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  • cell culture
  • Springer  (37)
  • Wiley-Blackwell  (7)
  • American Institute of Physics (AIP)
  • Cell Press
  • 1990-1994  (44)
  • 1991  (44)
  • 1
    ISSN: 1573-0832
    Keywords: Fumonisin B1 ; fumonisin B2 ; AAL toxin ; T-2 toxin ; mycotoxin ; Fusarium moniliforme ; bioassay ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Fumonisins B1 and B2 and AAL toxin are a series of structurally related mycotoxins. Fumonisins B1 and B2, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC50 values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 μg/ml for fumonisins B1, B2 and AAL toxin, respectively „in 100 μl cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC50 = 2.5, 2 and 5 μg/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B1 and B2 does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.
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  • 2
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    Springer
    Methods in cell science 13 (1991), S. 45-48 
    ISSN: 1573-0603
    Keywords: cell culture ; corneal epithelium ; primary explant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An improved procedure for the primary culture of pure human corneal epithelial cells from corneal explants is described. Confluent monolayers of epithelial cells can be consistently produced from small segments of donor corneas regardless of donor age and without feeder layers. Incubating segments on collagen at the air-liquid interface significantly improves the yield of cells per cornea and shortens cell migration time as compared to culturing on plastic. Fibroblast contamination is eliminated by serum-free medium and confirmed by indirect immunofluorescent staining for keratin.
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  • 3
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    Methods in cell science 13 (1991), S. 143-147 
    ISSN: 1573-0603
    Keywords: kidney ; renal cortex ; proximal tubule ; enzymatic dissociation ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A procedure is described for the establishment and propagation of epithelial cell rich cultures derived from normal human kidney cortex (NHK-C cells). Cells are harvested from tissue fragments of donor human kidney by progressive enzymatic dissociation. NHK-C cultures are morphologically heterogeneous but exhibit, predominantly, the functional characteristics of cells of the kidney proximal tubule.
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  • 4
    ISSN: 1432-203X
    Keywords: anthocyanin ; cell culture ; nitrate ; sucrose ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.
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  • 5
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    Molecular and cellular biochemistry 108 (1991), S. 151-156 
    ISSN: 1573-4919
    Keywords: cytochrome P-450 ; mitochondria ; heme ; hepatocytes ; mitochondrial DNA ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study examined various biochemical parameters such as mitochondria and mitochondrial DNA (mtDNA), total heme and cyto P450 content in fresh hepatocytes and dedifferentiated hepatocytes. These parameters were chosen in order to understand the dramatic decrease in drug metabolism in cultured hepatocytes. The data in this study shows a temporal decrease in cytochrome P450, total heme and also a decrease in mitochondria. Also, the ratio of mtDNA content to mitochondrial density was found to increase as hepatocytes underwent dedifferentiation. Stereological analysis of cell preparations provided a measure of mitochondrial density per cell area and mtDNA content was assessed by the use of a specific radiolabelled probe. This study demonstrates that a loss of the organelle which is partially responsible for synthesis of heme correlates with a decrease in cytochrome P450.
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  • 6
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    Molecular and cellular biochemistry 104 (1991), S. 35-43 
    ISSN: 1573-4919
    Keywords: α1-adrenergic receptors ; β-adrenergic receptors ; cardiac muscle ; cell culture ; gene expression ; protein kinase C
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The molecular mechanisms of cardiac myocyte growth are relevant to important problems in cardiovascular disease. A cell culture model has been developed to explore the role of adrenergic hormones in cardiac myocyte growth and gene expression. Activation of a cardiac myocyte α1-adrenergic receptor by catecholamines induces hypertrophic growth of neonatal rat cardiac myocytes and initiates selective increases in contractile protein gene transcription. These effects on growth and gene expression do not depend on contractile activity. The cardiac myocytes contain at least two subtypes of α1-adrenergic receptors and at least three isoforms of protein kinase C (PKC). A distinct α1 receptor subtype may mediate hypertrophy and gene transcription. Different isoforms of PKC are translocated to different intracellular sites on activation, and there is evidence that the β-PKC isoform may be an element in the signal transduction pathway from an α1 receptor at the surface to the cardiac myocyte nucleus. Growth regulation through a β-adrenergic receptor can also be demonstrated in the culture model. The growth response mediated through a β-adrenergic receptor differs in several respects from that transduced through an al adrenergic receptor.
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  • 7
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    Plant molecular biology 16 (1991), S. 167-169 
    ISSN: 1573-5028
    Keywords: Phaseolus vulgaris ; cell culture ; chalcone isomerase ; elicitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 8
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    Plant molecular biology 16 (1991), S. 739-740 
    ISSN: 1573-5028
    Keywords: Lupinus polyphyllus ; cell culture ; cDNA clone ; ribosomal protein ; rps 16
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 9
    ISSN: 1573-5028
    Keywords: tobacco ; 5-enolpyruvylshikimate-3-phosphate synthase ; cDNA clone ; gene expression ; gene amplification ; glyphosate ; cell culture ; tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.
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  • 10
    ISSN: 1573-0778
    Keywords: cell culture ; lymphocyte ; lymphokine-activated killer cell ; recombinant interleukin 2 ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (3H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.
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  • 11
    ISSN: 1573-0778
    Keywords: adoptive immunotherapy ; cell culture ; cell culture apparatus ; Interleukin-2 ; lymphokine-activated killer cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7×107 cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity. The JCC-device was able to reduce the consumption of basal medium, rIL-2 and serum by 20%, 84% and 96%, respectively compared to the conventional tissue culture systems. The JCC-device improved the routine performance of adoptive immunotherapy with LAK cells and rIL-2.
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  • 12
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    Cytotechnology 7 (1991), S. 121-130 
    ISSN: 1573-0778
    Keywords: cell culture ; cellulose sulphate ; encapsulation ; monoclonal antibodies ; poly-dimethyl-diallyl-ammoniumchloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A new encapsulation method was developed for the cultivation of mammalian cells. The capsules were produced using a solution of sodium cellulose sulphate (CS)(1.5%) and poly-dimethyl-diallyl-ammoniumchloride (PDMDAAC). When CS droplets fell into the precipitation bath consisting of a 2% solution of PDMDAAC, immediately a membrane at the interphase was built up. The influences of varying encapsulation process parameters on capsule characteristics, cell growth, and monoclonal antibody production were tested. This new method showed advantages when compared to other methods mainly due to time simplicity of the whole process.
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  • 13
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    Cytotechnology 5 (1991), S. 31-46 
    ISSN: 1573-0778
    Keywords: serum-free ; cell culture ; carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
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  • 14
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    Fish physiology and biochemistry 9 (1991), S. 261-269 
    ISSN: 1573-5168
    Keywords: Cortisol ; RU 486 ; temperature ; rainbow trout ; cell culture ; [3H]-Thymidine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The rainbow trout gonadal cell line, RTG-2, which survives temperatures from 0 to 28°C and proliferates at 5 to 26°C, responded to cortisol from 28°C to 0°C by influencing [3H]-thymidine incorporation into DNA. Over the normal temperature range of rainbow trout, 10–22°C, cortisol inhibited [3H]-thymidine incorporation. The antiglucocorticoid RU 486 had no effect on [3H]-thymidine incorporation at these temperatures and blocked the response to cortisol. Another antiglucocorticoid RU 362 also had no effect but was less effective in blocking the cortisol response. During incubation at 28°C this inhibitory response to cortisol was detected inconsistently during the first 24 h but was observed consistently during the second 24 h. At 0°C, cortisol and RU 486 had no effect during short treatments, but a 60 h exposure to either steroid stimulated [3H]-thymidine incorporation over a 48 h labelling period. These results suggest that temperature shifts between 10–22°C, do not change the direction of a response to cortisol and support the use of the upper portion (20–22°C) of the temperature range for studies on salmonid cells in culture.
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  • 15
    ISSN: 1573-5044
    Keywords: ammonium ; anthocyanin ; cell culture ; growth kinetics ; production medium ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p〈0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.
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  • 16
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    Plant and soil 130 (1991), S. 75-80 
    ISSN: 1573-5036
    Keywords: cell culture ; FeHEDTA ; Glycine max ; iron chelate reduction ; iron nutrition ; soybean
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (−Fe). Values for Km and Vmax, determined from a Lineweaver-Burk plot, were 57 μM and nmoles mg-1 dry weight for the +Fe cells and 50 μM and 22 nmoles mg-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The Km and Vmax values for Fe-deficient roots were 45 μM and 20 nmoles mg-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.
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  • 17
    ISSN: 1573-5117
    Keywords: callus ; cell culture ; domestication ; protoplast ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cellular biotechnology is a promising application in the propagation and selection of superior strains of seaweeds. Although axenic cultures, organogenetic tissue cultures, vegetative micro-propagation, callus induction and high yields of agar from calli have been described for several species of Gelidium, a number of basic problems remain to be solved. These include standardized methods for obtaining axenic cultures, identification of requirements for organic nutrients, PGR's, cellular disorganization and reorganization, somaclonal variation and somatic incompatibilities. Future progress in seaweed biotechnology will depend on the resolution of many of these problems.
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  • 18
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    Hydrobiologia 227 (1991), S. 187-192 
    ISSN: 1573-5117
    Keywords: Turbellaria ; planaria ; cell culture ; leucine uptake
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have tested various conditions for the culture of cells dispersed from planarians. The cells were procurred by digestion of planarian tissue in pronase and filtering through a nylon mesh. Using incorporation of L-[3H]leucine into protein as a gauge of cell growth, we found that the optimum salt concentration was about 50% of that for mammalian cells (about 160 mOsm) and that optimum pH was about 8. Sera from several species and a tetrapeptide (Arg-Gly-Asp-Ser, the cell-attachment sequence in fibronectin) greatly increased leucine uptake and extended cell survival up to a period of about two weeks. Various growth factors and some other substances tested had no effect on uptake of leucine, cell morphology, or survival. A few other compounds tested were cytotoxic. None of the experimental media promoted cell proliferation.
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  • 19
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    Plant cell, tissue and organ culture 26 (1991), S. 53-59 
    ISSN: 1573-5044
    Keywords: cell culture ; cell wall ; conditioned medium ; inoculum density ; low inoculum growth factor ; Pinus radiata
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pinus radiata cells in suspension culture abruptly lose their growth capacity when diluted below a critical inoculum density. This threshold density can be lowered by adding the supernatant (conditioned medium) from healthy cultures which have been grown separately at high densities. Fresh medium is conditioned rapidly indicating that the factor responsible is either potent or produced rapidly. Activity-response curves increase progressively with concentration indicating that still greater effect may be obtained if the factor can be concentrated following separation from other medium components. The effect is not mimicked by a number of candidate compounds (including auxins, cytokinins, polyamines and vitamins). Partial characterisation studies indicate that the factor is relatively small (〈1 000 dalton) and possibly an oligosaccharide. It is considered that the factor is an essential structural component of the walls of expanding cells where it is reversibly-bound.
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  • 20
    ISSN: 1573-6830
    Keywords: acetylcholinesterase ; asymmetric-form types ; chick hindlimb muscle ; development ; cell culture ; diisopropylfluorophosphate inactivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle,in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP)in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFPin vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.
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  • 21
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    Plant cell, tissue and organ culture 24 (1991), S. 49-54 
    ISSN: 1573-5044
    Keywords: anthocyanin ; cell culture ; osmotic potential ; reverse-phase HPLC ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from −0.43 MPa to −0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.
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  • 22
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    Plant cell, tissue and organ culture 24 (1991), S. 207-210 
    ISSN: 1573-5044
    Keywords: anthraquinone production ; cell culture ; Galium vernum ; polymeric adsorbents ; secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.
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  • 23
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    Plant cell, tissue and organ culture 25 (1991), S. 147-152 
    ISSN: 1573-5044
    Keywords: cell culture ; Daucus carota ; 2-deoxy-D-glucose ; invertase ; selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Variant carrot (Daucus carota L.) cell lines resistant to the growth inhibitory effects of the glucose analogue 2-deoxy-D-glucose (dGlc) were isolated utilizing a feeder plate technique. Growth of sensitive cells was less than 7.5% of controls on medium supplemented with 3.0 mM dGlc, whereas resistant variants achieved growth ranging from 15% to 70% of that in controls. Increased levels of acid invertase activity in variant cell lines in response to dGlc in the culture medium, together with decreased sensitivity of the acid invertase enzyme (EC 3.2.1.26) to dGlc, is proposed as one of several potential mechanisms contributing to the observed dGlc resistance.
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  • 24
    ISSN: 1573-0603
    Keywords: bronchial epithelium ; cell culture ; intermediate filaments ; carbohydrate cytochemistry ; lectins ; peroxidase-labeled lectins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A protocol is presented for correlating the morphology of human bronchial epithelial cells after Papanicolaou staining with their periodic acid schiff (PAS)/AB-reactivity, intermediate filament type, and the pattern of staining with lectins having specific affinities for glycoconjugates:griffionia simplicifolia (GSA-IB4; terminal alpha-d-galactose),Dolichos biflorus (DBA,N-acetyl-d-galactosamine). The combination of these stains identified differentiation markers in morphologic studies of normal, transformed, and malignant bronchial epithelial cells in culture.
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  • 25
    ISSN: 1573-0603
    Keywords: pH ; NH 4 + ; BCECF ; human kidney ; proximal tubule ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This protocol describes the use of the pH-sensitive, intracellularly trapped dye 2′,7′-bis (2-carboxyethyl), 5 (and -6) carboxyfluorescein (BCECF), to characterize the pH regulating mechanisms in cultured human kidney proximal tubule cells. This is a reliable method for intracellular pH measurements and is applicable to single cells, cell suspensions, and confluent cultures.
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  • 26
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    Methods in cell science 13 (1991), S. 285-288 
    ISSN: 1573-0603
    Keywords: DNA ; fluorimetry ; fibroblast ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An improved direct fluorimetric assay is described using bisbenzimidazol fluorescence at 356 nm excitation and 458 nm emission wavelengths. The turbidity of the medium was shown to have no effect on fluorescence under an absorbance of 0.2 at the excitation wavelength. The method was evaluated by comparisons with a colorimetric DNA assay and cell counting. The linearity of fluorescence was determined up to 15μg/ml of DNA. The method was very reproducible and sensitive to detect 10 ng/ml of DNA and may be used for cell suspensions containing around 5000 cells/ml or more.
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  • 27
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    Methods in cell science 13 (1991), S. 289-294 
    ISSN: 1573-0603
    Keywords: fetal bovine ; oviduct ; cell culture ; microscopy ; immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Bovine epithelial cell monolayers were obtained for culture from fetal oviduct after in situ trypsinization. Isolated ciliated and secretory cells obtained in high yield with good viability were suspended in B2-MENEZO'S medium supplemented with 7.5% fetal bovine serum FBS. The plated primary cultures reached confluency 2 days after initial seeding, producing a monolayer of cohesive polygonal cells with viability of 85 to 95%. Associated with this large epithelial cell population, ciliated cells as well as a few elongated spindle cells were observed. After the first subculture the ciliated cells disappeared and the epithelial cells in the monolayer grew more rapidly to confluence than adult-derived cultures. In addition, frozen-thawed oviduct epithelial cells also maintained a level of 75 to 85% viability during postthaw subculture. The epithelial cells maintained their secretory activity in culture as indicated by electron microscopy and immunocytochemistry. The cell culture monolayers contained keratin, a specific cytoskeletal component of epithelial cells. This culture system may offer benefits for in vitro culture of mammalian embryos.
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  • 28
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    Plant cell, tissue and organ culture 26 (1991), S. 195-201 
    ISSN: 1573-5044
    Keywords: Brassica napus ; cell culture ; diffusion ; liquid medium ; oxygen availability ; protoplast culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 μM O2, below 60 μM all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.
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  • 29
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    Plant cell, tissue and organ culture 27 (1991), S. 267-274 
    ISSN: 1573-5044
    Keywords: cell culture ; enzyme induction ; fungal elicitor ; l-phenylalanine ammonia-lyase ; Picea abies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate. In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light. With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.
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  • 30
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    Cellular and molecular neurobiology 11 (1991), S. 579-591 
    ISSN: 1573-6830
    Keywords: proteoglycans ; glycosaminoglycans ; mouse retina ; photoreceptors ; retinal neurons ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. In recent years, a number of histochemical and immunocytochemical studies have suggested that proteoglycans, particularly those in the inter-photoreceptor matrix, exhibit altered distributions in several murine models for retinal degenerations. We are using a cell culture system to characterize the proteoglycans synthesized by neurons and photoreceptors derived from mouse retina, with the long-term goal of analyzing their role in retinal degenerations. 2. In this study we describe initial studies using cells derived from the retinas of normal mice. Cultures of retinal neurons and photoreceptors, which were free of glial, epithelia, or endothelial cells, were labeled with3H-glucosamine and35SO4. Proteoglycans isolated from the medium and cell layer were analyzed on the basis of charge, relative hydrodynamic size, and glycosaminoglycan content. 3. The studies indicate that the cultures actively synthesize proteoglycans. The medium contained predominantly chondroitin sulfate/dermatan sulfate, while the cell layer had a higher proportion of heparan sulfate, indicating a differential distribution between the two compartments.
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  • 31
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    Cytotechnology 5 (1991), S. 15-30 
    ISSN: 1573-0778
    Keywords: high density ; cell culture ; serum-free medium ; hybridoma ; CHO cells ; virus production ; insect cells ; adoptive immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion At the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old. This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs. It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.
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  • 32
    ISSN: 1573-0778
    Keywords: cell culture ; kinetics ; Ig promoter/enhancer ; plasmacytoma ; recombinant protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.
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  • 33
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    Cytotechnology 6 (1991), S. 49-54 
    ISSN: 1573-0778
    Keywords: Bivalvia ; cell culture ; embryo ; mitosis ; scallop
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.
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  • 34
    ISSN: 1573-0778
    Keywords: cell culture ; endosteal human osteoblasts ; maxilla ; mandible ; titanium ; biocompatibility ; alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Fragments of cancellous and cortical bone from human maxilla and mandible were cultured by the explant technique. Cells isolated by trypsinization of primary cultures were characterized as osteoblasts on the basis of intracellular alkaline phosphatase activity, the constituents of the extracellular matrix, and response to human parathormone (PTH). In culture, the osteoblasts often gave rise to superposed clumps of large cells whose cytoplasm contained endoplasmic reticulum, numerous mitochondria, vacuoles, and a dense network of intermediate filaments, often at the level of the plasma membrane. In the presence of vitamin C and 1,25-dihydroxyvitamin D3, the osteoblasts produced an extracellular matrix composed of collagen type I and various non-collagenous proteins, including osteocalcin. Biochemical test results were comparable to those reported for osteoblasts of other origins (rat calvaria, human iliac crest), and namely elevated intracellular alkaline phosphatase activity and cAMP accumulation in response to stimulation by human PTH (1–34). Osteoblasts isolated in this manner were cultured in the presence of pure titanium disks to determine the effects of exposure to this metal. Electron microscopy revealed few significant differences in cell growth and specific enzyme activity compared to control osteoblasts grown on plastic dishes, reflecting the excellent biologic and biochemical relationship between the osteoblasts and pure titanium. This experimental system thus appears suitable for biocompatibility studies, and in particular, evaluation of dental implants.
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  • 35
    ISSN: 1573-904X
    Keywords: Caco-2 ; unstirred water layer ; intestinal permeability ; steroids ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 × 10−5 cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564µm. P and h in the stagnant Transwell were 3.08 × 10−5 cm/sec and 2597 µm, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.
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  • 36
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    Pharmaceutical research 8 (1991), S. 1453-1460 
    ISSN: 1573-904X
    Keywords: peptide ; transport ; permeability ; lipophilicity ; hydrogen bonding ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The relationship between structure and permeability of peptides across epithelial cells was studied. Using confluent monolayers of Caco-2 cells as a model of the intestinal epithelium, permeability coefficients were obtained from the steady-state flux of a series of neutral and zwitterionic peptides prepared from D-phenylalanine and glycine. Although these peptides ranged in lipophilicity (log octanol/water partition coefficient) from −2.2 to +2.8, no correlation was found between the observed flux and the apparent lipophilicity. However, a strong correlation was found for the flux of the neutral series and the total number of hydrogen bonds the peptide could potentially make with water. These results suggest that a major impediment to peptide passive absorption is the energy required to break water–peptide hydrogen bonds in order for the solute to enter the cell membrane. This energy appears not to be offset by the favorable introduction of lipophilic side chains in the amino acid residues.
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  • 37
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    Biotechnology and Bioengineering 38 (1991), S. 653-658 
    ISSN: 0006-3592
    Keywords: light irradiation ; anthocyanin production ; Perilla frutescens ; cell culture ; bioreactor cultivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: After a series of experiments on photoperiodicity and light intensity under daylight supplied by an ordinary fluorescent lamp in cultivations using a flask and a roux bottle, it was found that irradiation at 27.2 W/m2 for the whole period was effective for anthocyanin production by a suspended culture of Perilla frutescens (shiso). A high amount of anthocyanin pigments, 3.0 g/L, was obtained in a bubble column bioreactor after 10 days of cultivation at an aeration rate of 0.1 vvm with light irradiation at 27.2 W/m2, while 2 g/L was obtained at 13.6 W/m2 and very little at 54.4 W/m2. A high amount of anthocyanin pigments, 2.9 g/L, was also produced using an aerated and agitated bioreactor at an agitation speed of 130 rpm, an aeration rate of 0.1 vvm and light irradiation intensity of 27.2 W/m2. The amount of anthocyanin produced was more than twice that without light irradiation, Keeping the other cultivation conditions the same. The results obtained also showed that the amount of anthocyanin pigment accumulated in a shake flask could be rather well reproduced in bioreactors for both aerated culture, and aerated and agitated culture, by improving the conditions of light irradiation, which conspicuously affects metabolite formation.
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    Biotechnology and Bioengineering 38 (1991), S. 459-470 
    ISSN: 0006-3592
    Keywords: cell culture ; contact inhibition phenomena ; discrete mathematical model ; cell proliferation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We report the development of new class of discrete models that can accurately describe the contact-inhibited proliferation of anchorage-dependent cells. The models are based on cellular automata, and they quantitatively account for contact inhibition phenomena occurring during all stages of the proliferation process: (a) the initial stage of “exponential” growth of cells without contact inhibition; (b) the second stage where cell colonies form and grow with few colony mergings; and (c) the final stage where proliferation rates are dominated by colony merging events. Model prediction are presented and analyzed to study the complicated dynamics of large cell populations and determine how the initial spatial cell distribution, the seeding density, and the geometry of the growth surface affect the observed proliferation rates. Finally, we present a model variant that can simulate contact-inhibited proliferation of asynchronous cell populations with arbitrary cell cycle-time distribution. The latter model can also compute the percentage of cells that are in a specific phase of their division cycle at a given time.
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  • 39
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    Biotechnology and Bioengineering 38 (1991), S. 972-976 
    ISSN: 0006-3592
    Keywords: cell culture ; antibody production ; fermentation ; continuous culture ; cell growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day-1. The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day-1. Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.
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  • 40
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    Biotechnology and Bioengineering 38 (1991), S. 1020-1028 
    ISSN: 0006-3592
    Keywords: hybridoma ; cell culture ; continuous culture ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.
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    Biotechnology and Bioengineering 37 (1991), S. 755-761 
    ISSN: 0006-3592
    Keywords: cell culture ; fiber-bed bioreactor ; anchorage-dependent cell cultures ; airlift ; bioreactor design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A concentric-cylinder airlift reactor, in which the annulus is a packed bed of glass fibers, has been developed in order to facilitate the scaleup and enhance the volumetric productivity of anchorage-dependent animal cell cultures. In this bio-reactor, oxygen-containing gas is sparged through the inner draft tube, causing bubble-free medium to flow through the fiber bed in the outer cylinder and providing both oxygenation and convective nutrient transfer to the cells. Several other desirable features for reactor operation are also provided by this design. Cell cultivations in this bioreactor have been successfully carried out and provide data for the feasibility of the large-scale cell cultivation.
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  • 42
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    Bulletin of experimental biology and medicine 112 (1991), S. 1152-1156 
    ISSN: 1573-8221
    Keywords: experimental leprosy ; synthetic tuftsin ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 43
    ISSN: 0730-2312
    Keywords: epidermis ; skin ; skin graft ; cell culture ; in vitro ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.
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  • 44
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    Journal of Cellular Biochemistry 45 (1991), S. 258-260 
    ISSN: 0730-2312
    Keywords: chondrogenesis ; chondrocyte ; cell culture ; joint repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Articular cartilage is notoriously defective in its capacity for self-repair, making joints particularly sensitive to degenerative processes. However, methods are now available for the preparation of large numbers of differentiated chondrocytes from a small biopsy sample from any patient. The cells are amplified by proliferation as fibroblast-like cells that will re-express the cartilage phenotype when placed in suspension or gel culture. The chondrocytes can be collected from gel cultures after agarase treatment and reconstituted into cartilage tissue in pellet cultures. In addition, these chondrocytes can be suspended in an appropriate delivery vehicle and implanted into defect sites with a high reparative success rate in an animal model. Appropriate procedures can now be tested in appropriate patient populations.
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