Growth control in primary fetal rat liver cells in culture.

Primary fetal rat liver cells cultured in medium deficient in, but not free of, arginine in the presence of dialyzed fetal calf serum grow until the final cell density is attained and cells become quiescent in the Go phase of the cell cycle. When growing cells are transferred into arginine free medium, cells become reversibly arrested in Go. Fetal rat liver cells can be induced to synthesize DNA by addition of high levels of arginine to serum free medium. Low arginine levels in the culture medium do not induce cell growth unless serum is present. Serum stimulates arginine uptake in fetal rat liver cells suggesting that serum growth factor(s) act by increasing intracellular arginine levels high enough to initiate the growth cycle. Fractionation of fetal calf serum by gel filtration on G-200 Sephadex yields a partially purified arginine uptake stimulating activity which is eluted from the column in the same fractions that contain fetal rat liver cell growth promoting activity. Insulin induces DNA synthesis in quiescent fetal rat liver cells. Glucagon reverses the stimulatory effects of insulin. N-6,O-2-Dibutyryl adenosine 3:5-cyclic monophosphoric acid (But2c-AMP) (10-minus4 M) and theophilline (10-minus3 M) inhibit arginine uptake and the initiation of DNA synthesis by serum. The role of arginine in the control of DNA synthesis in fetal rat liver cells and the mechanism of action of serum growth factors are discussed.     

Growth factors and apoptosis

Abstract. Apoptosis is nowadays recognized as an important mechanism by which cells can be eliminated from the organism. In particular its role in tissue modelling during embryogenesis has been highlighted. The human teratoma cell line Tera 2, which in several respects acts as a human embryonic stem cell, can be induced to undergo apoptosis by reducing the serum content of the tissue culture medium. We report here that this process can be reversed by replacing serum with the heparin-binding growth factors, acidic FGF and basic FGF. In contrast, neither of the mammalian transforming growth factors (TGF-β-3) managed to exert any effect on growth or apoptosis in Tera 2 cells.     

Quantitative Growth of Naegleria in Axenic Culture

A strain of Naegleria gruberi, isolated from a Vero cell culture and designated TS-1, was axenically cultivated in monolayer and mass aerating suspension culture. Cultural conditions for constant growth parameters and high-exponential cell densities were defined. Serum or other supplemented fractions were found essential in both Trypticase-yeast extract-glucose (TYG) and Casitone (CAS)-based media. Monolayer cultures grown in the CAS medium required lower levels of serum to reach maximum stationary densities of amoebae than cultures grown in the TYG medium. Heat-killed (121 C, 10 min) whole cell and cell lysate bacterial fractions were capable of replacing the serum in both the TYG and CAS media. Heat-killed bacterial fractions provided the same levels of growth as attained with serum in TYG medium, whereas the bacterial lysate supported only minimal growth in the same medium. In the CAS medium, both bacterial fractions resulted in the same level of growth which was equal to that obtained in reduced serum content. Strain TS-1 was established in suspension culture with the CAS medium used in monolayer culture. The addition of sheep red blood cells (RBC) or RBC lysate greatly enhanced growth responses. Further modifications resulted in a final medium for suspension culture consisting of Casitone-yeast extract-glucose-vitamin base, supplemented with serum and RBC lysate. This medium supported growth with a mean generation time of 9 h at 30 C and a stationary phase yield of greater than 5 x 10(6) amoebae per ml.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Growth of cultured mammalian cells on secondary glucose sources

Mammalian cells can grow in culture at very low glucose concentrations. They can also grow using starch or maltose as secondary sources of glucose if hydrolytic enzymes (amylase and/or maltase) are available to release the glucose. The serum supplement in the culture medium provides these enzymes in amount adequate to permit growth at as rapid a rate as when free glucose is added. Owing to the relatively slow liberation of glucose from the secondary sources, the cells produce less lactic acid, and the culture medium does not become acidic.If the amount of hydrolytic enzyme in the serum supplement is reduced by heat inactivation, the rate of glucose liberation is further reduced. As a result, glucose continues to be released into the medium even at high cell densities, when all glucose added directly to control cultures has been consumed at a time. For this reason, the cells survive longer at high density on secondary glucose sources than on free glucose. Use of such a culture system should have important practical advantages in maintaining dense cultures of any mammalian cell type.Medium containing secondary glucose sources and serum whose hydrolytic enzymes have been completely inactivated should be a selective medium for the corresponding cellular enzymes. Attempts to select for cell lines able to grow using their own amylase or maltase were not successful, but calculations based on embryonic pancreatic cells, known to synthesize amylase, showed that the amount of enzyme required should be quite low in comparison with that present in the differentiated state. The possibilities of selection for a differentiated function in cell culture have been very little explored, and such an approach may be fruitful if applied to the right cell types.     

Growth of kidney epithelial cells in hormone-supplemented,serum-free medium

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors – insulin, transferrin, prostaglandin E₁, hydrocortisone and triiodothyronine – as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E₁ in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over-growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.     

Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.Abbreviations BSA bovine serum albumin - CHSE-214 Chinook samon embryo cell line - dFBS dialyzed fetal bovine serum - FBS fetal bovine serum - GFSk-S1 goldfish skin cell line - GS glutamine synthetase - L-15 Leibovitz's L-15 media - L929 mouse fibroblast cell line - MEM minimum essential medium Eagle - PBS phosphate buffered saline - RTL-W1 rainbow trout liver cell line - RTSp-W1 rainbow trout spleen cell line     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Growth and differentiation of epidermal cells from the rainbow trout established as explants and maintained in various media

Growth and a number of differentiated characteristics of cultured epidermal cells from the rainbow trout Oncorhynchus mykiss were compared using two commercially available serum–free media, a dermal substrate/serum free kit and a serum–containing medium which had been previously optimized for epidermal cell culture. Each medium supported short term growth over 15 days. Only the medium supplied for dermal substrate culture supported longer growth periods. This medium was supplied for use with a collagen/stromal substrate but gave good cultures even without the substrate. Differentiation, measured by examining mucous cells, cytokeratins, epidermal growth factor receptor, gap junction status and ultrastructure showed that serum–free media gave quantitatively and qualitatively superior expression and short term retention of differentiation over serum–containing medium. Epithelial cell growth with expression of differentiated characteristics can be maintained in primary culture in serum–free medium for at least as long as in serum–containing medium. This provides a useful technique for use when serum presence in medium is undesirable or proves toxic to the specialized cell type under investigation.     

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.     

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.     

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

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.     

Growth of Im-9 Human Lymphoblasts In Serum-Free Medium: Stimulation By Glucocorticoids

Abstract. The IM-9 human B-lymphoblast cell line grows well in a completely defined serum-free medium containing insulin, transferrin, low density lipoprotein and oleic acid in complex with fatty acid-free bovine serum albumin. Growth of the IM-9 cells is stimulated by addition of physiological concentrations of hydrocortisone to this medium. the order of growth stimulatory potency of several steroids is dexamethasone > hydrocortisone > aldosterone, whereas testosterone does not stimulate growth of the IM-9 cells. This order of potency suggests that the effect is mediated by binding to glucocorticoid receptors. Growth of the IM-9 cells is also stimulated by the neuropeptide substance P. the defined serum-free medium described in this report will be useful for further studies of the biological responses of the IM-9 cells to other hormones in the absence of interference from hormones and growth factors present in serum.     

Growth of IM-9 human lymphoblasts in serum-free medium: stimulation by glucocorticoids

The IM-9 human B-lymphoblast cell line grows well in a completely defined serum-free medium containing insulin, transferrin, low density lipoprotein and oleic acid in complex with fatty acid-free bovine serum albumin. Growth of the IM-9 cells is stimulated by addition of physiological concentrations of hydrocortisone to this medium. The order of growth stimulatory potency of several steroids is dexamethasone greater than hydrocortisone greater than aldosterone, whereas testosterone does not stimulate growth of the IM-9 cells. This order of potency suggests that the effect is mediated by binding to glucocorticoid receptors. Growth of the IM-9 cells is also stimulated by the neuropeptide substance P. The defined serum-free medium described in this report will be useful for further studies of the biological responses of the IM-9 cells to other hormones in the absence of interference from hormones and growth factors present in serum.     

Growth of dissociated rat cerebellar cells using serum-free supplemented media and varied transferrin concentrations

Dissociated neonatal rat cerebellar cells were grown on medium supplemented with 10% horse serum (HS) and compared with those grown using a serum-free supplemented (SFS) medium, modified from Bottenstein and Sato (1979). containing insulin, transferrin, progesterone, putrescine, and selenium (after an initial 24 hr in 10% horse serum). Cells survived for several weeks using either medium. Cells grown in SFS had higher levels of GABA uptake than cells grown in HS. Cellular morphology and the proportion of neurons to glial cells were similar under the two conditions. Transferrin concentrations of 0.5, 10, and 100 µg/ml were tested. Neither neuronal nor glial cells were sensitive to this 200-fold variation. The SFS medium supports survival and maturation of both neurons and glial cells from rat cerebellum. However, the medium is not completely defined since (1) one day of serum is still required and (2) the heterogeneous cell population is undoubtedly conditioning the medium to some extent.This work was supported in part by grants from the Scottish Rite Schizophrenia Research Program, N.M.J., USA, and by Biomedical Research Grant S 07-RR5394, from the National Institutes of Health, PHS/DHHS.     

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.     

Growth and morphology of rabbit marginal vessel endothelium in cell culture

A procedure for the isolation and cultivation of endothelium from the marginal vessels of the rabbit ear is described. Endothelial cells, isolated by slow perfusion with a trypsin solution, are cultured in minimal essential medium supplemented with 10% fresh rabbit serum for up to 6 mo. In primary culture, marginal vessel endothelial cells grow in an expanding circular pattern with closely apposed cell membranes. Weibel-Palade bodies, subcellular organelles unique to endothelial cells in situ, are present in both primary and in serially cultivated cells (12 passages). In intact skin, Weibel-Palade (W-P) bodies are observed in the perinuclear cytoplasm in close proximity to the cell membrane facing the vascular lumen. 8-16 tubules of 200 A diameter are present in each body. In primary and subcultured cells, W-P bodies of identical size are seen in the vicinity of the Golgi apparatus and in close proximity to the outer cell membrane. At the optimum serum concentration (10%), a cell doubling time of 72-96 h is observed. When growth in normal rabbit serum and in platelet-poor serum is compared, a slower growth rate is observed in the absence of platelets, suggesting that factors released by platelets affect endothelial cell proliferation. However, addition of crude platelet factor does not substitute for complete serum. Fibroblast growth factor is not mitogenic for rabbit marginal vessel endothelium in vitro.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.     

Delay of Poliovirus Plaque Formation with Use of Fresh Bovine Serum in the Growth Medium of Host Cellsd

The plaque formation of poliovirus in HeLa cell monolayers was decreased upto 95% when the host cells were subcultured several times in a growth medium containing fresh bovine serum prior to their inoculation with virus. Extended incubation of infected cell monolayers indicated that the inhibition was in the form of a delay in plaque formation rather than a permanent inhibition of it. The inhibitory factor, which was found in most bovine sera, was very unstable and disappeared after storage of the serum at -20 C for a few weeks or at 36 C within a few days. In HeLa-cell monolayers, the fresh serum brought about a decrease in the plaque formation of all three poliovirus types as well as that of poliovirus ribonucleic acid. The plaque formation of poliovirus in monkey heart and HEp-2-cell monolayers was decreased irregularly by the use of fresh serum in the growth medium of these cells. Speculations were made as to the possible mode of action of the bovine serum inhibitor.     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)