Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Continuous growth of human tumor cell lines in serum-free media

Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10⁻¹⁰ M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth. This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J. Hill) and grant no. CA-37138 from the National Cancer Institute.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

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.     

Three-dimensional growth and morphogenesis of mouse submandibular epithelial cells in serum-free primary culture

Mouse submandibular epithelial cells can be grown in primary culture using the collagen gel matrix and a chemically defined medium consisting of insulin, transferrin, cholera toxin, and BSA (or FGF). Sustained cell growth leading to a 5–10-fold increase in cell number was observed in less than 2 weeks. In the presence of these additives, clumps of cells proliferate by extending ‘star-like’ projections into the matrix, resulting in three-dimensional outgrowths. The morphology of these outgrowths can be modulated to form a ‘cyst-like’ appearance by deleting BSA and adding cortisol to the basal medium containing insulin, transferrin, cholera toxin and FGF. In brief, a serum-free medium for sustained growth has been devised and a simple manipulation of supplements can modulate the three-dimensional colony morphology in the collagen gel matrix. Finally, the resulting outgrowths can produce epidermal growth factor (EGF) in response to dihydrotestosterone.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

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.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

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.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

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.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Growth of human foreskin fibroblasts in a serum-free,defined medium without platelet-derived growth factor

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Wheresas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), trasferrin (10 μg/ml), thrombin (1 μg/ml), ascorbic acid (10 μg/ml), and hydrocortisone (5 × 10^?5M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblst growth factor, nor somatomedin activity increased proliferation. This serum-free medium designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.     

Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.     

Growth of mouse vaginal epithelial cells in culture: Effect of sera and supplemented serum-free media

Summary Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel metrix and a serum-free medium composed of a 1∶1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly, diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17?-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen. This investigation was supported by grants CA-05388 and CA-09041, awarded by the National Institutes of Health, DHHS, Bethesda, MD.     

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

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.