RESUMO
Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum as possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine-coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.
Assuntos
Divisão Celular , Células Clonais/fisiologia , Peptídeos , Polilisina , Polímeros , Proteínas Sanguíneas , Adesão Celular , Linhagem Celular , Colágeno , Gelatina , Histonas , ProtaminasRESUMO
An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK). Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures, for frozen storage, and for serial culture. The need for feeder layers, conditioned medium, serum, and specialized culture surfaces has been eliminated entirely. With an optimal level of calcium ion (0.3 mM), colony-forming efficiency is about 30 percent and cellular multiplication rate is 0.96 doublings per day in the defined medium. A high-calcium concentration (1.0 mM) induces stratification and terminal differentiation, which can be quantified by counting cornified envelopes that are resistant to boiling in sodium dodecyl sulfate plus dithiothreitol. Under optimal conditions with wBPE present, cellular senescence occurs after about 40 population doublings. Scanning electron microscopy (SEM) has verified the occurrence of stratification during differentiation in the defined medium with high calcium. High-voltage electron microscopy (HVEM) after detergent extraction of human epidermal keratinocyte (HK) colonies grown in the defined medium with low and high calcium has revealed specific changes in the intermediate filament network and keratohyalin granules corresponding to changes in cellular differentiation. Indirect immunofluorescence studies have verified that the intermediate filament network observed with HVEM is composed of keratin proteins.
Assuntos
Cálcio/farmacologia , Células Epidérmicas , Sangue , Diferenciação Celular/efeitos dos fármacos , Células Clonais/metabolismo , Meios de Cultura , Imunofluorescência , Humanos , Microscopia Eletrônica de VarreduraAssuntos
Meios de Cultura , Músculos/citologia , Células-Tronco/citologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Células Clonais/citologia , Humanos , Lactente , Músculos/transplante , Distrofias Musculares/patologia , Distrofias Musculares/cirurgia , Transplante de Células-TroncoRESUMO
A survey of commercially available media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 micrograms/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 micrograms/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from NCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca2+ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca2+ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDF 151 with 10 micrograms/ml HC and 1.0 mg/ml FBSP.
Assuntos
Proteínas Sanguíneas/farmacologia , Células Clonais/citologia , Células Epidérmicas , Adenina/farmacologia , Cálcio/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Substâncias de Crescimento/farmacologia , Hormônios/farmacologia , Humanos , Hidrocortisona/farmacologia , Queratinas/biossínteseRESUMO
An optimized basal nutrient medium, MCDB 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 microgram/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg2+ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS.
Assuntos
Endotélio/citologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais , Meios de Cultura , Fator de Crescimento Epidérmico/farmacologia , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Cinética , Magnésio/farmacologia , Camundongos , MicrocirculaçãoRESUMO
A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable "essential" role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly "defined" medium are discussed.
Assuntos
Células Clonais/citologia , Meios de Cultura , Animais , Divisão Celular , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Insulina , Pulmão , Masculino , Metilcelulose , Ovário , Selênio , TiroxinaRESUMO
The amount of serum protein required for clonal growth of normal diploid human and chicken fibroblasts has been reduced more than 90% by development of improved synthetic media and culture conditions. The following has contributed significantly to the reduction: (1) replacement of specific serum proteins with defined small molecular nutrients; (2) precise quantitative balance of all defined nutrients for optimal growth of single cells of each type under consideration; (3) modification of the culture substrate; (4) improvement of the cell harvesting technique. By combining these improvements, optimal growth of human fibroblasts (fetal lung, newborn foreskin, amniotic fluid) and chicken embryo fibroblasts occurs in synthetic media supplemented with 500 microgram/ml dialyzed fetal bovine serum protein [equivalent to 1% (v/v) whole serum]. The human fibroblasts form large colonies at a suboptimum rate at 25 microgram/ml dialyzed serum protein [equivalent to 0.05% (v/v) whole serum] and several divisions can be obtained in the complete absence of serum protein. Human and chicken fibroblasts differ both in their quantitative requirements for defined nutrients, and in the type and number of incompletely characterized serum components that they continue to require under the improved growth conditions. The improved systems provide a sensitive assay for the remaining undefined growth-promoting components of serum and other biological extracts.
Assuntos
Sangue , Células Cultivadas , Meios de Cultura , Animais , Proteínas Sanguíneas , Divisão Celular , Linhagem Celular , Células Clonais , Diploide , Patos , Métodos , PeleRESUMO
Clonal growth of primary cultures of rabbit ear chondrocytes in a defined medium without serum or other undefined additives has been achieved. The clonal inoculum is a suspension of fully differentiated chondrocytes prepared by collagenase digestion of rabbit ear cartilage and used with no prior adaptation or selection in culture. When inoculated into medium MCDB 104 supplemented with 100 ng/ml fibroblast growth factor (FGF), 1 microgram/ml insulin, and 5 micrograms/ml of a lipid supplement previously developed for human fibroblasts, the isolated chondrocytes undergo clonal multiplication to form large colonies of epithelial-like cells. Colonies grown in the defined medium for 14 days accumulate at their centers refractile cartilage-like matrix that is stained by acidified Alcian green, although the amount is significantly less than with undefined additives. This system opens the way for detailed studies, in a defined background medium, of factors that regulate phenotypic expression of cartilage-like differentiated properties.
Assuntos
Cartilagem/citologia , Lipídeos/farmacologia , Animais , Contagem de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais/citologia , Meios de Cultura , Fatores de Crescimento de Fibroblastos , Insulina/farmacologia , Peptídeos/farmacologia , CoelhosRESUMO
Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48-72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.
Assuntos
Divisão Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Substâncias de Crescimento/farmacologia , Hormônios/farmacologia , Lipídeos/farmacologia , Animais , Contagem de Células , Linhagem Celular , Meios de Cultura , DNA/biossíntese , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Fatores de Crescimento de Fibroblastos , Insulina/farmacologia , Interfase , Camundongos , Peptídeos/farmacologiaRESUMO
Swiss 3T3 cells can be made quiescent at low density by plating in medium MCDB 402 supplemented with dexamethasone (DEX), insulin (INS) and bovine plasma fibronectin (BPFn) for 3 days. One hour after stimulation of these cells by fibroblast growth factor (FGF), an increase in the rate of protein synthesis can be measured. Nine hours after stimulation by FGF, the rate at which the cells enter S phase increases abruptly. This increased rate of entry into S phase is delayed when methylamine is added to the medium before FGF treatment and later removed. The delay is only for the amount of time that the cells are exposed to methylamine, with no subsequent effect on the rate at which the cells enter S. The early increase in rate of protein synthesis caused by FGF is not blocked by concentrations of methylamine that stop the progression of FGF-treated cells toward S phase. The assay system that has been developed provides a means for detailed analysis of the prereplicative phase of Swiss 3T3 cells in a serum-free medium and in the absence of density-dependent inhibition.
Assuntos
Substâncias de Crescimento/farmacologia , Interfase/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Proteínas Sanguíneas/farmacologia , Linhagem Celular , Meios de Cultura , DNA/biossíntese , Fatores de Crescimento de Fibroblastos , Metilaminas/farmacologia , Camundongos , Biossíntese de ProteínasRESUMO
Fibroblasts from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB 202 supplemented with 1 microgram/ml bovine insulin, 1 microM dexamethasone, 100 ng/ml fibroblast growth factor and 20 micrograms/ml of the lipid preparation described by Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. [(1981) Proc. Natl Acad. Sci, USA, 78, 5588-5592]. When maintained as a confluent monolayer in this medium, the fibroblasts from the adipose tissues spontaneously underwent an adipose differentiation. This was accelerated by substituting medium F12 for medium MCDB 202, and by raising the CO2 tension from 2% to 7.5% in air over the cultures. The differentiation was inhibited by deleting FGF from the growth medium, or by coating the culture surface with fibronectin or poly-D-lysine. Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum. The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of [14C]acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol-3-phosphate dehydrogenase.
Assuntos
Tecido Adiposo/citologia , Fibroblastos/citologia , Animais , Dióxido de Carbono/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos , Fibroblastos/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Peptídeos/farmacologia , Ovinos , Triglicerídeos/biossínteseRESUMO
Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.
Assuntos
Cartilagem/citologia , Células Clonais/citologia , Sangue , Diferenciação Celular , Meios de Cultura , Dexametasona/farmacologia , Fatores de Crescimento de Fibroblastos , Humanos , Insulina/farmacologia , Lipídeos/farmacologia , Peptídeos/farmacologiaRESUMO
Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 microgram per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as "replaceable" (those that can be replaced by modifying the medium or the culture conditions) and "nonreplaceable" (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold.
Assuntos
Células Clonais/citologia , Meios de Cultura , Proteínas Sanguíneas , Adesão Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Transformação Celular Viral , Substâncias de Crescimento/isolamento & purificação , Polilisina , TripsinaRESUMO
Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations of serum protein at low as 125 micrograms/ml. Survival of the cells under completely protein-free conditions also is enhanced greatly. The improvements that made these results possible include: (a) use of medium MCDB 402, which was developed specifically for Swiss 3T3 cells by adjusting the concentrations of all components of Dulbecco's modified Eagle's medium to optimum values for clonal growth with minimal serum protein and by adding other nutrients such as trace elements and "nonessential" amino acids that were not in the original formula; (b) use of culture surfaces that are coated with a positively charged polymer, poly-D-lysine; and (c) use of gentle low temperature trypsinization technique that minimizes cellular damage and the need to neutralize residual trypsin.
Assuntos
Linhagem Celular , Meios de Cultura , Aminoácidos , Animais , Proteínas Sanguíneas , Adesão Celular , Células Clonais/citologia , Substâncias de Crescimento/sangue , Íons , Camundongos , Polilisina , TripsinaRESUMO
The trace element selenium is essential for clonal growth of diploid fibroblasts from human fetal lung (WI-38) in media containing small amounts of serum protein. Maximum growth stimulation is obtained when 30 nM neutralized selenious acid is added to a synthetic medium containing 1.5 mg/ml of dialyzed fetal bovine serum protein (equivalent to a 3% serum concentration). Serum appears to be a source of selenium in most culture media, since higher concentrations of serum protein or whole serum mask the selenium requirement of WI-38 cells. Selenium is also required by a Chinese hamster cell line that can be grown in a protein-free synthetic culture medium.
Assuntos
Linhagem Celular , Meios de Cultura , Selênio , Células Clonais , Fibroblastos , ProteínasRESUMO
Whether the growth-promoting activity of Pedersen fetuin is due to fetuin itself or to a contaminant(s) has been a long-standing puzzle. The possibility that the growth-promoting activity of Pedersen fetuin for human muscle satellite cells (HMSC) could be caused by some other component of fetal bovine serum (FBS) that remained in the fetuin as a contaminant has been investigated. One liter of FBS was first precipitated with 50% saturated ammonium sulfate, which leaves the serum albumin in solution, and then with 25% polyethylene glycol, which leaves the fetuin in solution, to generate a fraction 50 PEG 2x that was enriched 11-fold in growth-promoting activity for HMSC, with 68% recovery of total activity. Further purification with FPLC anion exchange chromatography achieved 99-fold enrichment of the activity with 30% overall recovery. The activity is heat labile and pH sensitive, suggesting that it is of protein nature, and the size of the activity is above 70 kDa. SDS-PAGE of the most active fractions shows that they are virtually free of fetuin. Thus, although the active fractions are not homogeneous, these studies demonstrate that the growth-promoting activity for HMSC can be fully separated from fetuin.