Methods for growth of cultured cells in serum-free medium

Mitochondria prepared from skeletal muscle are typically contaminated with sarcoplasmic reticulum fragments and salt-soluble proteins. These contaminants can be removed by density-gradient centrifugation in Percoll. The resulting mitochondria retain both their original state three respiratory rates and their respiratory control ratios. The method has also been adapted to prepare mitochondria from very small tissue samples.     

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.     

The growth of mouse melanoma cells in hormone-supplemented, serum-free medium.

A mouse melanoma line, M2R, derived from the B₁₆ transplantable tumor can be grown in a hormone-supplemented serum-free medium. Cells grown in medium supplemented with insulin, transferrin, testosterone (or progesterone), follicle-stimulating hormone, nerve growth factor (NGF) and leutinizing releasing hormone show growth rates equivalent to that seen in medium supplemented with 5% fetal calf serum (FCS). This hormone-supplemented medium will support the growth of cells indefinitely. In cells grown in hormone-supplemented medium, insulin is shown to increase incorporation of glucose into glycogen and fatty acids. Transferrin is shown to act in part as an iron transport protein, although another role in growth stimulation cannot be eliminated. It is suggested that progesterone stimulates growth via an androgenic breakdown product and thus acts in a manner similar to testosterone.     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.     

Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

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.     

Serum protease inhibitors promote anchorage-independent growth of transformed cells in serum-free medium.

The effect of three serum serine protease inhibitors on the serum-free agar growth of an SV40-transformed 3T3 cell line was investigated. Antithrombin III, alpha-2-macroglobulin and alpha-1-antitrypsin were found to potently stimulate colony growth in a semisolid medium because of their anti-proteolytic properties. These results indicate that protease inhibitors can facilitate tumor cell growth in serum-free agar cultures and suggest that the stimulatory effect of serum on the growth of certain transformed cells in agar may at least partially be due to the high levels of protease inhibitors found in serum.     

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 growth of factor-producing lymphoid and myeloid cells in serum-free medium

The ability to grow lymphoid and myeloid cells in serum-free culture medium allows researchers to analyze the factors and mechanisms required for hemopoietic cell growth and differentiation without the interference of undefined serum components. Therefore, we used a serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines. Mo was maintained in RITC for more than 8 months and had a mean viability of 59% and the same doubling times as in serum-containing medium (SCM). Under these conditions, Mo cells produced hemopoietic colony-stimulating activity that included production of a basophil/eosinophil differentiation factor of similar content to that produced in SCM. WEHI-3B cells grown for more than 12 months in RITC, or for more than 3 months in RITC without Tf and Ins, had a doubling time of 20 h, whereas cells maintained in protein-free RITC showed a 2-fold increase in doubling time then died within 3 months. The IL-3 production by WEHI-3B cells cultured in RITC was higher than the production by cells grown in SCM. When IL-3 was assayed in 32Dcl/H4 cells that had been maintained in RITC for more than 4 months, a lower response to IL-3 was found, an indication that components other than the BSA, Tf and Ins in fetal calf serum are required for optimal cell growth and differentiation.     

Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

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.     

Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone-supplemented medium

A serum-free hormone-supplemented medium able to support the growth of rodent adipose precursor cells has been used to characterize additional components from serum required for the differentiation of preadipose Ob17 cells into adipose-like cells. Fetuin is shown to behave as a growth-promoting agent for these cells. In addition to growth hormone, triiodothyronine and a low-molecular weight component(s) also purified from serum, fetuin is required for the full expression of the differentiation program. Other serum proteins as well as other mitogenic factors are unable to substitute for fetuin. A possible role of fetuin in the development of adipose tissue is discussed.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

Growth of distal fetal rat lung epithelial cells in a defined serum-free medium

Summary Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 μg/ml), endothelial cell growth supplement (20 μg/ml), bovine pituitary extract (100 μg/ml), bovine serum albumin (50 μg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 μg/ml), transferrin (5 μg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 μg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E₁ (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung. Supported by grants MT-7867 and PG-42 from the Medical Research Council of Canada, HL-40458 from the National Heart, Lung and Blood Institute, Bethesda, MD, and an equipment grant from the Ontario Thoracic Society. Dr. Caniggia is a recipient of a research fellowship from the Italian Ministry of Education.