A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium

Cell culture with serum-containing medium has potential problems associated with contamination of infectious agents. This study demonstrates for the first time the feasibility of regenerating cartilage tissues in vivo by implantation of chondrocytes cultured in vitro in a chemically-defined, serum-free medium. Chondrocytes cultured in the serum-free medium grew similarly to those in a serum-containing medium. Implantation of chondrocytes cultured in the serum-free medium and seeded on to polymer scaffolds resulted in the regeneration of cartilage tissues with histological aspects similar to those of cartilage tissues regenerated from chondrocytes cultured in serum-containing medium.     

Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium

Summary A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na⁺-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na⁺-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by Grant 9 RO1 DK40286-07 from the National Institutes of Health, Bethesda, MD, and NIH Research Career Development Award 1 K04 CA 0088-01 to M.T.     

A serum-free medium for hybridoma cell culture

The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA bovine serum albumin - CS calf serum - DMEM Dulbecco's modified Eagle's medium - ELISA enzyme-linked immunosorbant assay - McAb monoclonal antibody - PEG polyethylene glycol - SFM serum-free medium     

Serum-free,chemically defined medium to evaluate the direct effects of growth factors and inhibitors on proliferation and function of neonatal rat cardiac muscle cells in culture

Summary Neonatal rat cardiac myocytes were isolated and cultured to evaluate the effects of growth factors and inhibitors on proliferation, survival, and functions in a serum-free medium. Insulin and transferrin in MCDB 107 nutrient medium elicited DNA and protein synthesis in cells on a fibronectin-coated culture surface in serum-free medium. Insulin was most effective on both DNA and protein synthesis in serum-free culture conditions. The serum-free, hormone-supplemented medium eliminated the contamination of noncardiac myocytes and supported the long-term survival (over 18 d) of cardiac myocytes. Dexamethasone was required to induce optimal contractility with or without insulin and transferrin. Serum contained both negative and positive effectors of DNA and protein synthesis of the cardiac myocytes. Concentrations of serum (above 5%) inhibited DNA and protein synthesis. Low density lipoprotein (LDL) accounted in part for the inhibitory activity. The serum-free culture system provides a useful model to elucidate the role of hormones, growth factors, and drugs in heart cell regeneration and function.     

A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation

A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Development of a new serum-free medium,USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes

Summary A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. Ths new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum. This work was supported by grant AM18983 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Summary The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [³H]lathosterol to [³H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells. This work was supported by National Institute of Health grants CA40294 and CA37589 to G. H. Sato and by a grant from RJR nabisco Inc. Editor's Statement These results help identify the defect in myeloma cells leading to cholesterol auxotrophy. The use of these cells in hybridoma derivation adds practical utility to a detailed appreciation of cholesterol metabolism in these cultures.     

Adult cardiac muscle cells in long-term serum-free culture: Myofibrillar organization and expression of myosin heavy chain isoforms

Summary A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture. Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T₃), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria, degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the culture period. Cardiac myocytes grown, in serum-free medium expressed the predominant myosin isoform V1 similar to their counterparts in vivo. T₃ is essential for the expression of isomyosin V1. This study demonstrates that adult cardiac muscle cells can be maintained in long-term serum-free culture from seeding to termination. The cells in serum-free conditions maintain at least two differentiated characteristics of adult myocytes investigated, namely, abundant organized myofibrils and predominant myosin isoform V1. This work is supported by grant DCB-8709594 from the National Science Foundation, Washington, DC     

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor

Summary The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine “essential” amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco’s modified Eagle’s medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our “modified” DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called “essential,” but rather an optimal balance of amino acids is required for each major function of each cell type cultured.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures

Summary The cell-type composition of the initial cell population from protease-dispersed neonatal rat heart tissue has been evaluated using time lapse photography and identification of cell type-specific functions. The effects of two commonly employed plating densities on growth and cell division of the two major cell types were examined. Total protein synthesis rates were not affected by plating density but did change with age in culture. Maximum protein synthesis rates were observed during the period of maximum cell division and cell growth (increase in total cell protein), which was from 24 h in culture to the 4th d in culture. After 6 d in culture, synthesis rates for total proteins remained constant for at least 2 wk. Sizing of cells by Coulter counter analysis indicated that essentially all the cells were increasing in size with age in culture. Measurements of cell numbers and rate of DNA synthesis indicated that the extent of cell division was dependent on plating density. Cells disaggregated from neonatal rat hearts consisted of approximately 75% muslce cells and 25% nonmuscle cells. This composition approximates the cell-type composition of the intact neonatal rat heart. In high density cultures there is little cell division and the relative proportionsof the cell types are preserved with time in culture. In low density cultures, proliferation of nonmuscle cells is a significant process and the composition of the cell population changes drastically during the first 2 to 3 d in culture. These results suggest that the low plating density used by many researchers may limit correlation of data derived from such cultures with the physiological state. It also indicates that plating densities should be given in published accounts for comparisons to be made with results from other laboratories. This work was supported in part by U.S. Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station and was authorized for publication as Paper 5490 in the journal series of the Pennsylvania Agricultural Experiment Station.     

Development of a serum-free medium using computer-assisted factorial design and analysis

Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA Analysis of variance - BSA bovine serum albumin - CHO Chinese hamster ovary - FBS fetal bovine serum - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - PBS phosphate buffered saline - SFM serum-free medium     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.