnion stromal cells expressing stem cell markers can differentiate into mesoderm lineage and may be an alternative source to mesenchymal stem cells derived from adipose tissue and bone marrow for the use in tissue repair. Content Type Journal Article Category Brief Report Pages 1-7 DOI 10.1007/s10616-011-9398-x Authors Stefania Violini, Parco Tecnologico Padano, CERSA, Via Einstein, Loc. Cascina Codazza, 26900 Lodi, Italy Chiara Gorni, Parco Tecnologico Padano, CERSA, Via Einstein, Loc. Cascina Codazza, 26900 Lodi, Italy Laura Francesca Pisani, Parco Tecnologico Padano, CERSA, Via Einstein, Loc. Cascina Codazza, 26900 Lodi, Italy Paola Ramelli, Parco Tecnologico Padano, CERSA, Via Einstein, Loc. Cascina Codazza, 26900 Lodi, Italy Mario Caniatti, DIPAV, Veterinary Anatomo-Pathology and Avian Pathology Unit, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy Paola Mariani, Parco Tecnologico Padano, CERSA, Via Einstein, Loc. Cascina Codazza, 26900 Lodi, Italy Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 1

Description:    This study aimed to assess the efficiency and effects of insulin-like growth factor receptor-1 (IGF-IR) siRNA knockdown during bovine preimplantation embryonic development. In oocytes injected with IGF-IR siRNA, the relative IGF-IR mRNA levels compared to controls were 28% and 46% at 6 and 24 h after injection, respectively. With respect to the injection of IGF-IR siRNA in zygotes, 24 h after injection the relative levels of IGF-IR mRNA and protein in the two-cell embryos were 74% and 78% of those in the controls, respectively. IGF-IR siRNA reduced blastocyst formation (23.2%) compared to siRNA controls (33.0%) and uninjected oocytes (35.4%; P  〈 0.05) and the number of viable cells per IGF-IR siRNA-treated blastocyst (64 ± 3) was significantly reduced, compared to control siRNA and uninjected blastocysts (81 ± 3 and 116 ± 4; P  〈 0.01). In conclusion, IGF-IR siRNA knockdown reduces the development of bovine embryos, and microinjection in zygotes can decrease blastocyst cell number. Content Type Journal Article Category Original Research Pages 165-172 DOI 10.1007/s10616-011-9402-5 Authors L. M. Wang, Key Laboratory of China Education Ministry for Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, 010021 People’s Republic of China J. X. Wen, Key Laboratory of China Education Ministry for Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, 010021 People’s Republic of China J. L. Yuan, Key Laboratory of China Education Ministry for Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, 010021 People’s Republic of China Ming Cang, Key Laboratory of China Education Ministry for Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, 010021 People’s Republic of China D. J. Liu, Key Laboratory of China Education Ministry for Research of Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, 010021 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC. Content Type Journal Article Category Original Research Pages 203-216 DOI 10.1007/s10616-011-9413-2 Authors P. L. Mok, PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia S. K. Cheong, PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia C. F. Leong, Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia K. H. Chua, Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia O. Ainoon, Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences. Content Type Journal Article Category Original Research Pages 197-202 DOI 10.1007/s10616-011-9411-4 Authors Masoumeh Fakhr Taha, Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Mojtaba Rezazadeh Valojerdi, Department of Anatomy, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran Leili Hatami, Department of Anatomy, School of Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran Arash Javeri, Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Toll-like receptor 7 (TLR7) senses viral single-stranded RNA (ssRNA), induces the production of type I interferons (IFNs), IFN-α and -β, in macrophages such as dendritic cells (DCs), and its immune system protects the host from virus infection. Here, we found that a crude extract from immature green tea leaves (iTPS) containing a macromolecule with ssRNA fragments, induces IFN-α production in human macrophage-like cells. In addition IFN-α production was inhibited by treatment with TLR7 inhibitors or a phagocytosis inhibitor. Content Type Journal Article Category Brief Report Pages 145-148 DOI 10.1007/s10616-011-9412-3 Authors Manami Monobe, NARO Institute of Vegetable and Tea Science (NIVTS), 2769 Kanaya-Shishidoi, Shimada, Shizuoka, 428-8501 Japan Akiko Ogino, NARO Institute of Vegetable and Tea Science (NIVTS), 2769 Kanaya-Shishidoi, Shimada, Shizuoka, 428-8501 Japan Kaori Ema, NARO Institute of Vegetable and Tea Science (NIVTS), 2769 Kanaya-Shishidoi, Shimada, Shizuoka, 428-8501 Japan Yoshiko Tokuda, NARO Institute of Vegetable and Tea Science (NIVTS), 2769 Kanaya-Shishidoi, Shimada, Shizuoka, 428-8501 Japan Mari Maeda-Yamamoto, NARO Institute of Vegetable and Tea Science (NIVTS), 2769 Kanaya-Shishidoi, Shimada, Shizuoka, 428-8501 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified two-step method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time. Content Type Journal Article Category Original Research Pages 187-195 DOI 10.1007/s10616-011-9407-0 Authors Mariano Severgnini, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Jennifer Sherman, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Alfica Sehgal, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Narayanannair K. Jayaprakash, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Justin Aubin, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Gang Wang, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Ligang Zhang, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Chang G. Peng, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Kristina Yucius, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Jim Butler, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Kevin Fitzgerald, Alnylam Pharmaceuticals, 300 Third St, Cambridge, MA 02142, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to bring flow cytometry to current maturity. The aim of this paper is to present a complete retrospective of the technique and remind flow cytometry fundamentals before focusing on recent commercial instrumentation. Content Type Journal Article Category Review Pages 109-130 DOI 10.1007/s10616-011-9415-0 Authors Julien Picot, Institut National de la Transfusion Sanguine, 75739 Paris Cedex 15, France Coralie L. Guerin, University Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France Caroline Le Van Kim, Institut National de la Transfusion Sanguine, 75739 Paris Cedex 15, France Chantal M. Boulanger, University Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Voltage-gated Ca 2+ channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30 years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca 2+ currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca 2+ channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca 2+ channel currents, and of non-L-type Ca 2+ channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca 2+ currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture. Content Type Journal Article Category Original Research Pages 173-179 DOI 10.1007/s10616-011-9405-2 Authors Chen Zhou, State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, 100871 China Aiying Yang, Department of Cell Biology, Harbin Medical University, Harbin, 150086 China Zhen Chai, State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, 100871 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells. Content Type Journal Article Category Technical Note Pages 149-156 DOI 10.1007/s10616-011-9404-3 Authors Janette Contreras, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA Pang-Yu Hsueh, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA Hua Pei, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA Sarah F. Hamm-Alvarez, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Mercury, a xenobiotic metal, is a highly deleterious environmental pollutant. Moreover, in any form mercury is reported to be toxic. On the other hand, Thymbra spicata L. , a member of the Lamiaceae family, has long been investigated popularly of biological roles; mainly antimicrobial and antioxidant activities. However, there are very scarce data on the cytogenetic effects of thyme species. The purpose of this study was to investigate the genetic safety of different extracts from T. spicata (water extract, methanol extract, and ethanol extract) and the effects of T. spicata on mercury (as HgCl 2 ) induced genotoxicity. Sister chromatid exchange (SCE) and micronucleus (MN) assays were performed to assess DNA damages in cultured human lymphocytes (n = 5). Our results clearly revealed that, the SCE and MN rates induced by HgCl 2 were alleviated by the presence of T. spicata . As conclusion, this study demonstrated for the first time that the T. spicata provided increased resistance of DNA against HgCl 2 induced genetic damage in human lymphocytes. Based on the results of this study, it may be concluded that the T. spicata is a nontoxic material that could be used as a suppressor of heavy metal-induced genotoxicity. Content Type Journal Article Category Original Research Pages 181-186 DOI 10.1007/s10616-011-9406-1 Authors Ebubekir Dirican, Department of Microbiology, Medical Faculty, Kahramanmaraş Sütçü Imam University, 46100 Kahramanmaraş, Turkey Hasan Turkez, Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, 25240 Erzurum, Turkey Başak Toğar, Department of Biology, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form hybridoma, which may secrete McAbs against CAP. Hybridomas 1D 1 and 3G 12 secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 10 6 cells of hybridoma 1D 1 and 3G 12 into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb 3G 12 was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC 50 value was 50.8 ng/mL. Content Type Journal Article Category Original Research Pages 157-163 DOI 10.1007/s10616-011-9401-6 Authors Yu Yi, College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang road 18, Hangzhou, 310014 China Zhuhuan Wang, College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang road 18, Hangzhou, 310014 China Min Li, College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang road 18, Hangzhou, 310014 China Keyin Zhu, Biolink Biopharm, Co, Ltd, Hangzhou, 310018 China Guoqing Ying, College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang road 18, Hangzhou, 310014 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    WNIN/Ob, a mutant rat strain, developed at the National Center for Laboratory Animal Sciences (NCLAS) facility of National Institute of Nutrition (NIN), is a new animal model to study the metabolic syndrome. These animals have 47% fat in their body and isolation of islets from these animals were compounded due to the formation of amorphous viscous and jelly like material which reduced the islet yield. However, islets isolated from WNIN adult (≥12 months) control rats gave a good islet recovery, under standard isolation procedures using collagenase digestion. In the present study we optimized culture conditions in WNIN/Ob rats to isolate islets with higher yield, and also established primary islet cell cultures from these mutant rats, retaining cellular integrity and functionality. Content Type Journal Article Category Brief Report Pages 139-144 DOI 10.1007/s10616-011-9409-y Authors V. Venkatesan, National Centre for Laboratory Animal Science, National Institute of Nutrition, Jamai Osmania, Hyderabad, India M. Chalsani, National Centre for Laboratory Animal Science, National Institute of Nutrition, Jamai Osmania, Hyderabad, India S. S. Nawaz, National Centre for Laboratory Animal Science, National Institute of Nutrition, Jamai Osmania, Hyderabad, India R. R. Bhonde, Manipal Institute of Regenerative Medicine, Domulur Layout, Bangalore, India S. S. Challa, National Centre for Laboratory Animal Science, National Institute of Nutrition, Jamai Osmania, Hyderabad, India G. Nappanveettil, National Centre for Laboratory Animal Science, National Institute of Nutrition, Jamai Osmania, Hyderabad, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    Arrowroot ( Maranta arundinacea . L) is an underutilized local crop potentially to be developed as carbohydrate source and functional food in Indonesia. The objectives of this research are to evaluate the immunostimulatory effects of arrowroot extracts in vitro by using animal cell culture techniques, and in vivo by using BALB/c mice. The arrowroot tuber extracts were prepared by heat-treatment at 121 °C for 20 min in distilled water. The IgM production stimulatory activity of arrowroot tuber extracts against human hybridoma HB4C5 cells and mouse splenocytes was assessed. The result indicated that the arrowroot tuber extract stimulated IgM production by HB4C5 cells and immunoglobulin (IgG, IgA and IgM) production by splenocytes in vitro. In addition, the arrowroot tuber extracts strongly enhanced interferon γ production by splenocytes. In vivo study indicated that the diet containing arrowroot extracts increased the serum IgG, IgA and IgM levels in mice. These results revealed that the arrowroot tuber extracts have immunostimulatory effects in vivo as well as in vitro . Content Type Journal Article Category Brief Report Pages 131-137 DOI 10.1007/s10616-011-9403-4 Authors Ika Dyah Kumalasari, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Eni Harmayani, Faculty of Agricultural Technology, Gadjah Mada University, Jl. Socio Yusticia, Bulaksumur, Yogyakarta, 55281 Indonesia Lily Arsanti Lestari, Faculty of Medicine, Gadjah Mada University, Jl. Farmako, Sekip Utara, Yogyakarta, 55281 Indonesia Sri Raharjo, Faculty of Agricultural Technology, Gadjah Mada University, Jl. Socio Yusticia, Bulaksumur, Yogyakarta, 55281 Indonesia Widya Asmara, Faculty of Veterinary Medicine, Gadjah Mada University, Jl. Fauna No. 2, Karang Malang, Yogyakarta, 55281 Indonesia Kosuke Nishi, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Takuya Sugahara, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 2

Description:    P38 mitogen-activated protein kinases (p38 MAPK) and tumor necrosis factor-α (TNF-α) play important roles in oxidative stress-induced apoptosis in cardiac myocytes. However, the regulation and functional role of cross-talk between p38 MAPK and TNF-α pathways have not yet been fully characterized in cardiac myocytes. In this study, we found that inhibition of p38 MAPK with SB-203580 (SB) reduced H 2 O 2 -stimulated secretion of TNF-α, whereas pre-activation of p38 MAPK with sodium arsenite (SA) enhanced H 2 O 2 -stimulated secretion of TNF-α. In addition, pretreatment of cells with TNF-α increased basal and H 2 O 2 -stimulated p38 MAPK and apoptosis of cardiac myocytes, and p38 MAPK-associated apoptosis of cardiac myocytes induced by TNF-α was blocked by inhibition of p38 MAPK with SB. Finally, H 2 O 2 -induced apoptosis was attenuated by the inhibitors of p38 MAPK or reactive oxygen species (ROS), whereas it was enhanced by p38 MAPK agonist SA. These results suggest that H 2 O 2 -induced secretion of TNF-α increases apoptosis of cardiac myocytes through ROS-dependent activation of p38 MAPK. This may represent a novel mechanism that TNF-α partly interplays with p38 MAPK pathways during oxidative stress-modulated apoptosis in cardiac myocytes. Content Type Journal Article Category Original Research Pages 65-73 DOI 10.1007/s10616-011-9392-3 Authors Zhilong Chen, Department of Cardiology, Renmin Hospital of Wuhan University, 99 Zi Yang Road, Wuhan, 430060 Hubei, China Hong Jiang, Department of Cardiology, Renmin Hospital of Wuhan University, 99 Zi Yang Road, Wuhan, 430060 Hubei, China Yanwu Wan, Huangshi Central Hospital, Huangshi, 435000 Hubei, China Chaofang Bi, Huangshi Central Hospital, Huangshi, 435000 Hubei, China Yian Yuan, Huangshi Central Hospital, Huangshi, 435000 Hubei, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 1

Description: Erratum to: Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir Content Type Journal Article Category Erratum Pages 107-107 DOI 10.1007/s10616-011-9410-5 Authors Tsutomu Nagira, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Junko Narisawa, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Kiichirou Teruya, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Yoshinori Katakura, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Sun-Yup Shim, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Ken-ichi Kusumoto, American Type Culture Collection, Manassas, VA 20110, USA Sennosuke Tokumaru, Nihon Kefir Co. Ltd., 13-15 Asahi-machi, Fujisawa, 251-0054 Japan Koichiro Tokumaru, Nihon Kefir Co. Ltd., 13-15 Asahi-machi, Fujisawa, 251-0054 Japan David W. Barnes, American Type Culture Collection, Manassas, VA 20110, USA Sanetaka Shirahata, Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069 Journal Volume Volume 64 Journal Issue Volume 64, Number 1

Description:    The Drosophila melanogaster Schneider 2 (S2) cell line was established in 1972. Many studies have indicated that generation of recombinant proteins with S2 cells is more desirable than using other methods, since native proteins derived from S2 cells do not usually interact with those derived from mammalian cells. In order to minimize the duration for selections, we established an all-in-one single plasmid pMT-PURO, which enables to express the gene of interest as well as a selection gene “ pac ”. However, there is a weak point in the system. In order to verify the hallmark of the transformed cells, puromycin selection as well as verification of the gene of interests is still necessary. To improve this situation, we generated pMT-PURO2G and pMT-PURO2R, which enable to verify the hallmark of the transformed cells during the selections by the detection of enhanced green fluorescent protein (EGFP) or DsRED2. This new system gives reliable and reproductive results for recombinant protein synthesis and gets rid of some degree of uncertainty for the outcome of the transfection. Content Type Journal Article Category Brief Report Pages 1-6 DOI 10.1007/s10616-012-9473-y Authors Kotomi Nagahashi, Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Kazuo Umemura, Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Naohiro Kanayama, Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Takayuki Iwaki, Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Stem cells present an important tool in livestock assisted reproduction and veterinary therapeutic field such as tissue engineering. We report for the first time isolation of pluripotent stem cell-like cells expressing pluripotency markers (alkaline phospahatase, OCT-4, NANOG and SOX-2) from the amnion of water buffalo ( Bubalus bubalis ). The cells showed no apparent abnormalities in their chromosomal profiles before and after cryopreservation. The cytochemical staining revealed that pluripotent cells were capable of undergoing directed differentiation in vitro into osteocytes. It could be inferred that amnion-derived pluripotent stem cell-like cells can be isolated, cultured for many passages and differentiated into mesoderm lineage, and may be an alternative source to mesenchymal stem cells. These cells can have applications in assisted reproduction, developmental biological and regenerative medicine. Content Type Journal Article Category Brief Report Pages 1-8 DOI 10.1007/s10616-012-9464-z Authors A. Mann, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India R. P. Yadav, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India J. Singh, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India D. Kumar, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India B. Singh, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India P. S. Yadav, Buffalo Physiology and Reproduction Division, Central Institute for Research on Buffaloes, Hisar, 125001 Haryana, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format. Content Type Journal Article Category Review Paper Pages 1-16 DOI 10.1007/s10616-012-9481-y Authors Alexander P. Demchenko, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev, 01030 Ukraine Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Hybridoma HB-8696 produces monoclonal antibody (mAb) 520C9 (mouse IgG 1 ), which recognizes breast cancer oncoprotein c-erbB2. The objective of this study was to optimize the medium recipe of HB 8696 cell for production of mAb 520C9. The optimization consisted of two steps: (1) screening of significant nutrients to make subsequent experiments more efficient with less runs and (2) locating their optimal concentrations. 29 variables including essential and non-essential amino acids, glucose, serum and 6 salts, namely NaCl, KCl, CaCl 2 , NaH 2 PO 4 , MgSO 4 and Na-pyruvate were chosen in screening phase. The Plackett–Burman method was used to screen the variables influencing mAb production. Seven factors namely glucose, serum, asparagine, threonine, serine, NaCl and NaH 2 PO 4 were identified to have a positive influencing role on mAb production with a confidence level 〉90 % ( p  〈 0.1). Finally, Response surface methodology revealed the optimal level of the variables. The mAb production and average specific mAb production rate were enhanced by 111.05 and 105 %, respectively, compared to control medium. Content Type Journal Article Category Original Research Pages 1-20 DOI 10.1007/s10616-012-9480-z Authors Sucharita Sen, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Pradip K. Roychoudhury, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Recent studies have shown that, in numerous species, systemically administered bone marrow-derived mesenchymal stem cells undergo site-specific differentiation. This suggests that osteoblasts, by means of cytokine secretion, may promote dental pulp stem cells (DPSCs) to undergo osteogenesis. The objective of this study was to assess the potential synergistic interaction effect of osteoblasts on DPSCs for promotion of osteogenesis. Stem cells, derived from dental pulp of healthy human donors, were co-cultured with calvaria osteoblasts using a culture insert system. The proliferation rate, calcium deposition, osteogenic-related gene expression of induced DPSCs, including Runx-2, bone sialoprotein, osteocalcin and collagen-1, were assayed using MTT, Alizarin Red S staining and reverse transcriptase polymerase chain reaction, respectively. Co-cultured DPSCs had the highest rate of proliferation compared with those cultured in absence of osteoblasts. The morphology and ultrastructure of DPSCs in the co-cultures showed improvement, with co-cultured DPSCs becoming more osteoblast-like as compared with DPSCs cultured alone, and the mineralization potential of co-cultured DPSCs was enhanced compared with DPSCs cultured alone. Furthermore, osteogenic-related genes were significantly over-expressed in co-cultured DPSCs after osteogenic induction. The results demonstrate that DPSCs successfully differentiate towards osteoblasts and that the paracrine interaction of osteoblasts is likely to contribute to DPSC differentiation. It is believed that this study demonstrates certain useful applications for DPSCs in bone tissue engineering. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9479-5 Authors Yuying Wang, Department of Prosthodontics, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086 Heilongjiang Province, People’s Republic of China Jie Yao, Department of Thoracic Surgery, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009 People’s Republic of China Mengtong Yuan, Department of Prosthodontics, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086 Heilongjiang Province, People’s Republic of China Zhiwu Zhang, Department of Orthopaedics, The 117th Hospital of PLA, Hangzhou, 310013 Zhejiang Province, People’s Republic of China Weiping Hu, Department of Prosthodontics, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086 Heilongjiang Province, People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Human umbilical cord mesenchymal stem cells (hUMSC) are primitive multipotent cells capable of differentiating into cells of different lineages. They can be an alternative source of pluripotent cells since they are ethically and regulatory approved, are easily obtained and have low immunogenicity compared to embryonic stem cells which are dogged with numerous controversies. hUMSC can be a great source for cell and transplantation therapy. Content Type Journal Article Category Review Paper Pages 1-11 DOI 10.1007/s10616-012-9489-3 Authors Paulina Duya, Tianjin University of Traditional Chinese Medicine, 312 Anshan West Road, Nankai district, Tianjin, China Yuhong Bian, Tianjin University of Traditional Chinese Medicine, 312 Anshan West Road, Nankai district, Tianjin, China Xiaoqian Chu, Tianjin University of Traditional Chinese Medicine, 312 Anshan West Road, Nankai district, Tianjin, China Yanjun Zhang, Tianjin University of Traditional Chinese Medicine, 312 Anshan West Road, Nankai district, Tianjin, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    l -alanyl- l -glutamine (AlaGln) is dipeptide that has better solubility and stability than Glutamine (Gln). In this study, we evaluated the utility of this dipeptide during culture of POTELLIGENT™ Chinese hamster ovary (CHO) cells expressing anti-CD20 chimeric antibody. Although AlaGln in the culture medium lowered the specific growth rate, the MAb titer was maximized when Gln was completely replaced by AlaGln in both the basal and feed media. Moreover, AlaGln augmented production of antibody not only at flask scale but also at spinner scale, although the extent of this effect was dependent on the cell clone. To explore the mechanism responsible for the effect of AlaGln on cell growth, we measured apoptosis in the early phase of cell culture on days 8, 9, and 10. The apoptotic ratio was reduced in medium containing AlaGln. Ammonia was generated in medium containing Gln when it was maintained at 37 °C, which impeded the growth and productivity of the cells. In contrast, AlaGln produced less ammonia under these conditions, which may have been one of the properties associated with its beneficial effects. We conclude that certain dipeptides can serve as superior alternative sources of amino acids in cell culture and antibody production. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9468-8 Authors Yasufumi Imamoto, Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma, 370-0013 Japan Hisaya Tanaka, Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma, 370-0013 Japan Ken Takahashi, Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma, 370-0013 Japan Yoshinobu Konno, Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma, 370-0013 Japan Toshiyuki Suzawa, Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma, 370-0013 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    It has been demonstrated that hydrogen peroxide (H 2 O 2 ) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H 2 O 2 in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H 2 O 2 -induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH 2 -terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H 2 O 2 -treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect. Content Type Journal Article Category Original Research Pages 1-15 DOI 10.1007/s10616-012-9469-7 Authors Tomoya Kinjo, Division of Life Engineering, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Jun Ye, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Hanxu Yan, Division of Life Engineering, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Takeki Hamasaki, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Hidekazu Nakanishi, Division of Life Engineering, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Kazuko Toh, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Noboru Nakamichi, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Shigeru Kabayama, Nihon Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka, 531-0076 Japan Kiichiro Teruya, Division of Life Engineering, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Sanetaka Shirahata, Division of Life Engineering, Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Wharton’s jelly mesenchymal stem cells (WJMSCs) are important alternative source of pluripotent cells for several therapeutic purposes. Understanding of adhesion properties of such cells is necessary to regulate the attachment, growth and proliferation on targeted culture surfaces. BCP-K1, a line of WJMSCs, and polystyrene (PS) culture dishes were used as membrane samples. A 13.56 MHz inductively coupled discharge plasma reactor with a mixture of N-containing gas and noble gas was used. This was expected to introduce the more hydrophilic groups on PS surface and enhance the cell adhesion. The plasma-treated PS dishes with the mixed gas of N 2  + He at 50 W and NH 3  + He at 100 W were reactive towards BCP-K1. Cellular adhesion and proliferation was significantly twice as efficient on the treated surfaces than on PS dishes. BCP-K1 also secreted more focal adhesion kinase to adhere and proliferate when cultured on N 2 -treated PS dishes than on the NH 3 -treated PS dishes. Stable stemness markers were detected, including CD105, CD9 and SSEA-4, expressed on BCP-K1 growing on the modified PS dish surfaces, during 7 days of culturing. The presence of –NH 2 groups on the PS dish surface were revealed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. A large amount of oxygen- and nitrogen-containing functional groups, up to 9.0 %, were introduced by NH 3 plasma and N 2 plasma. The functional groups introduced on to the PS surfaces were clearly the key factors which enhanced WJMSCs attachment and stemness stability. Content Type Journal Article Category Original Research Pages 1-16 DOI 10.1007/s10616-012-9467-9 Authors Somruthai Tunma, The Graduate School, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Kewalin Inthanon, The Graduate School, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Chanokporn Chaiwong, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Jantrawan Pumchusak, Materials Science Research Center (MSRC) Faculty of Science, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Weerah Wongkham, Department of Biology, Faculty of Science, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Dheerawan Boonyawan, Materials Science Research Center (MSRC) Faculty of Science, Chiang Mai University, 239 Huaykaew Rd., Muang, 50200 Thailand Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9476-8 Authors Sandra Regina Lepri, General Biology Department, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 km 380, Campus Universitário, Cx. Postal 6001, Londrina, PR CEP 86051-980, Brazil Rodrigo Cabral Luiz, Pathological Sciences Department, State University of Londrina (UEL), Londrina, PR, Brazil Leonardo Campos Zanelatto, General Biology Department, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 km 380, Campus Universitário, Cx. Postal 6001, Londrina, PR CEP 86051-980, Brazil Patrícia Benites Gonçalves da Silva, General Biology Department, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 km 380, Campus Universitário, Cx. Postal 6001, Londrina, PR CEP 86051-980, Brazil Daniele Sartori, General Biology Department, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 km 380, Campus Universitário, Cx. Postal 6001, Londrina, PR CEP 86051-980, Brazil Lucia Regina Ribeiro, Institute of Biosciences, UNESP, Rio Claro, SP, Brazil Mario Sergio Mantovani, General Biology Department, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 km 380, Campus Universitário, Cx. Postal 6001, Londrina, PR CEP 86051-980, Brazil Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 °C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes ( cyp19a1 , fshr , lhr and star ). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle. Content Type Journal Article Category Original Research Pages 1-14 DOI 10.1007/s10616-012-9484-8 Authors B. Crespo, Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas (CSIC), Torre la Sal, Ribera de Cabanes s/n, 12595 Castellón, Spain S. Zanuy, Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas (CSIC), Torre la Sal, Ribera de Cabanes s/n, 12595 Castellón, Spain A. Gómez, Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas (CSIC), Torre la Sal, Ribera de Cabanes s/n, 12595 Castellón, Spain Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-012-9471-0 Authors M. Dhanasekaran, Stem Cell Department, Lifeline Multispeciality Hospital, Chennai, 600 096 India S. Indumathi, Department of Zoology and Biotechnology, Loyola College, Chennai, 600 096 India R. P. Lissa, Stem Cell Department, Lifeline Multispeciality Hospital, Chennai, 600 096 India R. Harikrishnan, Stem Cell Department, Lifeline Multispeciality Hospital, Chennai, 600 096 India J. S. Rajkumar, Stem Cell Department, Lifeline Multispeciality Hospital, Chennai, 600 096 India D. Sudarsanam, Department of Zoology and Biotechnology, Loyola College, Chennai, 600 096 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components. Content Type Journal Article Category Technical Note Pages 1-10 DOI 10.1007/s10616-012-9462-1 Authors Martin Jordan, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Damien Voisard, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Antoine Berthoud, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Laetitia Tercier, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Beate Kleuser, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Gianni Baer, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Hervé Broly, Biotech Process Sciences, Merck Serono Biotech Center, 1809 Fenil-sur-Corsier, Switzerland Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    β-glucan is an important polysaccharide due to its medicinal properties of stimulating the immune system and preventing chronic diseases such as cancer. The aim of the present study was to determine the anticlastogenic effect of β-glucan in cells exposed to ultraviolet radiation (UV). Chromosome aberration assay was performed in drug-metabolizing cells (HTC) and non drug-metabolizing cells (CHO-K1 and repair-deficient CHO-xrs5), using different treatment protocols. Continuous treatment (UV + β-glucan) was not effective in reducing the DNA damage only in CHO-xrs5 cells. However, the pre-treatment protocol (β-glucan before UV exposition) was effective in reducing DNA damage only in CHO-K1 cells. In post-treatment (β-glucan after UV exposition) did not show significative anticlastogenic effects, although there was a tendency toward prevention. The data suggest that β-glucan has more than one action mechanism, being capable of exerting desmutagenic as well as bio-antimutagenic action. The findings also suggest that the presence of the xenobiotic metabolizing system can reduce the chemopreventive capacity of β-glucan. Therefore, these results indicate that β-glucan from Saccharomyces cerevisiae can be used in the prevention and/or reduction of DNA damage. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9448-z Authors Ariane Fernanda da Silva, Departamento de Biologia Geral, Universidade Estadual de Londrina, Campus Universitário, Londrina, Paraná, Brazil Rodrigo Juliano Oliveira, Universidade Federal de Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil Andressa Megumi Niwa, Departamento de Biologia Geral, Universidade Estadual de Londrina, Campus Universitário, Londrina, Paraná, Brazil Gláucia Fernanda Rocha D’Epiro, Departamento de Biologia Geral, Universidade Estadual de Londrina, Campus Universitário, Londrina, Paraná, Brazil Lúcia Regina Ribeiro, Departamento de Biologia Celular, Universidade Estadual Paulista, Rio Claro, Brazil Mário Sérgio Mantovani, Departamento de Biologia Geral, Universidade Estadual de Londrina, Campus Universitário, Londrina, Paraná, Brazil Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    In this study, the anti-allergy potency of thirteen tannins isolated from the galls on buds of Carpinus tschonoskii (including two tannin derivatives) was investigated. RBL-2H3 (rat basophilic leukemia) cells were incubated with these compounds, and the release of β-hexosaminidase and cytotoxicity were measured. Of the thirteen tannins, tetragalloylglucose ( 2 ), pentagalloylglucose ( 3 ), casuarictin ( 4 ), and casuarinin ( 9 ) were the most potent inhibitors, and all the tannins showed no cytotoxic effect after 24 h of incubation. The results obtained suggest that tannins from C. tschonoskii are capable of inhibiting allergic reactions and may be useful for the treatment or prevention of type I allergic diseases. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9457-y Authors Parida Yamada, Alliance for Research on North Africa, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8572 Ibaraki, Japan Takako Ono, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8572 Ibaraki, Japan Hideyuki Shigemori, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8572 Ibaraki, Japan Junkyu Han, Alliance for Research on North Africa, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8572 Ibaraki, Japan Hiroko Isoda, Alliance for Research on North Africa, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8572 Ibaraki, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ m ) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ m did not always externalize PS, while some late apoptotic cells had polarized Δψ m . The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS–PI and Δψ m assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs. Content Type Journal Article Category Original Research Pages 1-12 DOI 10.1007/s10616-012-9455-0 Authors Yuhgi Suzuki, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Hiroo Hasegawa, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Tomohiro Tsuji, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Kazuto Tsuruda, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Daisuke Sasaki, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Kaori Ishihara, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Kazuhiro Nagai, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Katsunori Yanagihara, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Yasuaki Yamada, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Shimeru Kamihira, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki City, 852-8501 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, l -ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering. Content Type Journal Article Category Brief Report Pages 1-8 DOI 10.1007/s10616-012-9456-z Authors Naotaka Kishimoto, Department of Anesthesiology, Osaka Dental University, 8-1 Hanazonocho, Kuzuha, Hirakata, 573-1121 Japan Yoshihiro Momota, Department of Anesthesiology, Osaka Dental University, 8-1 Hanazonocho, Kuzuha, Hirakata, 573-1121 Japan Yoshiya Hashimoto, Department of Biomaterials, Osaka Dental University, 8-1 Hanazonocho, Kuzuha, Hirakata, 573-1121 Japan Kayoko Ando, Department of Anesthesiology, Osaka Dental University, 8-1 Hanazonocho, Kuzuha, Hirakata, 573-1121 Japan Takeshi Omasa, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima, 770-8506 Japan Junichiro Kotani, Department of Anesthesiology, Osaka Dental University, 8-1 Hanazonocho, Kuzuha, Hirakata, 573-1121 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The dried flower buds of Magnolia sp. are widely used as herbal medicines because of their anti-inflammatory, anti-malarial and anti-platelet activities. Here, we found that veraguensin and galgravin, lignan compounds derived from Magnolia sp., dose-dependently inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblastic cells. These compounds also inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells and bone marrow macrophages. In the RANKL-induced signaling pathway, veraguensin and galgravin reduced p38 phosphorylation and suppressed the expression of c-Fos, a key transcription factor for osteoclastogenesis. Veraguensin and galgravin also inhibited osteoclastic pit formation, which was accompanied by decreased mature osteoclast viability. In conclusion, these results indicate that veraguensin and galgravin can inhibit bone resorption and may offer novel compounds for the development of drugs to treat bone-destructive diseases such as osteoporosis. Content Type Journal Article Category JAACT Special Issue Pages 1-8 DOI 10.1007/s10616-011-9416-z Authors Midori Asai, Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Ji-Won Lee, Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549 Japan Yasunori Itakura, Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Bong-Keun Choi, Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Takayuki Yonezawa, Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Toshiaki Teruya, Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Byung-Yoon Cha, Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Motoko Ohnishi, Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Akira Yamaguchi, Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549 Japan Je-Tae Woo, Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Numerous challenges remain in the successful clinical translation of cell-based therapeutic studies for skeletal tissue repair, including appropriate cell sources and viable cell delivery systems. Poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) amphiphilic block copolymers have been extensively explored in microspheres preparation. Due to the introduction of hydrophilic PEG segments into PCL backbones, these copolymers have shown much more potentials in carrying protein, lipophilic drugs or genes than commonly used poly (ε-caprolactone) (PCL) and poly (lactic acid). The aim of this study is to investigate the attachment and osteogenic differentiation of human placenta derived mesenchymal stem cells (PMSCs) on PEG-PCL triblock copolymers nanofiber scaffolds. Here we demonstrated that PMSCs proliferate robustly and can be effectively differentiated into osteogenic-like cells on nanofiber scaffolds. This study provides evidence for the use of nanofiber scaffolds as an ideal supporting material for in vitro PMSCs culture and an in vivo cell delivery vehicle for bone repair. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9450-5 Authors Dongmei Zhang, State Key Laboratory of Biotherapy and Cancer Center West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041 People’s Republic of China Aiping Tong, State Key Laboratory of Biotherapy and Cancer Center West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041 People’s Republic of China Liangxue Zhou, Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041 People’s Republic of China Fang Fang, Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041 People’s Republic of China Gang Guo, State Key Laboratory of Biotherapy and Cancer Center West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals. Content Type Journal Article Category Method in Cell Science Pages 1-14 DOI 10.1007/s10616-012-9459-9 Authors Nuria Jiménez, Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Vincent J. D. Krouwer, Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Jan A. Post, Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Citral, 3,7-dimethyl-2,6-octadienal, is a key component of the essential oils extracted from several lemon-scented herbal plants. Besides its antifungal activity, the anticancer effect of citral was studied in recent years. In this study, we investigated the effect of citral on the acute promyelocytic leukemia cell line NB4. Citral treatment had an antiproliferative effect in NB4 cells via the induction of apoptosis assessed by morphology, proliferation assay, DNA electrophoresis, Annexin V-FITC/PI staining and caspase-3 activation. And citral induced apoptosis of NB4 cells in a dose- and time-dependent manner. In addition, citral treatment induced decreased mitochondrial membrane potential, indicating that citral induced apoptosis via the mitochondrial pathway. Bax up-regulation and Bcl-2 down-regulation on mRNA level and NF-κB down-regulation on protein level was found in this study, suggesting that Bcl-2, Bax and NF-κB may be involved in the mechanism of the apoptotic effect of citral on NB4 cells. These data suggest that citral has a potential therapeutic effect on leukemia. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9453-2 Authors Hailong Xia, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Wei Liang, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Qin Song, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Xiaowen Chen, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Xin Chen, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Jian Hong, The First Affiliated Hospital of Anhui Medical University, 218# Jixi Road, Hefei, 230022 Anhui, People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description: Erratum to: Suppressive effects of natural reduced waters on alloxan-induced apoptosis and type 1 diabetes mellitus Content Type Journal Article Category Erratum Pages 1-1 DOI 10.1007/s10616-012-9461-2 Authors Yuping Li, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Takeki Hamasaki, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Kiichiro Teruya, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Noboru Nakamichi, Hita Tenryosui Co. Ltd., Nakanoshima-machi, Hita, 877-0074 Japan Zbigniew Gadek, Centre for Holistic Medicine and Naturopathy, 57392 Nordenau, Germany Taichi Kashiwagi, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Hanxu Yan, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581 Japan Tomoya Kinjo, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581 Japan Takaaki Komatsu, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Yoshitoki Ishii, Hita Tenryosui Co. Ltd., Nakanoshima-machi, Hita, 877-0074 Japan Sanetaka Shirahata, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-011-9408-z Authors Muzi Jin, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Asga Wu, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Sergei Dorzhin, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Qunhua Yue, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Yuzhen Ma, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Dongjun Liu, Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, 010021 Hohhot, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Mesenchymal stem cells (MSCs) are multipotent, can be easily expanded in culture and hence are an attractive therapeutic tool for cardiac repair. MSCs have tremendous potential to transdifferentiate to cardiac lineage both in vitro and in vivo. The present study examined the differentiation capacity of conditioned media derived from ischemic cardiac tissue on human MSCs. Human Bone marrow-derived MSCs after due characterization by immunocytochemistry and flow cytometry for MSC specific markers were induced by culture media derived from ischemic (n = 13) and non-ischemic (n = 18) human cardiac tissue. Parallel cultures were treated with 5-azacytidine (5-azaC), a potent cardiomyogen. MSCs induced with ischemic conditioned media formed myotube like structures, expressed sarcomeric Troponin I, alpha myosin heavy chain proteins and were positive for cardiac specific markers (Nkx2.5, human atrial natriuretic peptide, myosin light chain-2a, GATA-4) as was observed in 5-azaC treated cells. However, uninduced MSCs as well as those induced with non-ischemic cardiac conditioned media still maintained the fibroblast morphology even after 3 weeks post-induction. Transmission electron microscopic studies of cardiomyocyte-like cells derived from MSCs revealed presence of sarcomeric bands but failed to show gap junctions and intercalated discs as of adult cardiomyocytes. These findings demonstrate that ischemic cardiac conditioned media induces morphological and molecular changes in MSCs with cardiac features, but at a primitive stage. Proteomics analysis of the ischemic conditioned media revealed differential expression of three relevant proteins (C-type lectin superfamily member 13, Testis-specific chromodomain protein Y2 and ADP/ATP translocase 1), whose exact role in cardiac regeneration needs further analysis. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9440-7 Authors Balasundari Ramesh, International Centre for Cardio Thoracic and Vascular Diseases, Frontier Lifeline and Dr. K.M.Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Mogappair, Chennai, 600101 India Dillip Kumar Bishi, International Centre for Cardio Thoracic and Vascular Diseases, Frontier Lifeline and Dr. K.M.Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Mogappair, Chennai, 600101 India Suneel Rallapalli, International Centre for Cardio Thoracic and Vascular Diseases, Frontier Lifeline and Dr. K.M.Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Mogappair, Chennai, 600101 India Sarasabarathi Arumugam, MIOT Hospital, 4/112, Mount Poonamalle Road, Manapakkam, Chennai, 600 089 India Kotturathu Mammen Cherian, International Centre for Cardio Thoracic and Vascular Diseases, Frontier Lifeline and Dr. K.M.Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Mogappair, Chennai, 600101 India Soma Guhathakurta, International Centre for Cardio Thoracic and Vascular Diseases, Frontier Lifeline and Dr. K.M.Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Mogappair, Chennai, 600101 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). MSCs are capable of differentiating into tissues of the three primary lineages and have the potential to enhance repair in damaged organs through the principals of regenerative medicine. Given the ease with which MSCs may be isolated from different species the aim of this study was to isolate and characterize putative bone marrow derived MSCs from the spiny mouse, Acomys cahirinus . MSCs were isolated from the spiny mouse in a traditional manner, and based on plastic adherence, morphology, colony forming unit-fibroblast assays and functional assessment (adipogenic, osteogenic and chondrogenic differentiation potential) a population of putative mesenchymal stem cells from the compact bone of the spiny mouse have been isolated and characterized. Such methodological approaches overcome the lack of species-specific antibodies for the spiny mouse and could be employed for other species where the cost of generating species-specific antibodies is not warranted. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9443-4 Authors Hayley Dickinson, Monash Immunology and Stem Cell Laboratories (MISCL), Melbourne, VIC, Australia Phillipa Milton, Monash Immunology and Stem Cell Laboratories (MISCL), Melbourne, VIC, Australia Graham Jenkin, Monash Immunology and Stem Cell Laboratories (MISCL), Melbourne, VIC, Australia Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Zinc plating is widely used to protect steels against corrosion. However, the possibility of a high environmental risk for zinc has been recently discussed among advanced countries and more environmentally-friendly substitutes are required urgently. Therefore, monitoring zinc concentration changes on metallic materials such as steel is very important. We chose to measure zinc concentration changes in some mammalian cells and confirmed that V79 cells were highly sensitive to changes in zinc concentrations. In this study, the following process was applied to the proprietary production for tin-zinc alloy films on steel using V79 cells. Specimens were immersed in PBS to produce extracts. Zinc concentrations in the extracts almost corresponded to zinc concentrations on steel surfaces. When extracts were added to a V79 cell culture, colony formation was inhibited, and inhibition increased with increases in zinc concentrations. Changes in zinc concentrations on steel surfaces with heat treatment could be monitored relatively well by V79 cells, even though the results were still semi-quantitative. Content Type Journal Article Category JAACT Special Issue Pages 1-7 DOI 10.1007/s10616-012-9433-6 Authors Akiko Ogawa, Department of Chemistry and Biochemistry, Suzuka National College of Technology, Suzuka, 510-0243 Japan Naoaki Okuda, Department of Chemistry and Biochemistry, Suzuka National College of Technology, Suzuka, 510-0243 Japan Katsuya Hio, Mie Prefecture Industrial Research Institute Metal Science Branch, Kuwana, 511-0937 Japan Hideyuki Kanematsu, Department of Materials Science and Engineering, Suzuka National College of Technology, Suzuka, 510-0243 Japan Hidekazu Tamauchi, Kitasato University School of Medicine, Sagamihara, 225-8555 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs’ protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-012-9428-3 Authors Tatiana Taís Sibov, Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, Hospital Israelita Albert Einstein, Avenida Albert Einstein, no 627/701, Morumbi, São Paulo, SP, CEP: 05651-901 Brazil P. Severino, Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil L. C. Marti, Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil L. F. Pavon, Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, Hospital Israelita Albert Einstein, Avenida Albert Einstein, no 627/701, Morumbi, São Paulo, SP, CEP: 05651-901 Brazil D. M. Oliveira, Universidade de Brasília, Brasília, DF, Brazil P. R. Tobo, Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil A. H. Campos, Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil A. T. Paes, Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil E. Amaro, Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, Hospital Israelita Albert Einstein, Avenida Albert Einstein, no 627/701, Morumbi, São Paulo, SP, CEP: 05651-901 Brazil L. F Gamarra, Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, Hospital Israelita Albert Einstein, Avenida Albert Einstein, no 627/701, Morumbi, São Paulo, SP, CEP: 05651-901 Brazil C. A. Moreira-Filho, Departamento de Pediatria, Faculdade de Medicina da Universidade de São Paulo-USP, São Paulo, SP, Brazil Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Omentum fat derived stem cells have emerged as an alternative and accessible therapeutic tool in recent years in contrast to the existing persuasive sources of stem cells, bone marrow and subcutaneous adipose tissue. However, there has been a scanty citation on human omentum fat derived stem cells. Furthermore, identification of specific cell surface markers among aforesaid sources is still controversial. In lieu of this existing perplexity, the current research work aims at signifying omentum fat as a ground-breaking source of stem cells by surface antigenic profiling of stem cell population. In this study, we examined and compared the profiling of cell surface antigenic expressions of hematopoietic stem cells, mesenchymal stem cells, cell adhesion molecules and other unique markers such as ABCG2, ALDH and CD 117 in whole cell population of human omentum fat, subcutaneous fat and bone marrow. The phenotypic characterization through flowcytometry revealed the positive expressions of CD 34, CD 45, CD 133, HLADR, CD 90, CD 105, CD 73, CD 29, CD 13, CD 44, CD 54, CD 31, ALDH and CD 117 in all sources. The similarities between the phenotypic expressions of omentum fat derived stem cells to that of subcutaneous fat and bone marrow substantiates that identification of ultimate source for curative therapeutics is arduous to assess. Nevertheless, these results support the potential therapeutic application of omentum fat derived stem cells. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9427-4 Authors M. Dhanasekaran, Lifeline Multispeciality Hospital, Perungudi, Chennai, India S. Indumathi, Loyola College, Nungambakkam, Chennai, India A. Kanmani, Lifeline Multispeciality Hospital, Perungudi, Chennai, India R. Poojitha, Lifeline Multispeciality Hospital, Perungudi, Chennai, India K. M. Revathy, Lifeline Multispeciality Hospital, Perungudi, Chennai, India J. S. Rajkumar, Lifeline Multispeciality Hospital, Perungudi, Chennai, India D. Sudarsanam, Loyola College, Nungambakkam, Chennai, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The aim of this study was to investigate the genotoxic and/or cytotoxic effects of Tamiflu, commercial form of the oseltamivir antiviral and most frequently prescribed for the treatment of influenza infections, on cultured human peripheral lymphocytes by using sister chromatid exchange (SCE), chromosomal aberration (CA), and cytokinesis-blocked micronucleus (CBMN) assays. Cells were treated with 0.5, 1, 2 μg/mL oseltamivir, the Tamiflu capsule ingredient, for 24 or 48 h in the absence or presence of an exogenous metabolic activation system (S9 mix). The test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. On the other hand, some concentrations of Tamiflu (2 μg/mL without S9 mix for 48 h and 1 μg/mL with S9 mix) induced SCE and also decreased significantly the proliferation index (PI) (48 h period) and the nuclear division index (NDI) (24 h period) ( P  〈 0.05) in the absence of S9 mix. Considering the results, Tamiflu did not induce significant increases of CA or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but weak SCE induction was observed. On the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the S9 mix. Content Type Journal Article Category Original Research Pages 1-7 DOI 10.1007/s10616-011-9422-1 Authors Hasan Basri Ila, Department of Biology, Faculty of Science and Letters, Cukurova University, 01330 Adana, Turkey Ahmet Ilhan, Department of Biology, Faculty of Science and Letters, Cukurova University, 01330 Adana, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-β was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-β to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7. Content Type Journal Article Category JAACT Special Issue Pages 1-9 DOI 10.1007/s10616-012-9442-5 Authors Arei Miyamoto, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Masamichi Takami, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Akifumi Matsumoto, Department of Prosthodontics, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Ayako Mochizuki, Department of Oral Physiology, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Takako Yamada, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Keita Tachi, Department of Prosthodontics, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Isao Shibuya, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Tomoya Nakamachi, Department of Anatomy, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Seiji Shioda, Department of Anatomy, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Kazuyoshi Baba, Department of Prosthodontics, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Tomio Inoue, Department of Oral Physiology, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Yoichi Miyamoto, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Mijung Yim, College of Pharmacy, Sookmyung Women’s University, Hyochangwongil 52, Yongsan-ku, Seoul, 140-742 Korea Ryutaro Kamijo, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Glycyrrhetinic acid (GA) is the active compound in Glycyrrhizae radix , a famous traditional Chinese medicine. Recently the anticancer activity of GA became the focus of scientific interest and many GA derivatives were developed as anti-tumor lead compounds. We previously reported that AEGA, a GA derivative, has proliferation inhibition and apoptosis-inducing activity in various human tumor cells. The present study was undertaken to further investigate the molecular mechanisms involved in AEGA-induced apoptosis in human leukemia K562 cells. AEGA can inhibit the growth of K562 cells in dose- and time-dependent manners determined by the MTT assay. Induction of apoptosis was evidenced by morphological changes and biochemical markers such as cell shrinkage, chromatin condensation and DNA ladder formation. Further mechanistic analysis revealed that AEGA induced apoptosis through the collapse of mitochondrial membrane potential, the accumulation of the cytosolic cytochrome c and the activation of caspase-9 and caspase-3. The apoptosis induction by AEGA was associated with the alteration in the ratio of Bcl-2/Bax protein expression. These results suggest that AEGA may induce apoptosis through a mitochondria-mediated pathway, and might have the therapeutic value against hematological malignancies. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-011-9419-9 Authors Zhenbei Gao, The Laboratory of Proteomics and Molecular Enzymology, College of Life Science, Zhejiang Sci-Tech University, Xiasha Higher Education Zone, Hangzhou, 310018 Zhejiang Province, China Xiao Kang, The Laboratory of Proteomics and Molecular Enzymology, College of Life Science, Zhejiang Sci-Tech University, Xiasha Higher Education Zone, Hangzhou, 310018 Zhejiang Province, China Jun Hu, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Haidian District, Beijing, 100084 China Yong Ju, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Haidian District, Beijing, 100084 China Chuanlian Xu, The Laboratory of Proteomics and Molecular Enzymology, College of Life Science, Zhejiang Sci-Tech University, Xiasha Higher Education Zone, Hangzhou, 310018 Zhejiang Province, China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage. Content Type Journal Article Category JAACT Special Issue Pages 1-8 DOI 10.1007/s10616-011-9400-7 Authors Hiroto Miwa, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku N13W8, Sapporo, 060-8628 Japan Yoshiki Hashimoto, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku N13W8, Sapporo, 060-8628 Japan Keiji Tensho, Department of Orthopedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621 Japan Shigeyuki Wakitani, Department of Orthopedic Surgery, Osaka City University School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, Osaka 545-8585, Japan Mutsumi Takagi, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku N13W8, Sapporo, 060-8628 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (~50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (~90%). Moreover dependency of hMSCs to size of transfected nucleic acid and time-dependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, which eventually improves nonviral cell-mediated gene therapy. Content Type Journal Article Category Original Research Pages 1-18 DOI 10.1007/s10616-012-9430-9 Authors Behnaz Bakhshandeh, Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran Masoud Soleimani, Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran Maryam Hafizi, Stem Cell Biology Department, Stem Cell Technology Research Center, Tehran, Iran Nasser Ghaemi, School of Chemistry, College of Science, University of Tehran, Tehran, Iran Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r 2 values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions. Content Type Journal Article Category JAACT Special Issue Pages 1-17 DOI 10.1007/s10616-011-9377-2 Authors Yoshinobu Konno, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Yuki Kobayashi, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Ken Takahashi, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Eiji Takahashi, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Shinji Sakae, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Masako Wakitani, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Kazuya Yamano, Tokyo Research Park, Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo, 194-8533 Japan Toshiyuki Suzawa, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Keiichi Yano, Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan Toshio Ohta, Fuji Research Park, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Suntou-gun, Shizuoka, 411-8731 Japan Masamichi Koike, Kyowa Hakko Kirin Pharma, Inc., 212 Carnegie Center, Suite 101, Princeton, NJ 08540, USA Kaori Wakamatsu, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, 376-8515 Japan Shinji Hosoi, Kyowa Hakko Kirin Co., Ltd., 1-6-1 Ohte-machi, Chiyoda-ku, Tokyo, 100-8185 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer’s disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran— S. choloroleuca , S. mirzayanii and S. santolinifolia —against H 2 O 2 -induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases. Content Type Journal Article Category Original Research Pages 1-17 DOI 10.1007/s10616-011-9418-x Authors Shabnam Zeighamy Alamdary, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Fariba Khodagholi, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Fatemeh Shaerzadeh, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Niloufar Ansari, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Ali Sonboli, Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Tehran, Iran Solaleh Khoramian Tusi, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Phytoestrogens are a group of naturally occurring compounds that have weak estrogenic activity. Genistein and daidzein are major phytoestrogens produced by soybeans. It has been reported previously that at high concentration, some phytoestrogens inhibit cell cycle progression of mouse germinal vesicle (GV) oocytes, but the environmentally relevant level is much lower. Here we show the effects of low concentrations of the isoflavones genistein, daidzein and the daidzein metabolite, equol, on mouse oocyte maturation. GV oocytes denuded of cumulus cells were cultured in TaM medium containing low levels (5 μM) of genistein, daidzein. or equol. In all cases, the oocytes underwent normal GV break down, first polar body extrusion and became arrested at metaphase II (mII). As judged by fluorescence microscopy, the treated mII oocytes exhibited normal distributions of actin microfilaments, cortical granules and metaphase spindle formation with condensed metaphase chromatin. Moreover, mRNA expression levels of the cytostatic factors Emi2 and Mos were similar to those of their respective controls. These data suggest that exposure of maturing GV oocytes to environmental levels of genistein, daidzein or equol in vitro do not cause negative effects on maturation to produce mII oocytes. Content Type Journal Article Category JAACT Special Issue Pages 1-7 DOI 10.1007/s10616-011-9369-2 Authors Naoko Yoshida, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577 Japan Katsushige Mizuno, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Eight intestinal cell lines, established from different animal species were submitted to DSMZ (German Collection of Microorganisms and Cell Cultures) in order to analyze their species of origin and their microbial contamination. Species identity was determined by PCR targeting mitochondrial genes and hence confirmed by sequencing the amplified PCR products. For three cell lines (CIEB, CLAB, PSI-1) we confirmed the species identity, whereas the species of origin of the three other cell lines (B6, B10XI and IPEC) was not the expected one: B6 and B10XI cells, which were supposed to be of chicken origin were identified as porcine cells. IPEC, allegedly a sub clone of the well-known porcine intestinal cell line IPEC-J2, was of bovine instead of porcine origin. However, two further IPEC-clones, namely IPEC-1 and IPEC-J2, provided by another source were shown to be derived from the correct species (i.e. pig). Furthermore, six out of these eight cell lines turned out to be highly contaminated with mycoplasma. Alerted by this high incidence of infected and false specified cell lines, we feel obliged to inform all those working with animal intestinal cell lines and we strongly recommend verifying the species identity before using them. Also, the presence of mycoplasma should be tested when taking the cells in culture for the first time, and this mycoplasma control should be repeated at regular time intervals (e.g. every 4 weeks). Content Type Journal Article Category Brief Report Pages 1-6 DOI 10.1007/s10616-011-9420-3 Authors Klaus G. Steube, Leibniz-Institut, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany Anne-Leena Koelz, Leibniz-Institut, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany Cord C. Uphoff, Leibniz-Institut, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany Hans G. Drexler, Leibniz-Institut, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany Jeannette Kluess, Medical Faculty, Institute of Anatomy, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany Pablo Steinberg, Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na + , osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors. Content Type Journal Article Category Original Research Pages 1-12 DOI 10.1007/s10616-012-9446-1 Authors Wei-Ting Hsu, Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Rigzen P. S. Aulakh, Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Donald L. Traul, TAP Biosystems, York Way, Royston, Hertfordshire SG8 5WY, UK Inn H. Yuk, Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The control of viral infections, especially those caused by influenza viruses, is of great interest in Public Health. Bio prospection has shown the presence of active principles in the hemolymph of arthropods, and in the salivary gland of ticks, and some of these are of interest for the development of new pharmacological drugs. Ticks lay their eggs in the environment, and to protect them from desiccation and microbial attack they involve the eggs in a waxy layer produced by an organ known as Gené’s Organ. In this study, the eggs wax from tick Amblyomma cajennense (Fabricius) was extracted using ice cold phosphate buffer. The antiviral activity was evaluated with picornavirus and influenza virus. In both cases egg wax was able to inhibit virus replication. For influenza virus, an amount as small as 12 μg/mL of crude egg wax suspension neutralized 128 UHA (hemaglutinant unit) of H 1 N 1 influenza virus. With picornavirus, egg wax led to a 256-fold reduction in virus production by L929 cells. Egg wax was not cytotoxic to VERO, MDCK and L929 cell, being observed that the cell morphology was preserved with concentration as high as 2 mg/mL. In addition no genotoxic effect was observed for Vero cells, suggesting a very interesting potential antiviral activity. Content Type Journal Article Category Original Research Pages 1-6 DOI 10.1007/s10616-012-9444-3 Authors Solange de Lima-Netto, Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Alessandro Pinheiro, Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Eliana Nakano, Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Rita Maria Zucatelli Mendonça, Laboratório de Virologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Darci Moraes Barros-Battesti, Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Ronaldo Zucatelli Mendonça, Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP CEP 05503-900, Brazil Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The effect of femtosecond laser irradiation on adherent mesenchymal stem cell was investigated with the aim to develop a novel noninvasive cell purification system. A single mesenchymal stem cell was irradiated with a femtosecond laser on the center of the cell using several energy values and the cell lost its replication capacity with one time irradiation with an energy of 2.0 μJ. Besides at a neighbor point in the major axis, when irradiated in the minor axis at a distance shorter than 10 μm, the cell stopped its replication capacity. The accumulation effect of cell damage caused by multiple laser shots at a neighbor point in the minor axis was correlated with the critical distance at which the cell lost its replication capacity. Finally, a novel equation of laser cell damage as a function of laser pulse energy and laser shot number is proposed. Content Type Journal Article Category JAACT Special Issue Pages 1-7 DOI 10.1007/s10616-012-9437-2 Authors Jun Sakai, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8 Kita-ku, Sapporo, 060-8628 Japan Daniel Roldán, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8 Kita-ku, Sapporo, 060-8628 Japan Kosei Ueno, Section of Informatics and Processing, Research Institute for Electronic Science, Hokkaido University, N21W10 Kita-ku, Sapporo, 001-0021 Japan Hiroaki Misawa, Section of Informatics and Processing, Research Institute for Electronic Science, Hokkaido University, N21W10 Kita-ku, Sapporo, 001-0021 Japan Yoichiroh Hosokawa, Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan Takanori Iino, Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan Shigeyuki Wakitani, Orthopedic Surgery, Osaka City University School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585 Osaka, Japan Mutsumi Takagi, Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8 Kita-ku, Sapporo, 060-8628 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Transient protein expression using polyethyleneimine as a transfection agent is useful for the rapid production of small amounts of recombinant proteins. It is known that an increase in extracellular DNA concentration during transfection can lead to a nonlinear increase in intracellular DNA concentration. We present an approach that hypothesizes that this nonlinearity can be used to decrease the amount of plasmid required for productive transfections. Through addition of non coding ‘carrier’ DNA to increase total DNA concentration during transfection, we report a statistically significant increase in protein (IgG) expression per unit plasmid used for transfection. This approach could be useful to increase protein yields for large scale transfections under conditions where plasmid availability is limited. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9435-4 Authors Ketaki Pradhan, Chemical Engineering and Process Development, CSIR - National Chemical Laboratory, Pune, 411008 India Mugdha Gadgil, Chemical Engineering and Process Development, CSIR - National Chemical Laboratory, Pune, 411008 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The genotoxic effects of antimicrobial food additive sodium sorbate (SS) was assessed by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), and micronucleus (MN) in cultured human lymphocytes and comet assay in isolated human lymphocytes. Lymphocytes were treated with four concentrations (100, 200, 400 and 800 μg/ml) of SS as well as a negative (sterile distilled water) and a positive control (Mitomycin-C: MMC for cultured lymphocytes and H 2 O 2 for isolated lymphocytes). The result of this study indicated that SS increased the frequency of CAs at both 24 and 48 h period compared to control. When gaps were included, this increase was significant at 200, 400 and 800 μg/ml concentrations at 24 h and, at all concentrations at 48 h treatment time. When gaps were excluded, this increase was significant at only 800 μg/ml concentration at both 24 and 48 h treatments. In addition, SS increased SCEs/cell and MN frequency at 400 and 800 μg/ml concentrations at both 24 and 48 h compared to negative control. Furthermore, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1 h in vitro exposure. The present results show that SS is genotoxic to the human peripheral blood lymphocytes in vitro at the highest concentrations. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9434-5 Authors Sevcan Mamur, Genetic Toxicology Laboratory, Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Deniz Yüzbaşıoğlu, Genetic Toxicology Laboratory, Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Fatma Ünal, Genetic Toxicology Laboratory, Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Hüseyin Aksoy, Department of Biology, Faculty of Art and Science, Sakarya University, Sakarya, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Recent studies have shown that the use of biomaterials and new biodegradable scaffolds for repair or regeneration of damaged tissues is of vital importance. Scaffolds used in tissue engineering should be biodegradable materials with three-dimensional structures which guide the growth and differentiation of the cells. They also tune physical, chemical and biological properties for efficient supplying of the cells to the selected tissues and have proper porosity along with minimal toxic effects. In this manner, the study of these characteristics is a giant stride towards scaffold design. In this study, Gelatin/Siloxane/Hydroxyapatite (GS-Hyd) scaffold was synthesized and its morphology, in vivo biodegradability, cytotoxic effects and ability for cell adhesion were investigated using mesenchymal stem cells (MSCs). The cells were treated with different volumes of the scaffold suspension for evaluation of its cytotoxic effects. The MSCs were also seeded on scaffolds and cultured for 2 weeks to evaluate the ability of the scaffold in promoting of cell adhesion and growth. To check the biodegradability of the scaffold in vivo, scaffolds were placed in the rat body for 21 days in three different positions of thigh muscle, testicle, and liver and they were analyzed by scanning electron microscopy (SEM) and weight changes. According to the results of the viability of this study, no cytotoxic effects of GS-Hyd scaffold was found on the cells and MSCs could adhere on the scaffold with expanding their elongations and forming colonies. The rate of degradation as assessed by weight loss was significant within each group along with significant differences between different tissues at the same time point. SEM micrographs also indicated the obvious morphological changes on the surface of the particles and diameter of the pores through different stages of implantation. The greatest amount of degradation happened to the scaffold particles implanted into the muscle, followed by testicle and liver, respectively. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-012-9426-5 Authors Zeinab Neshati, Cell and Molecular Biology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Ahmad Reza Bahrami, Cell and Molecular Biology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Hossein Eshtiagh-Hosseini, Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Maryam M. Matin, Cell and Molecular Biology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Mohammad Reza Housaindokht, Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Taymaz Tabari, Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Mohammad Amin Edalatmanesh, Cell and Molecular Biology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30 min, cultured for 12 days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120 days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank’s Buffered Salt Solution (HBSS) as a control. At 120 days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-012-9431-8 Authors Guang-Ping Ruan, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Jin-Xiang Wang, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Rong-Qing Pang, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Xiang Yao, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Xue-Min Cai, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Qiang Wang, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Li-Hua Ma, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Xiang-Qing Zhu, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Xing-Hua Pan, Research Center of Stem Cell, Tissue and Organ Engineering, Kunming General Hospital of PLA, Kunming, 650032 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on - line quantified up to 10 6  cell mL −1 . Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f c , which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-011-9421-2 Authors Emma Petiot, Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, Nancy-Université, 2 avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy Cedex, France Amal El-Wajgali, Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, Nancy-Université, 2 avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy Cedex, France Geoffrey Esteban, FOGALE Nanotech, 125 rue de l’Hostelerie, Ville Active Bâtiment A, Parc Acti plus, 30900 Nîmes, France Cécile Gény, Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy L’Etoile, France Hervé Pinton, Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy L’Etoile, France Annie Marc, Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, Nancy-Université, 2 avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy Cedex, France Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Permethrin is a common synthetic chemical, widely used as an insecticide in agriculture and other domestic applications. The previous reports indicated that permethrin is a highly toxic synthetic pyrethroid pesticide to human and environmental health. Therefore, the present experiment was undertaken to determine the effectiveness of olive leaf extract in modulating the permethrin induced genotoxic and oxidative damage in rats. The animals used were broadly divided into four (A, B, C and D) experimental groups. Group A rats served as control animals and received distilled water intraperitoneally (n = 5). Groups B and C rats received intraperitoneal injections of permethrin (60 mg kg −1  b.w) and olive leaf extract (500 mg kg −1  b.w), respectively. Group D rats received permethrin (60 mg kg −1  b.w) plus olive leaf extract (500 mg kg −1  b.w). Rats were orally administered their respective feed daily for 21 days. At the end of the experiment rats were anesthetized and serum and bone marrow cell samples were obtained. Genotoxic damage was assessed by micronucleus and chromosomal aberration assays. Total antioxidant capacity and total oxidant status were also measured in serum samples to assess oxidative status. Treatment of Group B with permethrin resulted in genotoxic damage and increased total oxidant status levels. Permethrin treatment also significantly decreased ( P  〈 0.05) total antioxidant capacity level when compared to Group A rats. Group C rats showed significant increases ( P  〈 0.05) in total antioxidant capacity level and no alterations in cytogenetic parameters. Moreover, simultaneous treatments with olive leaf extract significantly modulated the toxic effects of permethrin in Group D rats. It can be concluded that olive leaf extract has beneficial influences and could be able to antagonize permethrin toxicity. As a result, this investigation clearly revealed the protective role of olive leaf extract against the genetic and oxidative damage by permethrin in vivo for the first time. Content Type Journal Article Category Original Research Pages 1-6 DOI 10.1007/s10616-011-9424-z Authors Hasan Turkez, Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum, Turkey Başak Togar, Department of Biology, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey Elif Polat, Department of Biochemistry, Medical Faculty, Atatürk University, 25240 Erzurum, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    We purified and identified an IgE suppressor from the strawberry ‘Toyonoka’, based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15–48 kDa range and 〈10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15–48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0–9.2. We focused on the active fractions of pI 8.0–9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with 〉98% identical nucleotides and 〉99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health. Content Type Journal Article Category JAACT Special Issue Pages 1-6 DOI 10.1007/s10616-012-9432-7 Authors Akira Iwamoto, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, 808-0196 Fukuoka, Japan Kazuhiro Mitsuda, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, 808-0196 Fukuoka, Japan Aiko Inoue, The Cell Engineering Center, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu, 802-0985 Fukuoka, Japan Tamaki Kato, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, 808-0196 Fukuoka, Japan Yuichi Inoue, The Cell Engineering Center, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu, 802-0985 Fukuoka, Japan Hiroharu Kawahara, The Cell Engineering Center, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu, 802-0985 Fukuoka, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    A MATLAB ® toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fed-batch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-011-9425-y Authors Chetan T. Goudar, Cell Culture Development, Global Biological Development, Bayer HealthCare, 800 Dwight Way, Berkeley, CA 94710, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood. Considering that tolerogenic antigen-presenting cells (APCs) are likely to be a key factor in the acquisition of oral tolerance, in the present study, we examined the changes of dendritic cells (DCs) in the lamina propria (LP) on exposure to food proteins at weaning. C57BL/6 female mice were weaned at the age of 3 weeks and orally administered 10 mg of ovalbumin (OVA) for ten consecutive days after weaning. The administration led to a decrease in the plasma level of immunoglobulin specific for OVA, suggesting the acquisition of oral tolerance. The uptake of fluorescence-labeled OVA was significantly observed for CD11c + LPDCs. When we analyzed the changes of two types of LPDCs, PDCA-1 + MHC II + DCs and CD103 + MHC II + DCs, ten consecutive gavages of OVA marginally, but not significantly, augmented only the frequency of PDCA-1 + MHC II + DCs. Considering that the change of APCs likely appears immediately on the response to antigen intake, we found the statistically significant increase in the frequency of PDCA-1 + DCs, but not in that of CD103 + DCs, even after two treatments, indicating PDCA-1 + DCs to be recruited in the LP within 2 days of exposure to food proteins. These results suggest that the behavior of tolerogenic PDCA-1 + DCs may change at weaning with the removal of the immunoprotective components of maternal milk. Content Type Journal Article Category JAACT Special Issue Pages 1-10 DOI 10.1007/s10616-011-9351-z Authors Ryuji Ohue, Laboratory of Food Environmental Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, Kyoto 606-8502, Japan Masahiro Nakamoto, Laboratory of Food Environmental Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, Kyoto 606-8502, Japan Naofumi Kitabatake, Laboratory of Food Environmental Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, Kyoto 606-8502, Japan Fumito Tani, Laboratory of Food Environmental Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, Kyoto 606-8502, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    By means of a model predictive control strategy it was possible to ensure a high batch-to-batch reproducibility in animal cell (CHO-cell) suspensions cultured for a recombinant therapeutic protein (EPO) production. The general control objective was derived by identifying an optimal specific growth rate taking productivity, protein quality and process controllability into account. This goal was approached indirectly by controlling the oxygen mass consumed by the cells which is related to specific biomass growth rate and cell concentration profile by manipulating the glutamine feed rate. Process knowledge represented by a classical model was incorporated into the model predictive control algorithm. The controller was employed in several cultivation experiments. During these cultivations, the model parameters were adapted after each sampling event to cope with changes in the process’ dynamics. The ability to predict the state variables, particularly for the oxygen consumption, led to only moderate changes in the desired optimal operational trajectories. Hence, nearly identical oxygen consumption profiles, cell and protein titers as well as sialylation patterns were obtained for all cultivation runs. Content Type Journal Article Category Original Research Pages 1-12 DOI 10.1007/s10616-012-9438-1 Authors Mathias Aehle, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Weinbergweg, 22, 06120 Halle (Saale), Germany Kaya Bork, Institute of Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystrasse 1, 06114 Halle (Saale), Germany Sebastian Schaepe, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Weinbergweg, 22, 06120 Halle (Saale), Germany Artur Kuprijanov, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Weinbergweg, 22, 06120 Halle (Saale), Germany Rüdiger Horstkorte, Institute of Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystrasse 1, 06114 Halle (Saale), Germany Rimvydas Simutis, Institute of Automation and Control Systems, Kaunas University of Technology, Studentu g. 48, 3028 Kaunas, Lithuania Andreas Lübbert, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Weinbergweg, 22, 06120 Halle (Saale), Germany Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The most potent of the dioxins, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), is a persistent and ubiquitous environmental contaminant. And the health impact of exposure to TCDD is of great concern to the general public. Recent data indicate that l -glutamine (Gln) has antioxidant properties and may influence hepatotoxicity. The objective of the present study was undertaken to explore the effectiveness of Gln in alleviating the hepatotoxicity of TCDD on primary cultured rat hepatocytes. Gln (0.5, 1 and 2 mM) was added to cultures alone or simultaneously with TCDD (0.005 and 0.01 mM). The hepatocytes were treated with TCDD and Gln for 48 h. Then cell viability was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and lactate dehydrogenase (LDH) release, while total antioxidant capacity (TAC), total glutathione (TGSH) and total oxidative stress (TOS) levels were determined to evaluate the oxidative injury. The DNA damage was also analyzed by liver micronucleus assay (MN) and 8-oxo-2-deoxyguanosine (8-OH-dG). The results of MTT and LDH assays showed that TCDD decreased cell viability but not l -glutamine. TCDD also increased TOS level in rat hepatocytes and significantly decreased TAC and TGSH levels. On the basis of increasing doses, the dioxin in a dose-dependent manner caused significant increases of micronucleated hepatocytes (MNHEPs) and 8-OH-dG as compared to control culture. Whereas, in cultures exposured with Gln alone, TOS levels were not changed and TAC and TGSH together were significantly increased in dose-dependent fashion. The presence of Gln with TCDD modulated the hepatotoxic effects of TCDD on primary hepatocytes cultures. Noteworthy, Gln has a protective effect against TCDD-mediated DNA damages. As conclusion, we reported here an increased potential therapeutic significance of l -glutamine in TCDD-mediated hepatic injury for the first time. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9449-y Authors Hasan Turkez, Department of Molecular Biology and Genetics, Faculty of Sciences, Erzurum Technical University, Erzurum, Turkey Fatime Geyikoglu, Department of Biology, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey Mokhtar I. Yousef, Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, 21526 Egypt Kubra Celik, Department of Biology, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey Tulay O. Bakir, Department of Biology, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Cervical cancer is one of the most common gynecologic malignancies and poses a serious health problem worldwide. Identification and characterization of cervical cancer stem cells may facilitate the development of novel strategies for the treatment of advanced and metastatic cervical cancer. Breast cancer-resistance protein (Bcrp1)-positive cells were selected from a population of parent HeLa cells using flow cytometry. The invasion capacity of Bcrp1-positive and -negative cells was analyzed with a Boyden chamber invasion test. The tumorigenicity of these cells was determined by in vivo transplantation in non-obesity diabetes/severe combined immunodeficiency (NOD/SCID) mice. The Bcrp1-positive subpopulation accounted for about 7% of the parent HeLa cell population. The proliferative capacity of the Bcrp1-positive cells was greater than that of the Bcrp1-negative cells ( P  〈 0.05). In the invasion assay, the Bcrp1-positive cells demonstrated a greater invasive capacity through the artificial basement membrane than their Bcrp1-negative counterparts. Following transplantation of 10 4 cells, only the Bcrp1-positive cells formed tumors in NOD/SCID mice. When 10 5 or 10 6 cells were transplanted, the tumor incidence and the tumor mass were greater in the Bcrp1-positive groups than those in the Bcrp1-negative groups ( P  〈 0.05). The Bcrp1-positive subpopulation cervical cancer stem cells. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9436-3 Authors Song-Ling Zhang, Department of Gynecology and Obstetrics, First Hospital of Jilin University, Xinmin Street 71-1, Changchun, 130021 People’s Republic of China Yi-Shu Wang, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Xinmin Street 126, Changchun, 130021 People’s Republic of China Tong Zhou, Norman Bethune College of Jilin University, Changchun, 130021 People’s Republic of China Xiao-Wei Yu, Department of Gynecology and Obstetrics, First Hospital of Jilin University, Xinmin Street 71-1, Changchun, 130021 People’s Republic of China Zhen-Tong Wei, Department of Gynecology and Obstetrics, First Hospital of Jilin University, Xinmin Street 71-1, Changchun, 130021 People’s Republic of China Yu-Lin Li, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Xinmin Street 126, Changchun, 130021 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit + stem cells (c-kit + AFS) were compared with the c-kit − (unselected) stem cells (c-kit − AFS). Our findings revealed that the optimal period to obtain c-kit + AFS cells was between 16 and 22 weeks of gestation. Following cell sorting, c-kit + AFS cells shared similar morphological and proliferative characteristics as the c-kit − AFS cells. Both c-kit + and c-kit − AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit + and c-kit − AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit + AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit − AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit + AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9441-6 Authors Jing Bai, Department of Cardiology, First Affiliated Hospital of Chinese PLA General Hospital, 51 Fucheng Road, Haidian District, Beijing, 100048 China Yiru Wang, Institute of Geriatric Cardiology, Chinese PLA General Hospital, 28 Fuxing Road, Haidian District, Beijing, 100853 China Lifeng Liu, Institute of Geriatric Cardiology, Chinese PLA General Hospital, 28 Fuxing Road, Haidian District, Beijing, 100853 China Jie Chen, Institute of Geriatric Cardiology, Chinese PLA General Hospital, 28 Fuxing Road, Haidian District, Beijing, 100853 China Wenlan Yang, Direction Center of Family Planing, Haidian Maternal and Children’s Hospital, 33 Haidian Southroad, Haidian District, Beijing, 100080 China Lianru Gao, Department of Cardiology, Navy General Hospital, 6 Fucheng Road, Haidian District, Beijing, 100048 China Yu Wang, Institute of Geriatric Cardiology, Chinese PLA General Hospital, 28 Fuxing Road, Haidian District, Beijing, 100853 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Several lichen species have been used for medicinal purposes throughout the ages, and they are reported to be effective in the treatment of different disorders including ulcer and cancer. It is revealed that lichens may be easily accessible sources of natural drugs and possible food supplements after their safety evaluations. The main objective in this study was to evaluate the roles of aqueous extracts of Xanthoria elegans (at 25, 50 and 100 μg/ml) upon mitomycin C (MMC; at 10 −7 M) induced genotoxic and oxidative damages in cultured human lymphocytes. X. elegans were collected from the Erzurum and Artvin provinces (in Turkey) during August 2010. After the application of MMC and X. elegans extract (XEE), separate and together, human whole blood cultures were assessed by four genotoxicity end-points including chromosomal aberration, micronucleus, sister chromatid exchange (SCE) and 8-oxo-2-deoxyguanosine (8-OH-dG) assays. In addition, biochemical parameters [total antioxidant capacity (TAC) and total oxidative stress (TOS)] were examined to determine oxidative effects. According to our results, the frequencies of cytogenetic endpoints and 8-OH-dG levels were significantly increased by MMC compared with controls in human peripheral lymphocytes. MMC caused oxidative stress by altering TAC and TOS levels. On the contrary, XEE led to increases of TAC level without changing TOS level. XEE had no genotoxic effect. Furthermore, our findings revealed that MMC induced increases in the mean frequencies of four genotoxic indices were diminished by XEE in dose dependent manner, indicating its protective role towards cells from MMC exerted injury. In conclusion, the results obtained in the present study indicate for the first time that XEE is a potential source of natural antigenotoxicants. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9447-0 Authors Hasan Turkez, Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum, Turkey Elanur Aydin, Department of Biology, Faculty of Science, Atatürk University, Erzurum, Turkey Ali Aslan, Department of Biology, Kazım Karabekir Education Faculty, Atatürk University, Erzurum, Turkey Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm 3 after 3 days, 1.24 ± 0.76 mm 3 after 7 days, 0.33 ± 0.03 mm 3 after 14 days, and 0.09 ± 0.05 mm 3 after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-011-9417-y Authors Yinghua Guo, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Longxiang Su, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Junlou Wu, From Shandong Provincial Hospital, Shandong University School of Medicine, Jinan, 250021 China Dong Zhang, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Xiaojun Zhang, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Guizhi Zhang, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Tianzhi Li, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Junfeng Wang, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Changting Liu, Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853 People’s Republic of China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins. Content Type Journal Article Category JAACT Special Issue Pages 1-13 DOI 10.1007/s10616-011-9397-y Authors Yoshinori Kawabe, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Hirokatsu Makitsubo, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Yujiro Kameyama, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Shuohao Huang, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Akira Ito, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Masamichi Kamihira, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Insulin-producing cells express limited activities of anti-oxidative enzymes. Therefore, reactive oxygen species (ROS) produced in these cells play a crucial role in cytotoxic effects. Furthermore, diabetes mellitus (DM) development is closely linked to higher ROS levels in insulin-producing cells. Hita Tenryosui Water ® (Hita T. W., Hita, Japan) and Nordenau water (Nord. W., Nordenau, Germany), referred to as natural reduced waters (NRWs), scavenge ROS in cultured cells, and therefore, might be a possibility as an alternative to conventional pharmacological agents against DM. Therefore, this study aimed to investigate the role of NRWs in alloxan (ALX)-induced β-cell apoptosis as well as in ALX-induced diabetic mice. NRWs equally suppressed DNA fragmentation levels. Hita T. W. and Nord. W. ameliorated ALX-induced sub-G 1 phase production from approximately 40% of control levels to 8.5 and 11.8%, respectively. NRWs restored serum insulin levels ( p  〈 0.01) and reduced blood glucose levels ( p  〈 0.01) in ALX-induced mice. Hita T. W. restored tissue superoxide dismutase (SOD) ( p  〈 0.05) activity but not tissue catalase activity. Hita T. W. did not elevate SOD or catalase activity in HIT-T15 cells. Nord. W. restored SOD ( p  〈 0.05) and catalase ( p  〈 0.05) activity in both cultured cells and pancreatic tissue to normal levels. Even though variable efficacies were observed between Hita T. W. and Nord. W., both waters suppressed ALX-induced DM development in CD-1 male mice by administering NRWs for 8 weeks. Our results suggest that Hita T. W. and Nord. W. protect against ALX-induced β-cell apoptosis, and prevent the development of ALX-induced DM in experimental animals by regulating ALX-derived ROS generation and elevating anti-oxidative enzymes. Therefore, the two NRWs tested here are promising candidates for the prevention of DM development. Content Type Journal Article Category JAACT Special Issue Pages 1-17 DOI 10.1007/s10616-011-9414-1 Authors Yuping Li, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Takeki Hamasaki, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Kiichiro Teruya, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Noboru Nakamichi, Hita Tenryosui Co. Ltd., Nakanoshima-machi, Hita, 877-0074 Japan Zbigniew Gadek, Centre for Holistic Medicine and Naturopathy, 57392 Nordenau, Germany Taichi Kashiwagi, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Hanxu Yan, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581 Japan Tomoya Kinjo, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581 Japan Takaaki Komatsu, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Yoshitoki Ishii, Hita Tenryosui Co. Ltd., Nakanoshima-machi, Hita, 877-0074 Japan Sanetaka Shirahata, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Polyphenols are known to exhibit wide spectrum of benefit for brain health and to protect from several neurodegenerative diseases. The present study was sought to determine the neuroprotective effects of Rosmarinus officinalis ’ polyphenols (luteolin, carnosic acid, and rosmarinic acid) through the investigation of stress-related proteins. We carried out measurement of the expression of heat-shock protein (Hsp) 47 promoter in heat stressed Chinese hamster ovary transfected cells. We performed proteomic analysis and confirmed gene expression by real time PCR in PC12 cells. Results showed that these compounds modulated significant and different effects on the expression of 4 stress-related proteins: heat shock protein 90 α ( Hsp90 ), Transitional endoplasmic reticulum ATPase ( VCP/p97 ), Nucleoside diphosphate kinase ( NDK ), and Hypoxia up-regulated protein 1 ( HYOU1 )) at translational and post translational levels in PC12 cells and they downregulated the expression of Hsp47 activity in Chinese hamster transformed cells. These findings suggest that luteolin, carnosic acid, and rosmarinic acid may modulate the neuroprotective defense system against cellular stress insults and increase neuro-thermotolerance. Content Type Journal Article Category JAACT Special Issue Pages 1-10 DOI 10.1007/s10616-011-9352-y Authors Abdelfatteh E. L. Omri, Graduate School of Life and Environmental Sciences, Alliance for Research on North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8572, Japan Junkyu Han, Graduate School of Life and Environmental Sciences, Alliance for Research on North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8572, Japan Manef Ben Abdrabbah, Unité de Recherche Physico-chimie et Moléculaire, Institut Préparatoire aux Etudes Scientifiques et Techniques IPEST, La Marsa, University of Carthage, 2070 Tunis, Tunisia Hiroko Isoda, Graduate School of Life and Environmental Sciences, Alliance for Research on North Africa (ARENA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8572, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description: Galangin and its emerging anti-neoplastic effects Content Type Journal Article Category Letter to the Editor Pages 1-2 DOI 10.1007/s10616-012-9507-5 Authors Shailendra Kapoor, University of Illinois at Chicago, Chicago, IL, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague–Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9498-2 Authors Patricia E. Engels, Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Basel, University Basel, Spitalstrasse 21, 4031 Basel, Switzerland Mathias Tremp, Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Basel, University Basel, Spitalstrasse 21, 4031 Basel, Switzerland Paul J. Kingham, Department of Integrative Medical Biology, Section of Anatomy, Umeå University, Umeå, Sweden Pietro G. di Summa, Division of Plastic, Reconstructive and Aesthetic Surgery, CHUV, University Hospital of Lausanne, Lausanne, Switzerland René D. Largo, Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Basel, University Basel, Spitalstrasse 21, 4031 Basel, Switzerland Dirk J. Schaefer, Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Basel, University Basel, Spitalstrasse 21, 4031 Basel, Switzerland Daniel F. Kalbermatten, Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Basel, University Basel, Spitalstrasse 21, 4031 Basel, Switzerland Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Stem bark of Oroxylum indicum (L) (SBOI) is used by ethnic communities of North East India as health tonic and in treating diseases of humans and animals. The objective of this research was to carry out a detailed investigation including total phenolic and flavonoid content, antioxidant, antimicrobial, cytotoxic and apoptotic activities of different solvent extracts of SBOI and to establish correlation between some parameters. Among petroleum ether (PE), dichloromethane and methanol (MeOH) extract of SBOI, MeOH extract contained the highest amount of total phenolic (320.7 ± 34.6 mg Gallic acid equivalent/g extract) and flavonoid (346.6 ± 15.2 mg Quercetin equivalent/g extract) content. In vitro antioxidant activity (IC 50 22.7 μg/ml) was highest in MeOH extract ( p  〉 0.05) and also a significant inverse correlation was observed between phenolic (r = 0.886)/flavonoid (r = 0.764) content and corresponding DPPH IC 50 . Only MeOH extract inhibited both bacteria and fungi. Although, individual extract showed cytotoxicity on HeLa cells with characteristic features of apoptosis, PE extract caused maximum cytotoxicity (IC 50 of 112.3 μg/ml, p  〈 0.05) and apoptotic activity (33.2 % sub-G0/G1 population) on HeLa cells. But, there was a significant non-inverse correlation of the MTT IC 50 with total phenolic (r = 0.812, p  〈 0.05)/flavonoid (r = 0.998, p  〈 0.05) content in the three solvent extracts. TLC analysis showed three unique compounds in PE extract which may have a role in apoptosis mediated cytotoxicity. These results called for futher chemical characterisation of MeOH and PE extract of SBOI for specific bioactivity. Content Type Journal Article Category Original Research Pages 1-13 DOI 10.1007/s10616-012-9463-0 Authors Dinesh Singh Moirangthem, Department of Biotechnology, Institute of Bioresources and Sustainable Development, Government of India, Takyelpat Institutional Area, Imphal, 795001 Manipur, India Narayan Chandra Talukdar, Department of Biotechnology, Institute of Bioresources and Sustainable Development, Government of India, Takyelpat Institutional Area, Imphal, 795001 Manipur, India Utpal Bora, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039 Assam, India Naresh Kasoju, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039 Assam, India Ratul Kumar Das, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039 Assam, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Our previous study reported that the saponin-rich fraction from Clematis chinensis Osbeck roots (SFC) could effectively alleviate experimental osteoarthritis induced by monosodium iodoacetate in rats through protecting articular cartilage and inhibiting local inflammation. The present study was performed to investigate the preventive effects of SFC on articular chondrocyte, and explore the underlying mechanisms. Primary rabbit chondrocytes were cultured and exposed to sodium nitroprusside (SNP), a NO donor. After treatment with different concentrations of SFC (30, 100, 300, 1,000 μg/ml) for 24 h, nucleic morphology, apoptotic rate, mitochondrial function and caspase-3 activity of chondrocytes were examined. The results showed that SNP induced remarkable apoptosis of rabbit chondrocytes evidenced by Hoechst 33258 staining and flow cytometry analysis, and SFC prevented the apoptosis in a concentration-dependent manner. Further studies indicated that SFC could prevent the depolarization of mitochondrial membrane potential (∆ψm) in SNP-treated chondrocytes and suppress the activation of caspase-3. It can be concluded that the protection of SFC on articular chondrocytes is associated with the anti-apoptosis effects via inhibiting the mitochondrion impairment and caspase-3 activation. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9485-7 Authors Wenjun Wu, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Xinghua Gao, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Xianxiang Xu, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Yubin Luo, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Mei Liu, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Yufeng Xia, State Key Laboratory of Natural Medicines, Department of Chinese Materia Medica Analysis, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Yue Dai, State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Osteosarcoma (OS) is the most frequent malignant bone tumour in children and adolescents. In metastatic patients, the most common site of metastasis is the lung. There are relatively few cell lines of metastatic OS reported in the literature and the cytogenetic aspects of OS metastases are still controversial and inconclusive. Here we describe the establishment of a new OS cell line, M-OS, from a pulmonary metastasis of a typical osteoblastic OS of an 11-year-old boy with metastatic OS at diagnosis. M-OS cells have been maintained in culture for over 50 passages for more than 1 year. M-OS was characterized by immunohistochemistry, conventional cytogenetics and fluorescence in situ hybridization (FISH). In order to evaluate in vitro cell modification, the immunohistochemical analysis was performed in three different moments of the cell line: 10th, 30th and 50th passages. The conventional cytogenetic analysis revealed the ploidy of M-OS cell line as near-diploid, with most metaphases hyperdiploid and tetraploid. We found a copy number gain of MDM2 gene as the most frequent alteration in the FISH analysis. The immunohistochemical analysis confirmed that M-OS cell line maintained the osteogenic nature even after all passages for the cell line establishment in vitro. Content Type Journal Article Category Brief Report Pages 1-7 DOI 10.1007/s10616-012-9487-5 Authors Carolina Salinas-Souza, Genetics Laboratory, Department of Pediatrics, Pediatric Oncology Institute (IOP/GRAACC/UNIFESP), Federal University of São Paulo, Rua Botucatu, 743-8° andar, São Paulo, SP 04023-062, Brazil Indhira Dias Oliveira, Genetics Laboratory, Department of Pediatrics, Pediatric Oncology Institute (IOP/GRAACC/UNIFESP), Federal University of São Paulo, Rua Botucatu, 743-8° andar, São Paulo, SP 04023-062, Brazil Renato de Oliveira, Department of Thoracic Surgery, Federal University of São Paulo, Rua Napoleão de Barros, 715-4º andar, São Paulo, SP 04024-002, Brazil Maria Teresa de Seixas Alves, Department of Pathology, Federal University of São Paulo, Rua Botucatu, 740, São Paulo, SP 04023-900, Brazil Antonio Sergio Petrilli, Department of Pediatrics, Pediatric Oncology Institute (IOP/GRAACC/UNIFESP), Federal University of São Paulo, Rua Botucatu, 743, São Paulo, SP 04023-062, Brazil Silvia Regina Caminada Toledo, Genetics Laboratory, Department of Pediatrics, Pediatric Oncology Institute (IOP/GRAACC/UNIFESP), Federal University of São Paulo, Rua Botucatu, 743-8° andar, São Paulo, SP 04023-062, Brazil Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Testicular cell culture of crab, Scylla serrata (Forskal) was used to study the effects of White spot syndrome virus (WSSV). We are showing the susceptibility of cell culture of crabs to WSSV. The proliferating cell culture of testes were maintained for more than 4 months in a medium prepared from L15 and crab saline supplemented with epidermal growth factor. The cell cultures inoculated with different concentrations of virus showed distinct cytopathic effects such as change in cell appearance, shrinkage and cell lysis. WSSV infection of cultured cells was confirmed by Nested PCR technique. The incorporation of viral DNA in cultured cells was shown by RAPD profile generated using 10-mer primers. The controls that were not exposed to WSSV did not show cytopathic effects. This work shows the usefulness of proliferating testicular cell culture for studying WSSV infection using molecular tools. Thus, this report gains significance as it opens new vistas for diagnostics and drugs for WSSV. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9482-x Authors Anumol Shashikumar, Department of Zoology, Goa University, Taleigao Plateau, Goa, 403206 India P. V. Desai, Department of Zoology, Goa University, Taleigao Plateau, Goa, 403206 India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors. Content Type Journal Article Category Original Research Pages 1-9 DOI 10.1007/s10616-012-9486-6 Authors Daniel Mueller, Biochemical Engineering Institute, Saarland University, Geb. A1 5, 66123 Saarbruecken, Germany Georg Tascher, Biochemical Engineering Institute, Saarland University, Geb. A1 5, 66123 Saarbruecken, Germany Georg Damm, Department of General-, Visceral- and Transplantation Surgery, Charité Medical University Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Andreas K. Nüssler, Department of Trauma Surgery, Eberhard-Karls University Tubingen, Schnarrenbergstr. 95, 72076 Tubingen, Germany Elmar Heinzle, Biochemical Engineering Institute, Saarland University, Geb. A1 5, 66123 Saarbruecken, Germany Fozia Noor, Biochemical Engineering Institute, Saarland University, Geb. A1 5, 66123 Saarbruecken, Germany Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    The efficacy of hepatocellular carcinoma (HCC) treatment is very low because of the high percentage of recurrence and resistance to anticancer agents. Hepatic cancer stem cells (HCSCs) are considered the origin of such recurrence and resistance. Our aim was to evaluate the stemness of doxorubicin and 5-fluorouracil resistant hepatic cancer cells and establish the new method to isolate the HCSCs from primary cultured HCC tumors. HCC biopsies were used to establish primary cultures. Then, primary cells were selected for HCSCs by culture in medium supplemented with doxorubicin (0, 0.1, 0.25, 0.5 or 1 μg/mL), 5-fluorouracil (0, 0.1, 0.25, 0.5 or 1 μg/mL) or their combination. Selection was confirmed by detection of HCSC markers such as CD133, CD13, CD90, and the side population was identified by rhodamine 123 efflux. The cell population with the strongest expression of these markers was used to evaluate the cell cycle, gene expression profile, tumor sphere formation, marker protein expression, and in vivo tumorigenesis. Selective culture of primary cells in medium supplemented with 0.5 μg/mL doxorubicin and 1 μg/mL 5-fluorouracil selected cancer cells with the highest stemness properties. Selected cells strongly expressed CD13, CD133, CD90, and CD326, efflux rhodamine 123 and formed tumor spheres in suspension. Moreover, selected cells were induced to differentiate into cells with high expression of CD19 and AFP (alpha-fetoprotein), and importantly, could form tumors in NOD/SCID mice upon injection of 1 × 10 5 cells/mouse. Selective culture with doxorubicin and 5-fluorouracil will enrich HCSCs, is an easy method to obtain HCSCs that can be used to develop better therapeutic strategies for patients with HCC, and particularly HCSC-targeting therapy. Content Type Journal Article Category Method in Cell Science Pages 1-13 DOI 10.1007/s10616-012-9511-9 Authors Ngoc Bich Vu, Laboratory of Stem Cell Research and Application, University of Science, VNU-HCM, HCM City, Vietnam Tam Thanh Nguyen, Laboratory of Stem Cell Research and Application, University of Science, VNU-HCM, HCM City, Vietnam Long Cong-Duy Tran, University of Medical Center, Ho Chi Minh University of Medicine and Pharmacy, HCM City, Vietnam Cong Dinh Do, University of Medical Center, Ho Chi Minh University of Medicine and Pharmacy, HCM City, Vietnam Bac Hoang Nguyen, University of Medical Center, Ho Chi Minh University of Medicine and Pharmacy, HCM City, Vietnam Ngoc Kim Phan, Laboratory of Stem Cell Research and Application, University of Science, VNU-HCM, HCM City, Vietnam Phuc Van Pham, Laboratory of Stem Cell Research and Application, University of Science, VNU-HCM, HCM City, Vietnam Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Pluripotent stem cells derived from testis is a new, natural, and unlimited source for cell therapy in regenerative medicine and represent a possible alternative to replacing of all cells in the body. Here, we designed a simple co-culture system of spermatogonia cells with Sertoli cells for the generation of embryonic stem-like cells from mouse testis. The importance of our simple method will be clear when we compared it with other complex and time-consuming methods. Embryonic stem-like colonies with sharp border confirmed by real-time PCR, immunocytochemistry and flow cytometry assessments. Embryonic stem-like colonies were immunopositive for pluripotency markers. Transition of spermatogonia cells to embryonic stem-like cells was accompanied by extensive changes in gene expression. These changes included significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. Also, we proved the differentiation capacity of embryonic stem-like cells to neuroepithelial-like cells which were immunoreactive to Nestin and Neurofilament 68. Evaluation of genes expression during in vitro differentiation into neuroepithelial-like cells showed high-level expression of Nestin whether this gene approximately has no expression in undifferentiated embryonic stem-like cells. Also, expression of pluripotency genes has significantly decreased in neuroepithelial-like cells compared with embryonic stem-like cells. This study shows that embryonic stem-like cells derived from testis are capable to differentiate into neuroepithelial-like cells that may provide a cellular reservoir usable for neurodegenerative disorders. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9465-y Authors Maryam Nazm Bojnordi, Department of Anatomical Sciences, Medical Sciences Faculty, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran Mansoureh Movahedin, Department of Anatomical Sciences, Medical Sciences Faculty, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran Taki Tiraihi, Department of Anatomical Sciences, Medical Sciences Faculty, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran Mohamad Javan, Department of Physiology, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Photopolymerizable hydrogels offer great potential in cartilage tissue engineering due to their ability to conform to irregular defect shapes and be applied in a potentially minimally invasive manner. An important process requirement in the use of photopolymerizable hydrogels is the ability of the suspended cells to withstand low intensity ultraviolet light (UV) exposure (4–5 mW/cm 2 ) and photoinitiator concentrations. For cartilage integration with underlying subchondral bone tissue, robust localized osteoblast activity is necessary. Yet, while it is known that osteoblasts do not respond well to UV light, limited work has been conducted to improve their survivability. In this study, we evaluated the cellular cytotoxicity of five different human cell sources at different UV exposure times, with and without a commercially used photoinitiator. We were able to confirm that human osteoblasts were the least tolerant to varying UV exposure times in comparison to bone marrow stem cell, periodontal ligament cell, smooth muscle and endothelial cell lineages. Moreover osteoblasts cultured at 39 °C did not deteriorate in terms of alkaline phosphatase expression or calcium deposition within the extracellular matrix (ECM), but did reduce cell proliferation. We believe however that the lower proliferation diminished osteoblast sensitivity to UV and the photoinitiator. In fact, the relative survivability of osteoblasts was found to be augmented by the combination of a biochemical factor and an elevated incubation temperature; specifically, the use of 50 mg/l of the anti-oxidant, ascorbic acid significantly ( P  〈 0.05) increased the survivability of osteoblasts when cultured at 39 °C. We conclude that ascorbic acid at an incubation temperature of 39 °C can be included in in vitro protocols used to assess cartilage integration with bone ECM. Such inclusion will enhance conditions of the engineered tissue model system in recapitulating in vivo osteoblast activity. Content Type Journal Article Category Original Research Pages 1-10 DOI 10.1007/s10616-012-9512-8 Authors Rupak Dua, Tissue Engineered Mechanics, Imaging and Materials Laboratory (TEMIM Lab), Department of Biomedical Engineering, College of Engineering and Computing, Florida International University, 10555 W. Flagler Street, EC 2612, Miami, FL 33174, USA Sharan Ramaswamy, Tissue Engineered Mechanics, Imaging and Materials Laboratory (TEMIM Lab), Department of Biomedical Engineering, College of Engineering and Computing, Florida International University, 10555 W. Flagler Street, EC 2612, Miami, FL 33174, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    α-Linolenic acid (ALA), a major fatty acid in flaxseed oil, has multiple functionalities such as anti-cardiovascular and anti-hypertensive activities. In this study, we investigated the effects of ALA on lipid metabolism and studied the possible mechanisms of its action in differentiated 3T3-L1 adipocytes using DNA microarray analysis. From a total of 34,325 genes in the DNA chip, 87 genes were down-regulated and 185 genes were up-regulated at least twofold in differentiated 3T3-L1 adipocyte cells treated with 300 μM ALA for a week, 5–12 days after induction of cell differentiation, compared to ALA-untreated 3T3-L1 adipocytes (control). From the Reactome analysis results, eight lipid metabolism-related genes involved in cholesterol and triacylglycerol biosynthesis pathway and lipid transport were significantly down-regulated by ALA treatment. Furthermore, ALA significantly decreased the mRNA expressions of sterol regulatory element binding protein ( SREBP) - 2 , SREBP - 1a, SREBP - 1c and fatty acid synthase ( FAS ) in 3T3-L1 adipocyte cells. On the other hand, the average levels of the gene expressions of carnitine palmitoyltransferase 1a ( CPT - 1a ) and leptin in 300 μM ALA treatment were increased by 1.7- and 2.9-fold, respectively, followed by an increase in the intracellular ATP content. These results show that ALA is likely to inhibit cholesterol and fatty acid biosynthesis pathway by suppressing the expression of transcriptional factor SREBPs. Furthermore, ALA promotes fatty acid oxidation in 3T3-L1 adipocytes, thereby increasing its health benefits. Content Type Journal Article Category JAACT Special Issue Pages 1-9 DOI 10.1007/s10616-012-9510-x Authors Satoshi Fukumitsu, Central Laboratory, Nippon Flour Mills Co., Ltd., Midorigaoka, Atsugi, Kanagawa, Japan Myra O. Villareal, Alliance for Research on North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki, Japan Shoko Onaga, Alliance for Research on North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki, Japan Kazuhiko Aida, Central Laboratory, Nippon Flour Mills Co., Ltd., Midorigaoka, Atsugi, Kanagawa, Japan Junkyu Han, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Hiroko Isoda, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Nitrocellulose membranes, one of the most important and oldest cellulose derivatives, are commonly used for nucleic acid and protein detection in research and diagnostic applications. However, a limited number of studies have explored whether they can act as scaffolds for cell growth. In this study, we investigated this polymeric material for its ability to support the growth of human cells. Eight established cell lines were examined for adherence, growth, spread, and survival on nitrocellulose membranes by optical microscopy after hematoxylin and eosin and/or immunocytochemical staining and by scanning electron microscopy. Apoptosis and leakage of lactate dehydrogenase (LDH) were also assessed. All cells readily adhered to and spread on the surface of nitrocellulose membranes as well as coverslips, and the cells maintained the expression of digestive system-specific genes. No significant change was detected in apoptosis or leakage of LDH from cells grown on nitrocellulose membranes. These results suggested that nitrocellulose membranes have a suitable cytocompatibility towards human cells and that they might be used for tissue-engineering scaffolds. Moreover, we demonstrate an additional and underused property of nitrocellulose of specific relevance to microscopic imaging, as it can be rendered virtually transparent, thus the cells growing on such membranes can be observed directly under an optical microscope after staining. Content Type Journal Article Category Original Research Pages 1-11 DOI 10.1007/s10616-012-9458-x Authors Aimin Li, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Yadong Wang, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Lijuan Deng, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Xinmei Zhao, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Qun Yan, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Yidong Cai, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Jianhua Lin, Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Yang Bai, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Side Liu, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Yali Zhang, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description: Erratum to: Preface Content Type Journal Article Category Erratum Pages 1-1 DOI 10.1007/s10616-012-9478-6 Authors Mutsumi Takagi, Hokkaido University, Sapporo, Japan Masashi Fujiwara, Hokkaido University, Sapporo, Japan Takeshi Omasa, University of Tokushima, Tokushima, Japan Sanetaka Shirahata, Kyushu University, Fukuoka, Japan Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs. Content Type Journal Article Category Original Research Pages 1-8 DOI 10.1007/s10616-012-9477-7 Authors Il Soo Moon, Department of Anatomy, Dongguk Medical Institute, College of Medicine, Dongguk University, Gyeongju, 780-714 Korea HyunSook Lee, Department of Anatomy, Dongguk Medical Institute, College of Medicine, Dongguk University, Gyeongju, 780-714 Korea Randall S. Walikonis, Department of Physiology and Neurobiology, University of Connecticut, 75 N. Eagleville Road, U-3156, Storrs, CT 06269, USA Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069

Description:    Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae , V. mimicus , V. albensis and uncultured clones of Vibrio . Phenotypically also it resembled both V. cholerae and V. mimicus . Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administering APV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios . Content Type Journal Article Category Original Research Pages 1-14 DOI 10.1007/s10616-012-9472-z Authors K. Manjusha, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India P. Jayesh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India Divya Jose, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India B. Sreelakshmi, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India P. Priyaja, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India Prem Gopinath, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India A. V. Saramma, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India I. S. Bright Singh, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, 682016 Kerala, India Journal Cytotechnology Online ISSN 1573-0778 Print ISSN 0920-9069