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

Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 microgram per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as "replaceable" (those that can be replaced by modifying the medium or the culture conditions) and "nonreplaceable" (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold.     

Cerebral microvascular smooth muscle in tissue culture

Summary Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco’s modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a “hill and valley” pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-³⁵]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, α-actin. The identity of α-actin is confirmed by analysis of NH₂-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of α-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation. This work was supported by Veteran’s Administration Research Funds, National Institutes of Health Grant HL 14230 (Arteriosclerosis Specialized Center of Research, sponsored by the National Heart, Lung, and Blood Institute), and Cardiovascular Research Program Project Grant from the National Institutes of Health to the University of Iowa (HL-14388). A. R. S. is a postdoctoral fellow of the National Institute of General Medical Sciences (GM-09020). P. A. R. is an established investigator of the American Heart Association.     

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     

Requirements for maintaining the embryonic state of avian tendon cells in culture

Summary Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen. However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the inability of serum at low concentrations to remain a potent mitogen after a few subcultures. The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and “older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells are triggered to mature by a mechanism that correlates with high cell density. This investigation was supported in part by National Science Foundation Grant PCM 77-14982; in part by the Division of Biomedical and Environmental Research of the Department of Energy under contract W-7405-ENG-48; and by a National Institutes of Health Fellowship IF32 CA 05807-01, from the National Cancer Institute to R. I. S.     

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.     

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).     

Analysis of growth dynamics and the relationship to the fluctuations in alkaline phosphatase in two strains of cells of HeLa S3 derivation

Summary Studies have been carried out to determine an association between glucocorticoid-induced changes in the pattern of growth and the fluctuations of alkaline phosphatase in two HeLa strains. The results showed that growth arrest in steroid-treated cells did not have the characteristics of density-induced growth inhibition. Alkaline phosphatase increased with increased cell density, the increase being greater than control in steroid-treated cells of the “inducible” strain (HeLa S3G, HeLa₆₅) and less than control in the “suppressible” strain (HeLa S3K, HeLa₇₁). Increased serum concentration in the growth medium (0 to 20%) caused an increase in alkaline phosphatase in S3G strain and a decrease in the S3K strain. This investigation was supported by the Veterans Administration and by USPHS Research Grant CA-08315 from the National Cancer Institute.     

A defined medium for and the effect of insulin on the growth,amino acid transport,and morphology of chinese hamster ovary cells,CHO-K1 (CCL 61) and the isolation of insulin “independent” mutants

Summary Insulin, FeSO₄, or transferrin are major requirements together with HEPES buffer and selenium for the growth of CHO-K1 (CCL 61) in a modified F12 medium (M-F12). Insulin stimulates growth at 1 ng/ml to 10 μg/ml. In the defined medium minus insulin, CHO-K1 grows slowly as elongated, elliptical cells in parallel arrays typical of normal diploid fibroblasts in contrast to round-to-cuboid cells in loosely overlapping arrays in the presence of serum or insulin. During prolonged incubation in the absence of insulin the cells gather up into a large spherical cluster of viable cells. Insulin “independent” mutants have been isolated whose growth rate during exponential phase in the absence of insulin (48 h to 84 or 96 hrs) is 2.7 to 3.6 times that of the parental culture. Insulin stimulates the growth of these variants only during the first 48 h and is inhibitory at 50 to 500 ng/ml during the exponential phase. Insulin induction of the A system of amino acid transport occurs in about 8 h and requires both protein and RNA synthesis. This work was supported in part by National Science Foundation Grant PCM 7903242 and by the University of California.     

The nonexistence of “hermaphoroditic” tracer systems

This communiction argues that so-called “hermaphroditic” tracer systems, which are neither open nor closed, do not exist physically. The argument is based on the assumption that any observable (possibly nonhomogeneous) macroscopic compartment can be approximated by a compartmentC with a finite number of entry points for the tracer, each associated with an abstract subcompartment ofC. It is shown that the “hermaphroditic” property requires that the mean waiting time be infinite in at least one of the subcompartments, or in a subcompartment elsewhere in the system. A subcompartment with infinite mean waiting time must have some sort of memory, of infinite duration, which knows how long a given particle has been retained, however long that is, and thereby determines its probability of departure. Assuming, as seems likely, that no physical basis exists for such an infinite memory, it follows that “hermaphroditic” systems do not exist. Supported in part by U.S. Public Health Service Research Grant GM 21269 from the National Institute of General Medical Sciences, and in part by Biomedical Research Support Grant S07 RR 05392 from the National Institutes of Health.     

Cultivation of mammalian pineal cells: Retention of organization and function in tissue culture

Summary By means of a newly developed method of cultivating pineal tissue in vitro, the types of cells which comprise rat pineal glands have been identified. Previous in vitro studies have involved short-term culture more suitably called “organ culture” and provide no means of assessing the contribution of a putative “pineal” cell versus any other cell type found in the cultures. Short-term outgrowths of minced rat pineal glands provided a reproducible and easily dissociated source of pineal-derived cells. In monolayer culture these cells continued to have pineal enzyme activities which were sensitive to pineal-activating substances, and the cells aggregated to mimic the lobular organization of intact glands. Two types of aggregates were found, each composed of a single morphological cell type. In addition to the transient appearance of skeletal muscle straps, connective tissue and neural/glial tissue was consistently found. The cell types are discussed in relation to their in vivo counter-parts. Supported by NSF Grant GB-43215 to S.B. and NSF Grant GB-20919 to S.R.H.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Study of the dynamics of sertoli cell secretions in a new superfusion,two-compartment culture system

Summary A new superfusion, two-compaatment culture system recently developed in our laboratory was used to investigate the dynamic changes in bidirectional secretion of transferrin, (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10⁻⁶ M; follicle stimulating hormone, 0.1 μg/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to 3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers and for the study of bidirectional secretions under more “physiologic” conditions than those provided by static cultures. This work was supported in part by grant HD 17802 (AS) from the National Institutes of Health, Bethesda, MD.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

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.     

Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

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.     

Growth in primary culture of mouse submandibular epithelial cells embedded in collagen gels

Summary Mouse submandibular glands were dissociated and the epithelial cells embedded in a collagen gel matrix. A characteristic and reproducible pattern of growth was seen resulting in three-dimensional outgrowths with ductlike structures projecting into the matrix. A sustained cell growth leading to a 5 to 10-fold increase in cell number was observed in less than 2 wk. The extent of this growth was found to be dependent on serum concentration. Of the three sera tested, swine serum was found to promote greater growth compared to fetal bovine serum or horse serum. Swine serum dose response studies have shown that a concentration of 2 to 5% in the medium elicited only a modest increase, if any, in cell number compared to the initial value within a period of 2 wk. Various hormones and growth factors were then added to this “maintenance” medium. Insulin was found to stimulate growth consistently and reproducibly in a dose-dependent manner. Ultrastructurally, the resulting outgrowths were comprised of polarized cells joined by apical tight junctions and desmosomes. These outgrowths produced epidermal growth factor in response to dihydrotestosterone, triiodothyronine, and cortisol. The present system provides a method for sustaining growth and functional differentiation in primary culture of mouse submandibular gland epithelial cells. This investigation was supported by PHS Grants CA05388 and CA09041, awarded by the National Cancer Institute, Department of Health and Human Services.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Inhibition of collagen synthesis by α, α′-dipyridyl in human skin fibroblasts in culture

Summary In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[¹⁴C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[¹⁴C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[¹⁴C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation. This work was supported in part by a grant from the Easter Seal Research Foundation, and by Project 236, Health Services and Mental Health Administration, Department of Health, Education and Welfare, Grant HD-03110 from the National Institute of Child Health and Human Development, an American Cancer Society Institutional Grant (1N 15-J), a General Research Support Award (5-S01-FR-05406) from the National Institutes of Health, a University Research Council Grant, a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Disease (G.K.S.).