ALBERT

Description: Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.

Description: Gills cells of the freshwater mussel Lasmigona costata and the seawater clam Mesodesma mactroides were isolated (mussel: chemical dissociation; clam: mechanical dissociation) and fractionated (Percoll gradient) into Fractions I and II. Mitochondrial dyes (DASPEI: mussel; MitoTracker ® : clam) and Na + , K + -ATPase activity measurement were used to distinguish between cells of Fractions I and II. For mussel and clam, 80.5 ± 1.5 and 48.3 ± 3.2 % of cells were in Fraction II, respectively. For both species, cells of Fraction II had higher fluorescence emission and higher enzyme activity than those of Fraction I, being characterized as ‘cells rich in mitochondria’. Cells of Fraction II were kept in saline solutions approximating the ionic composition of hemolymph either under control conditions (no Cu addition) or exposed (3 h) to copper (Cu: 5, 9 and 20 μg Cu/L). Cell viability and Cu and Na + content were measured. For both species, Cu content was higher and Na + content was lower in cells exposed to 20 μg Cu/L. Furthermore, a strong negative correlation was observed between cell Na + and Cu content in the two bivalve species, indicating a possible competition between Cu and Na + for ion-transporting mechanisms or binding sites at gill cells of Fraction II. Considering that Cu is an ionoregulatory toxicant in aquatic invertebrates, these preliminary toxicological data support the idea of using isolated gill cells rich in mitochondria to study the mechanisms underlying the acute toxicity of waterborne Cu in freshwater and marine bivalves.

Description: To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4 -overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host cell line. Clone CHO-ATF4-16 did not show any change in growth rate compared with the parental cells or mock-transfected CHO-DP12-SF cells. The expression levels of mRNAs encoding both the antibody heavy and light chains in the CHO-ATF4-16 clone were analyzed. This analysis showed that ATF4 overexpression improved the total production and specific production rate of antibody without affecting the mRNA transcription level. These results indicate that ATF4 overexpression is a promising method for improving recombinant IgG production in CHO cells.

Description: The purpose of the present study was to investigate the osteoprotective effects of soybean oil (SbO) and sesame oil (SO) in ovarictomized (OVX) rats. The results indicated that the OVX rats exhibited a significant decrease in Ca and P level in both serum and bone, the activities of the antioxidant enzymes SOD and CAT and the antioxidant biomarker GSH accompanied with a marked increase in the oxidative stress markers MDA and PC, the inflammatory indices (TNF-α, CRP levels, WBCs counts and ACP activity) in, both, bone and serum. Supplementating the diet of the OVX rats with SbO (15 % w/w) or SO (10 % w/w) for 2 months to resulted in modulation of the alterations in all tested parameters and succeeded to restore minerals, antioxidant enzymes, antioxidant biomarkers, oxidative stress markers, inflammatory indices, and WBCs counts. It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.

Description: Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.

Description: The immense potency of nutritional components of human breast milk and importance of breastfeeding is known worldwide. Recent researches had identified stem cells as integral component of human breast milk. Nevertheless, there is little proof of evidence on the stem cell constituents of breast milk. It is imperative to explore the cellular constituents of human breast milk, including of stem cells, to open new avenue in child’s development and regeneration. Thus, we aimed at identifying the cellular constituents of human breast milk by phenotypic characterisation of diverse cell surface markers of hematopoietic stem cells (CD 34, CD 133, CD 117), mesenchymal stem cells (CD 90, CD 105, CD 73), myoepithelial cells (CD 29, CD 44), Immune cells (CD 209, CD 86, CD 83, CD 14, CD 13, HLADR, CD 45), as well as cell adhesion molecules (CD 31, CD 54, CD 166, CD 106, CD 49d), and other markers (ABCG2, CD140b) using flowcytometry. We found a lower expression of CD 34 (13.07 ± 2.0 %), CD 90 (7.79 ± 0.8 %) and CD 73 (2.19 ± 0.41 %), indicating scanty hematopoietic and mesenchymal stem cell population in human breast milk. On contrary, myoepithelial progenitors, cell adhesion molecules, immune cells and growth factors were identified as the major constituents of breast milk. Overall, this study illuminates the benefits of breast feeding as breast milk encompasses heterogeneous cellular components that benefits child’s growth, immunity and development. However, further research on these constituents of human breast milk will widen their applicability in treatment of neonatal disorders.

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.

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.

Description: The nasal pathway represents an alternative route for non-invasive systemic administration of drugs. The main advantages of nasal drug delivery are the rapid onset of action, the avoidance of the first-pass metabolism in the liver and the easy applicability. In vitro cell culture systems offer an opportunity to model biological barriers. Our aim was to develop and characterize an in vitro model based on confluent layers of the human RPMI 2650 cell line. Retinoic acid, hydrocortisone and cyclic adenosine monophosphate, which influence cell attachment, growth and differentiation have been investigated on the barrier formation and function of the nasal epithelial cell layers. Real-time cell microelectronic sensing, a novel label-free technique was used for dynamic monitoring of cell growth and barrier properties of RPMI 2650 cells. Treatments enhanced the formation of adherens and tight intercellular junctions visualized by electron microscopy, the presence and localization of junctional proteins ZO-1 and β-catenin demonstrated by fluorescent immunohistochemistry, and the barrier function of nasal epithelial cell layers. The transepithelial resistance of the RPMI 2650 cell model reached 50 to 200 Ω × cm 2 , the permeability coefficient for 4.4 kDa FITC-dextran was 9.3 to 17 × 10 −6  cm/s, in agreement with values measured on nasal mucosa from in vivo and ex vivo experiments. Based on these results human RPMI 2650 cells seem to be a suitable nasal epithelial model to test different pharmaceutical excipients and various novel formulations, such as nanoparticles for toxicity and permeability.

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”.

Description: A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett–Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.

Description: The therapeutic rationale for tissue repair and regeneration using stem cells is at its infancy and needs advancement in understanding the role of individual component’s innate capability. As stem cells of adipose tissue reside in a more heterogeneous population of stromal vascular fractions, cell separation or sorting becomes an eminent step towards revealing their unique properties. This study elucidates the comparative efficacy of lineage depleted adipose derived stromal vascular fraction (SVF) and their innate ability using magnetic activated cell sorter (MACS). To this end, isolated SVF from human adipose tissue was lineage depleted according to the manufacturer’s instructions using specific antibody cocktail through MACS. The enriched lineage negative (lin−) and lineage positive (lin+) cell fractions were cultured, phenotypically characterized for the panel of cell surface markers using flowcytometry and subjected to osteoblastic and adipogenic differentiation. The expression profile obtained for lin− cells was CD34−/CD45−/HLADR−/CD49d−/CD140b−/CD31−/CD90+/CD105+/CD73+/CD54+/CD166+/CD117− when compared to Lin+ cells expressing CD34+/CD45+/HLADR−/CD49d−/CD140b+/CD31−/CD90+/CD105+/CD73+/CD54+/CD166+/CD117+ (CD—cluster of differentiation). These results, thus, advances our understanding on the inherent property of the individual cell population. Furthermore, both the fractions exhibited mesodermal lineage differentiation capacity. To conclude, this research pursuit rationalized the regenerative therapeutic applicability of both lin− and lin+ cultures of human adipose tissue for disorders of mesodermal, haematological and vascular origin.

Description: Carvacrol (CVC) is a phenolic monoterpene present in many essential oils of medicinal and aromatic plants and has attracted attention because of its beneficial biological activities. To date, although various biological activities of CVC have been demonstrated, its neurotoxicity on cultured primary rat neurons and N2a neuroblastoma cells has never been explored. Therefore, in this present study, we aimed to describe in vitro antiproliferative and/or cytotoxic properties (by 3-(4,5 dimetylthiazol -2-yl)-2,5 diphenlytetrazolium bromide (MTT) test), genotoxic damage potentials (by single cell gel electrophoresis (SCGE) or Comet assay) and antioxidant activities (by total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis) of CVC in vitro. Dose (0–400 mg/L) dependent effects of CVC were tested on both cultured primary rat neurons and N2a neuroblastoma cells. Statistical analysis of MTT assay results indicated significant ( p  〈 0.05) decreases of cell proliferation rates in both cell types treated with CVC at 200 and 400 mg/L. On the other hand, the mean values of the total scores of cells showing DNA damage (for comet assay) was not found significantly different from the control values for both cells ( p  〉 0.05). In addition, our results indicated that 10, 25 and 50 mg/L of CVC treatment caused increases of TAC levels in cultured primary rat neurons but not in the N2a cell line. However, CVC treatments led to increases of TOS levels in cultured primary rat neurons at only 400 mg/L while they led to increases of TOS levels in N2a neuroblastoma cells at 200 and 400 mg/L. The present findings demonstrated that CVC could be a source of antioxidant and chemopreventive activities to be studied on cancer diseases.

Description: As rice bran contains various nutrients and other proteins of which a part has biological effects on animal cells, we tested the effect of rice bran extract on rat mesenchymal stem cells (rMSCs) obtained from bone marrow. These rMSCs are pluripotent and can be readily induced to differentiate into a number of cell types, including bone and cartilage. rMSC was aggregated by culturing in serum-free condition with rice bran extract, but was not aggregated by culturing in serum-free condition or in serum-containing medium. Moreover, the longer aggregates of rMSCs were cultured in serum-free condition with rice bran extract, the more the aggregates grew. After two passages in serum-free conditions, rMSCs lost their potency for differentiation into osteogenic cells; however, the addition of rice bran extract to serum-free medium successfully prevented the loss of this ability for differentiation. In addition, MSC makers CD105 and CD166 gene expression in serum-free condition with rice barn extract corresponded to these expressions in serum-containing medium. This result suggests that certain factors in rice bran could be bioactive and contribute toward retaining the ability of MSCs to differentiate into osteogenic cells after passaging.

Description: Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

Description: Patients with a decrease in limb perfusion with a potential threat to limb viability manifested by ischemic rest pain, ischemic ulcers, and/or gangrene are considered to have critical limb ischemia (CLI). Because of this generally poor outcome, there is a strong need for attempting any procedure to save the affected limb. The aim of this work is to evaluate the possibility to use stem cell therapy as a treatment option for patients with chronic critical lower limb ischemia with no distal run off. This study includes 20 patients with chronic critical lower limb ischemia with no distal run off who are unsuitable for vascular or endovascular option. These patients underwent stem cell therapy (SCT) by autologous transplantation of bone marrow derived mononuclear cells. 55 % of patients treated with SCT showed improvement of the rest pain after the first month, 60 % continued improvement of the rest pain after 6 months, 75 % after 1 year and 80 % after 2 years and continued without any deterioration till the third year. Limb salvage rate after STC was 80 % after the first year till the end of the second and third years. SCT can result in angiogenesis in patients with no-option CLI, providing a foundation for the application of this therapy to leg ischemia.

Description: Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit’s pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins , Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

Description: Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

Description: Recent ultrahigh-density tiling array and large-scale transcriptome analysis have revealed that large numbers of long non-coding RNAs (lncRNAs) are transcribed in mammals. Several lncRNAs have been implicated in transcriptional regulation, organization of nuclear structure, and post-transcriptional processing. However, the regulation of expression of lncRNAs is less well understood. Here, we show that the exogenous and endogenous expression of an oncogenic form of small GTPase Ras (called oncogenic Ras) decrease the expression of lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), which is involved in the regulation of cellular senescence. We also show that forced expression of oncogenic Ras increases the expression of lncRNA PANDA (p21 associated ncRNA DNA damage activated), which is involved in the regulation of apoptosis. Microarray analysis demonstrated that expression of multiple lncRNAs fluctuated by forced expression of oncogenic Ras. These findings indicate that oncogenic Ras regulates the expression of a large number of lncRNAs including functional lncRNAs, such as ANRIL and PANDA .

Description: The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo ( Bubalus bubalis ) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences ( P  〉 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences ( P  〈 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly ( P  〈 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

Description: The use of food additives has increased enormously in modern food technology but they have adverse effects in human healthy. The aim of this study was to investigate the DNA damage of some food additives such as citric acid (CA), benzoic acid (BA), brilliant blue (BB) and sunset yellow (SY) which were investigated in human male germ cells using comet assay. The sperm cells were incubated with different concentrations of these food additives (50, 100, 200 and 500 μg/mL) for 1 h at 32 °C. The results showed for CA, BA, BB and SY a dose dependent increase in tail DNA%, tail length and tail moment in human sperm when compared to control group. When control values were compared in the studied parameters in the treatment concentrations, SY was found to exhibit the highest level of DNA damage followed by BB 〉 BA 〉 CA. However, none of the food additives affected the tail DNA%, tail length and tail moment at 50 and 100 μg/mL. At 200 μg/mL of SY, the tail DNA% and tail length of sperm were 95.80 ± 0.28 and 42.56  ±  4.66, for BB the values were 95.06 ± 2.30 and 39.56 ± 3.78, whereas for BA the values were 89.05 ± 2.78 and 31.50 ± 0.71, for CA the values were 88.59 ± 6.45 and 13.59 ± 2.74, respectively. However, only the highest concentration of the used food additives significantly affected the studied parameters of sperm DNA. The present results indicate that SY and BB are more harmful than BA and CA to human sperm in vitro.

Description: Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Description: Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

Description: The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72 h after intraperitoneal injection of 12 ml thioglycollate. Cells (1 × 10 6 ) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10 min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21 days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

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: Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.

Description: Pancreatic islet transplantation is a promising therapy for Type I Diabetes. For many years the method used worldwide for islet purification in both rodent and human islet isolation has been Ficoll-based density gradients, such as Histopaque. However, it is difficult to purify islets in laboratories with staff limitations when large scale isolations are required. We hypothesized that filtration could be a more simple and fast alternative to obtain good quality islets. Four separate islet isolations were performed per method, comparing filtration and Histopaque purification with handpicking as the gold standard method for islet purity. Different parameters of quality were assessed: yield in number of islets per pancreas, purity by dithizone staining, viability by Fluorescein Diacetate/Propidium Iodide vital staining and in vitro functionality assessed by Glucose Stimulated Insulin Secretion. Time efficiency and cost were also analyzed. The overall quality of the islets obtained both by Histopaque and filtration was good. Filtration saved almost 90 % of the time consumed by Histopaque purification, and was also cheaper. However, one-third of the islets were lost. Since human and rodent islets share similar size but different density, filtration appears as a purification method with potential interest in translation to clinic.

Description: Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (〉140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO 2 − induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

Description: Siglecs are immunoglobulin lectin group proteins that recognize the sialic acid moiety. We previously reported that the expression of Siglec-9 on the macrophage cell line RAW264 markedly enhanced Toll-like receptor (TLR)-induced interleukin (IL)-10 production and inhibited the production of proinflammatory cytokines. In this study, we examined the lectin-dependent anti-inflammatory activities of Siglec-9. IL-10 production was modestly reduced by a mutation that disrupted the lectin activity of Siglec-9, while the reduction in tumor necrosis factor-α was not affected. Membrane fractionation experiments revealed that a part of Siglec-9 resided in the detergent-insoluble microdomain, the so-called lipid raft fraction. The amount of Siglec-9 in the lipid raft fraction rapidly increased following TLR2 stimulation by peptidoglycan and peaked after 3–10 min. This time course was similar to that of TLR2. The double tyrosine mutant in immunoreceptor tyrosine-based inhibitory motifs moved to lipid rafts in a similar manner, while lectin-defective Siglec-9 was not detected in the lipid raft fraction. The production of IL-10 was partially reduced by cholesterol oxidase that disturbed lipid raft organization. Taken together, these results suggest that Siglecs exhibit lectin-dependent changes in cellular localization, which may be partly linked to its control mechanism that increases the production of IL-10.

Description: The MDR1 gene encodes for P-glycoprotein (P-gp), which is an efflux transporter at the cell membrane. The P-gp has wide substrate specificity for multiple medications including the lipid lowering drug, atorvastatin. In this study, we investigated the possible association between three common MDR1 gene polymorphisms (G2677T, C3435T, and C1236T), and the lipid lowering effect of atorvastatin among Jordanians. Lipid and lipoproteins were measured in blood samples collected from patients (n = 201) at baseline and during atorvastatin treatment. MDR1 polymorphisms were genotyped using polymerase chain reaction–restriction fragment length polymorphism. Both the TT genotype of G2677T and the TT genotype of the C3435T polymorphisms were associated with lower levels of low-density lipoproteins after atorvastatin treatment. However, the effects of atorvastatin on the levels of total cholesterol, triglycerides, and high-density lipoprotein, were not correlated with any of the genotypes in both polymorphisms. Finally, the C1236T polymorphism was not associated with the lipid lowering effect of atorvastatin. In conclusion, the MDR1 gene polymorphisms G2677T, and C3435T, but not C1236T were associated with the lipid lowering effect of atorvastatin among Jordanians.

Description: The aim of the study was to evaluate the biological activities with toxic properties of the methanol, hexane, and chloroform extracts of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the Coast of Urla in the Aegean Sea. The extracts of C. compressa were tested for their antimicrobial and antioxidant activities in this study. Cytotoxic and mutagenic potentials of the extracts were also evaluated using cell culture and mutagenicity assays. Hexane extract was found to have higher total flavonoid and phenolic contents than the other extracts and exerted higher antioxidant activity than other extracts. All extracts exhibited moderate antimicrobial activity against tested microorganisms (minimum inhibitory concentration ranges are 32–256 μg/mL). The results indicated that the extracts had no significant cytotoxic activity against human hepatocellular carcinoma Hep 3B cell line in all treated concentrations (5–50 μg/mL) and did not show mutagenicity in the Ames test. Lethality was not observed among mice treated with oral doses of the extracts. In conclusion, results of investigations indicate that brown alga C . compressa is a natural source of antioxidant. It has moderate antimicrobial activities with no toxicity.

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.

Description: The effect of natural IgG antibody recognizing β-galactosyl epitope on hepatoma cell invasion was investigated. Anti-β-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-α-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-β-galactosyl antibody was completely canceled by adding lactose (galactose-β-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with β-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48 h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-β-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-β-galactosyl specificity may recognize the β-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.

Description: Megalocytiviruses are important emerging pathogens in both freshwater and marine finfish aquaculture. However, a limited number of piscine cell lines are persistently susceptible to these viruses, which greatly limits the study of megalocytiviruses. In this study, a new fibroblast-like cell line was established from an early primary culture from mandarin fish fry by a single cell cloning and was designated as MFF-8C1. The MFF-8C1 cells grow well in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and had been subcultured more than 60 passages since the initial recovery culture in October 2009. Chromosomal analysis revealed that 91 % of the MFF-8C1 cells maintained a normal diploid chromosome number (2n = 48) in the 46th passage. Infection experiments showed that both freshwater-borne and marine-borne megalocytiviruses induce severe cytopathic effects in infected MFF-8C1 cells characterized by the rounding and enlargement of cells, which are highly consistent with the previous description of the infection in other susceptible cells with megalocytivirus. Megalocytivirus infections were further confirmed by a transmission electron microscopy. Furthermore, the MFF-8C1-cultured megalocytiviral suspension was highly virulent to infected mandarin fish. In summary, a new fibroblast cell line from mandarin fish fry that was highly permissive to megalocytiviruses was established. The MFF-8C1 cell line is a promising cellular substrate candidate for cell-cultured vaccine production of megalocytivirus.

Description: Taif rose ( Rosa damascena trigintipetala Dieck) is a sort of damask rose, which is considered as one of the most important economic products of Taif. In this study, the authors investigated the possible cytotoxic, genotoxic, antimutagenic and anticancer effect of concrete and absolute rose oils. The results showed that both concrete and absolute rose oils were cytotoxically and genotoxically safe at a dose of 10 μg/ml when tested on cultures of normal human blood lymphocytes. Also, the results showed significant antimutagenic activity at p  〈 0.001 for absolute rose oil at the same dose level when tested on cultures of normal human blood lymphocytes supplemented with 300 ng/ml mitomycin C (MMC). On the other hand, concrete and absolute oils exerted a cytotoxic activity against two kinds of human cancer cell lines: HepG2 and MCF7. Concrete oil showed cytotoxic activity against HepG2 and MCF7 with a half maximal inhibitory concentration (IC 50 ) of 16.28 and 18.09 μg/ml, respectively, whereas absolute rose oil showed its cytotoxic activity against HepG2 and MCF7 with an IC 50  of 24.94 and 19.69, respectively. From this study, it is concluded that concrete and absolute rose oils are cytotoxically and genotoxically safe at a dose of 10 μg/ml when tested on cultures of normal human blood lymphocytes. In addition, absolute oil has an antimutagenic activity at the same dose. Further investigations are needed to study the activity of higher doses of both oils in vitro and in vivo in experimental animals in order to evaluate the capability of using these oils as therapeutic for treatment of some kinds of cancers.

Description: Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α 6 and laminin α 5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α 6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.

Description: To develop new anticancer agents has been considered as a useful and necessary strategy to suppress highly-metastatic lung cancer, the leading cause of cancer-related deaths in the world. In this study, we synthesized a new compound ethyl 6-bromocoumarin-3-carboxylyl L-theanine (TBrC) and studied the anticancer activity of TBrC and its molecular mechanisms of action. Our results show that TBrC remarkably inhibits the proliferation and migration in highly-metastatic lung cancer cells by inducing apoptosis and cell cycle arrest as well as regulating related protein expressions. Further study indicated that TBrC not only enhances the protein levels of Bax, cytosolic cytochrome c , caspase-3 and PARP-1 but also reduces the protein expressions of Bcl-2, cyclin D1, VEGFR1 and NF-κB as well as inhibits the phosphorylation and expressions of VEGFR2 and Akt in the cancer cells. More importantly, TBrC displays strong suppression of highly-metastatic tumor growth and reduces the tumor weight by 61.6 % in tumor-bearing mice without toxicity to the mice. Our results suggest that TBrC suppresses the proliferation and migration of lung cancer cells via VEGFR-Akt-NF-κB signaling pathways; TBrC may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of lung cancer.

Description: Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.

Description: Achyranthes bidentata ( A. bidentata ) Blume is a medicinal herb with the property of strengthening bones and muscles and ensuring proper downward flow of blood in terms of the therapeutic theory of traditional medicine. In the present study, the effect of A. bidentata root extract (AE) on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. AE caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, and mineralization in the cells ( P  〈 0.05). AE also decreased the production of TNF-α, IL-6, and RANKL induced by antimycin A, mitochondrial electron transport inhibitor. Exposure of MC3T3-E1 cells to antimycin A caused significant reduction of cell viability and mineralization. However, pretreatment with AE prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, ATP loss, ROS release, and nitrotyrosine increase, suggesting that AE may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, AE increased the phosphorylation of cAMP-response element-binding protein inhibited by antimycin A. Our study demonstrates that A. bidentata could significantly prevent osteoblast damage in aged patients.

Description: The present investigation was carried out to evaluate the effect of stress hormone cortisol on the myogenic markers in the C2C12 cells co-cultured with 3T3-L1 preadipocytes. Co-culturing was achieved by transwell inserts with a 0.4 μm porous membrane. C2C12 and 3T3-L1 cells were grown independently on the transwell plates. After differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates for co-culturing. 10 μg/μl of cortisol was added to the medium. After 72 h of treatment, C2C12 cells which were in the lower well were harvested for analysis. RT-PCR analysis of myogenic markers such as of myogenin, MyoD, Myf5, PAX3 and PAX7 showed a significant reduction in the mRNA expression of these myogenic markers. In addition, cortisol increased calpain activity, which led to accelerated protein degradation, which in turn reduced the myogenic rate. In conclusion, cortisol treatment reduced mRNA expression of myogenic markers in the co-cultured C2C12 cells, which is quite distinct from one dimensional mono-cultured C2C12 cells.

Description: Facial epidermal pigmentation and skin tumors can be caused by UV exposure and other physical and chemical irritations. In this report we describe the primary culture of melanocytes from human face skin. The ability to culture these melanocytes will enable their morphological and biological properties to be investigated. Skin specimens were obtained from patients who had undergone lower blepharoplasty procedures. Digestion with neutral protease and trypsin was used to obtain single cell suspensions of epidermal cells. The cells were cultured in M254 medium supplemented with human melanocyte growth solution. Cell morphology was observed using inverted microscopy. Melanocytes were positively identified using both l -DOPA staining and S-100 protein immunohistochemical staining. Immunofluorescence was used to confirm the expression of tyrosinase-related protein-1, a melanocyte-specific protein. The cellular ultrastructure of the melanocytes was observed by transmission electron microscopy. The cultured human melanocytes from face skin were multi-dendritic, and many mature melanosomes were observed. Therefore, using a specific culture medium, melanocytes from face skin can be successfully cultured and made available for further investigations.

Description: Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell artificially derived from non-pluripotent cells by overexpressing the transcription factors Oct4, Sox2, Klf4 and Nanog. These transcription factors play a pivotal role in stem cells; however, the function of these factors are not fully characterized. In this study, we analyzed Oct4, Sox2, Klf4 and Nanog in ten different species using bioinformatics, to provide more knowledge of the function of these genes. Nanog does not exist in the invertebrates Caenorhabditis elegans and Drosophila melanogaster , indicating that the absence of Nanog may be responsible for the developmental differences between vertebrates and invertebrates. Construction of phylogenetic trees confirmed that the function of Nanog is conserved from fish to mammals. The effect of alternative splicing on the protein domains present in Oct4, Sox2, Klf4 and Nanog were also analyzed. Examination of the expression patterns in human stem cells, iPS cells and normal tissues showed that Oct4, Sox2, Klf4 and Nanog are expressed at similar levels in iPS cells and embryonic stem cells, and expression of all four transcription factors decreases after differentiation. Expression of Klf4 reduced to the least during differentiation, and Klf4 was found to be specifically expressed in several normal tissues, especially the salivary gland. In this paper, we systematically indentified the family proteins of the four transcription factors used to induce pluripotent stem cells, and then analyzed their evolution status, composed of those protein domains, alternative splicing translation, expression status and interaction networks. Those analysis could shed a light for further research of iPS.

Description: We have already reported that lactate dehydrogenase (LDH) activates lymphocytes in vitro and in vivo. In this paper, we report the activating effects of LDH on the macrophage-like cell line J774.1. LDH was found to enhance production of IL-6 and TNF-α by J774.1 cells in a dose-dependent manner. Transcription levels of IL-6 and TNF-α in J774.1 cells were also enhanced by supplementation with LDH. From immunoblot analysis, it was revealed that LDH enhances the phosphorylation level of JNK in J774.1 cells. Moreover, the JNK inhibitor SP600125 decreased production of IL-6 and TNF-α induced by LDH. NF-κB translocation to the nucleus was also facilitated by LDH. These results was revealed that LDH enhances production of IL-6 and TNF-α by J774.1 cells via the increase of JNK phosphorylation and NF-κB translocation to the nucleus. Our data indicated that macrophages may be activated by LDH released from damaged tissues and cells in our body.

Description: The aims of the current work were to evaluate the hepatoprotective effect of calendula flowers and/or thyme leave extracts on aflatoxins (AFs)-induced oxidative stress, genotoxicity and alteration of p53 bax and bcl2 gene expressions. Eighty male Sprague–Dawley rats were divided into eight equal groups including: the control group, the group fed AFs-contaminated diet (2.5 mg/kg diet) for 5 weeks, the groups treated orally with thyme and/or calendula extract (0.5 g/kg b.w) for 6 weeks and the groups pretreated orally with thyme and/or calendula extract 1 week before and during AFs treatment for further 5 weeks. Blood, liver and bone marrow samples were collected for biochemical analysis, gene expression, DNA fragmentation and micronucleus assay. The results showed that AFs induced significant alterations in oxidative stress markers, increased serum AFP and inflammatory cytokine, percentage of DNA fragmentation, the expression of pro-apoptotic gene p53 and bax accompanied with a decrease in the expression of bcl2. Animals treated with the extracts 1 week before AFs treatment showed a significant decrease in oxidative damage markers, micronucleated cells, DNA fragmentation and modulation of the expression of pro-apoptotic genes. These results suggested that both calendula and thyme extracts had anti-genotoxic effects due to their higher content of total phenolic compounds.

Description: Human induced pluripotent stem (iPS) cells have great value for regenerative medicine, but are facing problems of low efficiency. MicroRNAs are a recently discovered class of 19–25 nt small RNAs that negatively target mRNAs. miR302/367 cluster has been demonstrated to reprogram mouse and human somatic cells to iPS cells without exogenous transcription factors, however, the repetition and differentiation potentiality of miR302/367 -induced pluripotent stem (mirPS) cells need to be improved. Here, we showed overexpression of miR302/367 cluster reprogrammed human embryonic kidney 293T cells into mirPS cells in serum-free N2B27-based medium. The mirPS cells had similar morphology with embryonic stem cells, and expressed pluripotent markers including Oct4, Sox2, Klf4, and Nanog. In addition, through formation of embryoid bodies, various cells and tissues from three germ layers could be determined. Moreover, we examined the potential of mirPS cells differentiating into germ cells both in vitro and in vivo. Taken together, these data might provide a new source of cells and technique for the investigation of the mechanisms underlying reprogramming and pluripotency.

Description: Daphnoretin is a bicoumarin compound isolated from a natural product, Wikstroemia indica , which has been used to treat many diseases. It has strong antiviral and anti-tumor activities. Taking the anti-tumor activity of daphnoretin as a starting point, the present study aimed to test the pro-apoptotic effect of daphnoretin and its underlying mechanism in HeLa cells. The inhibitory effects of daphnoretin on viability and proliferation of HeLa cells were determined by the MTT assay. Daphnoretin-induced apoptotic morphological changes were analyzed by mitochondrial membrane potential and Hoechst staining. The number and stage of apoptotic HeLa cells were determined by flow cytometry. Gene expression was determined by reverse-transcription polymerase chain reaction. Protein expression was determined by western blot. The caspase activity of HeLa cells was detected by a caspase-3 and caspase-9 colorimetric assay kit. We found that daphnoretin significantly inhibited HeLa cells’ viability by the MTT assay and flow cytometry. The nuclei of the apoptotic cells exhibited strong, blue fluorescence in Hoechst staining. Bax mRNA and protein levels were increased while bcl - 2 mRNA levels were decreased after daphnoretin treatment. Daphnoretin also activated both caspase-3 and caspase-9. These findings suggest that daphnoretin promotes apoptosis of HeLa cells in a mitochondria-mediated way. Daphnoretin therefore has potential to be a promising drug to treat uterine cervix cancer.

Description: The activation of phase-specific cyclin-dependent kinases is associated with ordered cell cycle transitions. Among the mammalian Cdks, Cdk2 is essential for liver cancer cell proliferation. The related cycling protein CDK2 was analyzed by 2D-gel and MALDI-TOF/TOF MS mass assay in liver cancer cells, which CDK2 was silenced. The results showed four significantly different spots in cell ribonucleoprotein (similar to ribosomal protein S12, chaperonin 10-related protein, beta-actin and zinc finger protein 276) and four in plasmosin (aldolase A protein, hCG, anonymous protein and tubulin, gamma complex associated protein 2). In the plasmosin, aldolase A catalyzes the production of tublin and actin. Together they regulate the cell cycle and arrest the cell in the S phage. In the cell ribonucleoprotein, proteins with homology to ribosomal protein S12 and chaperonin 10 play a similar role in cell cycle regulation.

Description: Desorption electrospray ionization may be used as a fast and convenient method for analysis and identification of lipids in the cell culture. Oxidative stress, which usually involves changes in lipids, was used as a model of pathology to show the utility of this analysis methodology. This paper addresses the surface preparation of cell culture slides, induction of oxidative stress, and cell monolayer culture preparation as well as optimization of the analysis. Advantages and drawbacks of the method were also discussed.

Description: The multipotent and immunosuppressive capacities of mesenchymal stem cells (MSCs) attract several scientists worldwide towards translational research focusing on treatment of diseases including liver failure. Though MSC’s have been isolated from different sources, researchers do not concur on the best source for expansion and clinical translation. In this study, we have compared the isolation, proliferation and expansion of MSCs from umbilical cord blood (UCB), Wharton’s Jelly (WJ), bone marrow (BM) and adipose tissue (AT). MSCs were isolated by density gradient separation from UCB, BM and AT and by both enzymatic and explant method for WJ. The MSCs are characterized by their ability to adhere to plastic, expression of positive (CD105, CD73, CD90, CD29, CD44) and negative (CD45, CD14, CD34) markers by flow cytometry and also by their in vitro adipogenic, osteogenic and chondrogenic differentiation. This comprehensive study clearly shows that WJ is better than UCB both in terms of rapidity, yield and ease of procedure. AT and BM are autologous sources for MSC’s but the specimen collection involves cumbersome and painful procedures and an invasive approach. However being autologous, they are safe and probable candidates for therapeutic future applications.

Description: The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O 2 ) and hypoxic (1 % O 2 ) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2 . Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.

Description: The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L −1 and neuron cells at concentrations over 150 mg L −1 . In addition, ZBN treatments at higher doses (≤50 mg L −1 ) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L −1 of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L −1 did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.

Description: Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.