Search PubMed⌕ Search

Biomedical subjects

R G Ham

Publications and source records attributed to R G Ham.

At least 37 records · Page 2Linked to original sources
PubMed1983

Control of entry of Swiss 3T3 cells into S phase by fibroblast growth factor under serum-free conditions.

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.

Animals↗
PubMed1983

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium.

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.

Blood↗
PubMed1983

Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.

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.

Blood↗
PubMed1982

Clonal growth of normal human epidermal keratinocytes in a defined medium.

Colony formation by normal human epidermal keratinocytes (HK) has been achieved in a medium that contains no deliberately added undefined supplements. The term "defined" is used to describe this medium, although the possibility that trace contaminants in its components could be contributing to the multiplication that it supports cannot yet be ruled out completely. The defined medium consists of a basal medium, MCDB 152, supplemented with 5 ng/ml epidermal growth factor (EGF), 10 micrograms/ml transferrin, 5 micrograms/ml insulin, 1.4 X 10(-6) M hydrocortisone, 1.0 X 10(-5) Methanolamine, 1.0 X 10(-5) M phosphoethanolamine, and 2.0 X 10(-9) M progesterone. MCDB 152 differs from MCDB 151, previously developed for multiplication of HK with small amounts of dialyzed serum (Peehl and Ham, 1980b), only by addition of the trace element mixture from human fibroblast medium MCDB 104 (McKeehan et al., 1977). Most of the requirement for transferrin, which is the least defined component of the defined medium, can be replaced by adding freshly dissolved and sterilized ferrous sulfate to the final medium after it has been filter sterilized. Insulin and EGF are clearly needed for optimal multiplication and hydrocortisone is mildly beneficial. Either ethanolamine or phosphoethanolamine must be present in the defined medium for HK multiplication. There is a greater need for EGF and less for hydrocortisone in the defined medium than in previous partially defined systems that we have worked with. Very large colonies of flattened epithelial cells are obtained in the defined medium, which has a low calcium concentration (0.03 mM) and does not favor keratinocyte differentiation. Less growth and more differentiation are obtained with higher calcium concentrations. The defined medium is highly selective for keratinocyte growth from a mixed inoculum of keratinocytes and fibroblasts.

Animals↗
PubMed1981

Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells.

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.

Amino Acids↗
PubMed1981

Protein-free medium for C-1300 mouse neuroblastoma cells.

An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1 x 10(4) cells/cm2 or greater, several different clones of C1300 mouse neuroblastoma cells can be cultured serially in Medium MCDB 411 with no serum or other undefined supplementation and with a doubling time of about 24 h. At clonal densities it is necessary to supplement the medium with 1.0 micrograms/ml insulin. Alternately, good clonal growth can be obtained without direct supplementation by coating the culture dishes with insulin and rinsing off all that is not tightly bound. Primary cultures of cells from serially transplanted C1300 tumors that have never been cultured previously in vitro can be established directly in unsupplemented Medium MCDB 411 with rapid initiation of multiplication and no apparent crisis or selection for minority cell types. The availability of a synthetic medium that supports growth of neuroblastoma cells without supplementation should facilitate the use of these cells as model systems for the study of neuronal function and differentiation.

Amino Acids↗
PubMed1981

Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone.

A serum-free, hormone-supplemented medium, enriched with a mixture of lipids, has been developed that supports rapid clonal growth of human diploid fetal lung fibroblasts (Flow 2000, WI-38, MRC-5, and IMR-90) and of low-passage human foreskin fibroblasts. The medium, which contains less than 1 microgram of total protein per ml, also supports serial passage of Flow 2000 cells under totally serum-free conditions. It provides lipid at a total of 10 micrograms/ml as a liposome prepared from a mixture of soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. The soybean lecithin, which contains a variety of naturally occurring phospholipids, can be replaced with a mixture of highly purified phospholipids. Except for possible contaminants in the substances used in its preparation, the serum-free medium is fully defined chemically. It consists of an optimized basal nutrient medium, MCDB 110, supplemented with insulin, epidermal growth factor, dexamethasone, prostaglandins E1 and F2 alpha, phosphoenolpyruvate, dithiothreitol, glutathione, and the lipids listed above.

Cell Division↗
PubMed1980

Clonal growth of human keratinocytes with small amounts of dialyzed serum.

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.

Adenine↗
PubMed1978

Development of improved media and culture conditions for clonal growth of normal diploid cells.

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.

Blood Proteins↗