Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum

Summary An optimized basal nutrient medium, MCBD 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 μg/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. This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD.     

Protein-free medium for C-1300 mouse neuroblastoma cells

Summary An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1×10⁴ 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 μg/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 MCBD 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. This work was supported by Grant CA-15305 from the National Cancer Institute.     

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

Summary Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations of serum protein as low as 125μg/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. Portions of this work were reported at the Thirtieth Annual Meeting of the Tissue Culture Association in Seattle, Washington. This work was supported by Grant CA-15305 from the National Cancer Institute     

Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein

Summary A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 μg per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium. This work was supported by Grant No. AG00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, U.S. Food and Drug Administration.     

Selective growth of normal adult human urothelial cells in serum-free medium

Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca²⁺ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10⁻⁶ M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca²⁺ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis. This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells

Summary Clonal growth of WI-38 cells with a plating efficiency of 45% has been achieved in a synthetic nutrient mixture (MCDB 102) supplemented with either whole or dialyzed fetal bovine serum. For optimum growth, the concentration of cysteine in the medium must be adjusted precisely. Deviation by a factor of three in either direction from the optimum concentration (9.0×10⁻⁵M) eliminates essentially all clonal growth. A high concentration of glutamine (2.5×10⁻³M) is also needed for, optimum clonal growth. Presented in preliminary form at the 26th Annual Meeting of the Tissue Culture Association, June 4, 1975. This work was supported by Grant No. HD-08181 from the National Institute of Child Health and Human Developement, Grant No. AG-00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, Food and Drug Administration.     

Nutritional requirements of papiliomavirus-transformed mouse cells and an unifected parent line in serum-free culture

Summary A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium used for this study was an MCDB 151-based medium (MCDB 151+S+I), supplemented with epidermal growth factor, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free culture required the addition of 250 μg/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 151+S+I, whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirements than the uninfected parent line. This work was supported in part by grant #1-P01 NS19214 from the National Institutes of Health, Bethesda, MD, NSF grant #R11-8217798 from the National Science Foundation, Washington, DC, and by a grant from the Otolaryngology Foundation.     

Isolation,growth requirements,cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium

Summary Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived growth factor (5 μg/ml), epidermal growth factor (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term serial culture (17.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell growth that varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by aracidonate and thrombin by 17 to 20 and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult endothelial cells were 2 to 3 times that of human adult smooth muscle cells and 20 to 30 times that of human fibroblasts. The work was supported by Public Health Service Grant AGO3275 and Grant No. 1718 from the Council for Tobacco Research. Editor's statement This paper provides an opportunity for relatively rapid, easy growth and cloning of endothelial cells from various human specimens which are more difficult to deal with than those obtained from an intact artery or intact umbilical vein. Russel Ross     

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. I. Effect of liposome B components on colony growth

Summary An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64–24 with far less protein supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 μg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 μg/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 μg/ml liposome B (a mixture of soy lecithin, sphingomyelin, cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine or sphingomyelin. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by B.A.VdH. in partial fulfillment of the requirements for the Ph.D. degree. This research was supported by grant CA-15305 to R.G.H. and grant CA-30545 to T.K.-S., both from the National Institutes of Health, Bethesda, MD.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Clonal growth of Chinese hamster cell lines in protein-free media

Summary 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. This research was supported by Grant No. CA15305 from the National Cancer Institute.     

The modulation of growth of normal rat liver epithelial cells in calcium-poor medium by epidermal growth factor,phenobarbital, phorbol ester,and retinoic acid

Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca²⁺, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast, phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal cells, but such an effect does not seem to be a universal property of tumor promoters. This research was supported by National Institutes of Health Grant CA 29323.     

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO₅ medium). Growth in WO₅ medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO₁₆₀ medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO₅ medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×10⁷ plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO₅ medium. This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation.     

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

Summary 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 μg 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. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by Contract 223-74-1156 from the Bureau of Biologics, U.S. Food and Drug Adminsstration, Grant AG 00310 from the National Institute on Aging, and Grant CA 15305 from the National Cancer Institute.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Control of the production of two protein hormones by rat pituitary cells in culture

Summary A clonal strain of rat pituitary tumor cells (GH₃) has continued to produce the pituitary protein hormones growth hormone (GH) and prolactin during 5 years of continued growth in monolayer culture. Studies of the effects of external stimuli have indicated that, in spite of the physical similarity of these protein hormones (each is a single polypeptide of molecular weight ≈23,000), their production is controlled by different mechanisms. Addition of hydrocortisone (HC) (3×10⁻⁶ m) to the growth medium leads, after a lag of 12 to 24 hr, to an increased relative rate (rate in experimental cells divided by rate in control cells) of GH production. The relative rate reaches a maximum of 5 to 8 at 30 to 100 hr. Stimulation by HC of GH production is observed in cells growing in either the stationary or the exponential phase of growth. Indirect estimates indicate that, in exponentially growing cells, GH represents about 2% and 14% of the total protein synthesized by control and fully stimulated cells, respectively. Maintenance of the stimulated state requires HC. HC decreases both the growth rate of GH₃ cells and their incorporation of amino acids into acid-insoluble material. At the same time that HC stimulates GH production it decreases the relative rate of prolactin production to about 0.2 to 0.3. On the other hand, addition of acid extracts of bovine hypothalamus, cerebral cortex, kidney, or liver (0.3 to 1.0 mg of protein per ml) to the medium leads to an increase of the relative rate of prolactin production to 6 to 9, while decreasing the relative rate of GH production to about 0.5. Chromatographic fractionation of simple extracts of bovine liver has yielded a macromolecular, heatlabile fraction exhibiting these effects at a concentration as low as 20 μg per ml. GH₃ cells which have been adapted to growth in suspension culture produce both GH and prolactin. HC is observed to stimulate GH production and suppress prolactin production by cells growing in this state, without affecting the growth rate of the cells. This investigation was supported in part by Research Grant AM 11011 from the National Institute of Arthritis and Metabolic Diseases.