Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

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).     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

Summary Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na⁺-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. This project was supported by grant 2-R01-DK15440-16A1 from the National Institutes of Health, Bethesda, MD, and by grant N0001 4-88-K-0427 from the Department of the Navy, Washington, DC.     

Growth inhibition and morphologic modulation of human fibroblastlike cells by erythromycin

Summary In vitro exposures of mass cultures and clones of human diploid fibroblastlike cells to erythromycin, in concentrations of 50 to 400 μg/ml, result in increasing degrees of growth inhibition and augmented cell volume, with a shift toward larger proportions of cells of the epithelioid type and fewer of the fibroblast type. These alterations were reversed upon subculture in the absence of the antibiotic. This research was supported by National Institutes of Health grants AG 00257 and RR 08139 (Division of Research Resources and National Institute on Aging) and National Science Foundation grant PCM-8003728.     

Isolation of myoblastic, fibro-adipogenic, and fibroblastic clonal cell lines from a common precursor and study of their requirements for growth and differentiation

C17-S1-D-T984 (to be referred to as T984) is a myogenic clonal cell line isolated from a mouse teratocarcinoma. T984 exhibits phenotypic instability since it gives rise not only to myogenic but also to fibro-adipogenic and fibroblastic clones. A cell line of each clone type has been established and studied with respect to (1) phenotypic expression and stability; and (2) growth and differentiation in serum-free and serum-supplemented media. In both respects, marked differences between the three cell lines were observed. All three cell lines respond by increased growth in serum-free media to insulin, transferrin, fibroblast growth factor (FGF) and the serum-spreading factor of Holmes. The fibroblastic and the fibro-adipogenic cell lines can both be grown indefinitely in a serum-free medium which contains the above factors. The fibro-adipogenic cell line, which differentiates in serum-supplemented medium, exhibits very limited differentiation in the absence of serum; the serum factor(s) required for adipogenic differentiation is (are) probably proteins of molecular weight superior to 10 000. In direct contrast, the myogenic cell line exhibits limited growth in serum-free medium but readily differentiates under these conditions. Moreover, myogenic differentiation could be obtained in the defined medium at very low densities and was not influenced by the addition of medium conditioned by cells seeded at high densities. Thus, in this system, muscular differentiation is apparently independent of diffusible endogenous or exogenous factors and is probably triggered by the arrest of growth. While our results do not explain the reason why T984 exhibits phenotypic instability, they do indicate that this clonal cell line and its clonal derivatives could be used to identify the factors that influence the growth and the differentiation of cells of different mesenchymal phenotypes. The possible relationship of phenotypic instability to muscular dystrophies is also discussed.     

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.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

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.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

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.     

Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin c) related to the commercial source of fetal bovine serum in the culture medium

Summary The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P<0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, β-glucosidase, β-glucuronidase, andN-acetyl-β-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera. This work was supported in part by grants from the National Institutes of Health, Bethesda, MD (NS 16287).     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

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.     

The effect of donor age on the in vitro life span of cultured human arterial smooth-muscle cells

Summary The number of population doublings of cultured human arterial smooth-muscle cells decreased as a function of donor age (0.5 to 82 years). Cells from older donors als showed longer latent periods for outgrowth from explants. These results extend other comprable observations with human skin fibroblasts to another cell type, and may have relevance to the pathogenesis of atherosclerosis with aging in vivo. This study was supported by a reserach program project grant (AG00299) from the National Institutes of Health.     

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium

Summary Cultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10⁻⁶ M insulin, 5μg/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4′,6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions. This work was supported in part by National Institutes of Health grants HL31854, HL38130, and RR07048.     

Establishment of an adult rat fibroblast cell line for studies of collagenase regulation

Summary Several dermal fibroblast lines have been established from explants taken from adult rats. The cells have been cultured for 2 yr and possess stable and well-defined growth characteristics through subculture 18. The cells are readily stored in liquid nitrogen with good viability after thawing. Collagenase activity secreted into the culture medium of the cells at different periods of growth has been examined. There is an 88% drop in total enzyme activity present in the medium between 4 and 14 d of culture, when the cells were plated to reach confluence at Day 8 to 10. A more pronounced fall is noted at earlier times when the cells are plated at a higher density. The correlation between DNA content of the cell monolayer and enzyme activity was −0.895, indicating a possible relationship between the growth of cells and collagenase release. This study was supported in part by grant AM 30856 from the National Institutes of Health, Bethesda, MD.     

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.