Summary
An optimized basal nutrient medium, MCBD 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 μg/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg2+ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS.
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Agy, P. C.; Shipley, G. D.; Ham, R. G. Protein-free medium for C-1300 mouse neuroblastoma cells. In Vitro 17:671–680; 1981.
Bennian, S.; Fitzpatrick, J.; harbell, J., et al. The effect of UVB on 6-keto-PGF1 production by cultured human endothelial cells. J. Invest. Dermatol. 82:428 (abstract); 1984.
Bettger, W. J.; Boyce, S. T.; Walthall, B. J., et al. Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone. Proc. Natl. Acad. Sci. USA 78:5588–5592; 1981.
bjornthorp, P.; Hansson, G. K.; Jonasson, L., et al. Isolation and characterization of endothelial cells from the epididymal fat pad of the rat. J. Lipid Res. 24:105–112; 1983.
Booyse, F. M.; Sedlak, B. J.; Rafelson, M. E. Culture of arterial endothelial cells. Characterization and growth of bovine aortic cells. Thromb. Diath. Haemorrh. 34:825–839; 1975.
Bowman, P. D.; Betz, L. A.; Ar, D., et al. Primary culture of capillary endothelium from rat brain. In Vitro 17:353–362; 1981
Bowen-Pope, D. F.; Vidair, C.; Sanui, H., et al. Separate roles for calcium and magnesium in their synergistic effect on uridine uptake by cultured cells: significance for growth control. Proc. Natl. Acad. Sci. USA 76:1308–1312; 1979.
Boyce, S. T.; Ham, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined culture and serum-free serial culture. J. Invest. Dermatol. 81 (Suppl):33s-40s; 1983.
Davison, P. M.; Bensch, K.; Karasek, U. A. Isolation and growth of endothelial cells from the microvessels on the newborn human foreskin in cell culture. J. Invest. Dermatol. 75:316–321; 1980.
Diglio, C. A.; Grammas, P.; Giacomelli, F., et al. Primary culture of rat cerebral microvascular endothelial cells; isolation, growth and characterization. Lab. Invest. 46:554–563; 1982.
Folkman, J.; Haudenschild, C. C.; Zetter, B. R. Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76:5217–5221; 1979.
Frank, R. N.; Kinsey, V. E.; Frank, K. W., et al. In vitro proliferation of endothelial cells from kitten retinal capillaries. Invest. Ophthalmol. Vis. Sci. 18:1195–1200; 1979.
Gimbrone, M. A. Culture of vascular endothelium. Prog. Hemost. Thromb. 3:1–28; 1976.
Goetz, I. E.; Warren, J.; Estrada, C., et al. Long-term serial cultivation of arterial and capillary endothelium from adult bovine brain. In Vitro 21:172–180; 1985.
Goldsmith, H. S.; Griffith, A. L.; Kupferman, A., et al. Lipid angiogenic factor from omentum. JAMA 252:2034–2036; 1984.
Gospodarowicz, D.; Brown, K. D.; Birdwell, C. R., et al. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin. J. Cell Biol. 77:744–788; 1978.
Gospodarowicz, D.; Greenburg, G.; Birdwell, C. R. Determination of cellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res. 38:4155–4171; 1978.
Graves, D.; Ham, R. G. Serum-free clonal growth of chicken embryo fibroblasts. In Vitro 18:305 (Abstract #127); 1982.
Ham, R. G. Clonal growth of mammalian cells in chemically defined synthetic medium. Proc. Natl. Acad. Sci. USA 53:288–293; 1965.
Ham, R. G. Nutritional requirements of primary cultures. A neglected problem of modern biology. In Vitro 10:119–129; 1974.
Ham, R. G. Survival and growth requirements of non-transformed cells. Hdbk. Exp. Pharmacol. 57:13–88; 1981.
Ham, R. G.. Importance of the basal nutrient medium in the design of hormonally defined media. Cold Spring Harbor Conf. Cell Prolifer. 9:39–60; 1982.
Ham, R. G. Growth of normal human cells in defined media. In: Fischer, G.; Weiser, R. G., eds. Hormonally defined media—A tool in cell biology. Berlin: Springer Verlag; 1983:16–30.
Ham, R. G. Selective media. In: Pretlow, T. G., II; Pretlow, T. P., eds. Cell separation: Methods and selected applications, vol. 3. New York: Academic Press; 1984:209–236.
Ham, R. G. Methods for formulation of basal nutrient media. In: Barnes, D.; Sirbasku, D.; Sato, G., eds. Cell culture methods for molecular and cell biology, vol. 1. New York: A. R. Liss; 1984:3–21.
Ham, R. G.; McKeehan, W. L. Development of improved medium and culture conditions for clonal growth of normal human diploid cells. In Vitro 14:11–22; 1978.
Ham, R. G.; McKeehan, W. L. Nutritional requirements for clonal growth of non-transformed cells. In: Katsuta, H., ed. Nutritional requirements of cultured cells. Tokyo: Japan Scientific Societies Press; 1978:63–115.
Ham, R. G.; McKeehan, W. L. Media and growth requirements. Methods Enzymol. 48:44–93; 1979.
Hamilton, W. G.; Ham, R. G. Clonal growth of Chinese hamster cell lines in protein-free media. In Vitro 13:537–547; 1977.
Hammond, S. L.; Ham, R. G.; Stampfer, M. L. Serum-free growth of human mammary epithelial cells: Rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc. Natl. Acad. Sci. USA 81:5435–5439; 1984.
Hoshi, H.; McKeehan, W. L. Isolation, growth requirements, cloning, prostacyclin production and life span of human adult endothelial cells in low serum culture medium. In Vitro 22:51–56; 1986.
Jaffe, E. A.; Nachman, R. I.; Becker, C. G., et al. Culture of human endothelial cells from umbilical veins. J. Clin. Invest. 52:2745; 1973.
Jennings, S. D.; Ham, R. G. Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium. Cell Biol. Int. Rep. 7:149–159; 1983.
Kern, P. A.; Knedler, A.; Eckel, R. H. Isolation and culture of microvascular endothelium from human adipose tissue. J. Clin. Invest. 71:1822–1829; 1983.
Knedler, A.; Tonnesen, M. G.; Haslett, C., et al. Rabbit microvascular endothelial cells: Isolation, characterization, and interaction with neutrophils. Fed. Proc. 44:1494 (Abstract No. 6338); 1985.
Marks, R. M.; Czerniecki, M.; Penny, R. Human dermal microvascular endothelial cells: An improved method for tissue culture and a description of some singular properties in culture. In Vitro 21:627–635; 1985.
Maruyama, Y. The human endothelial cell in culture. Z. Zellforsch. 60:69–79; 1963.
McAuslan, B. R.; Reilly, W. Selenium-induced cell migration and proliferation: Relevance to angiogenesis and microangiopathy. Microvasc. Res. 32:112–120; 1986.
McAuslan, B.R.; Bender, V.; Reilly, W., et al. New functions of epidermal growth factor: Stimulation of capillary endothelial cell migration and matrix dependent proliferation. Cell Biol. Int. Rep. 9:175–182; 1985.
McKeehan, W.L.; Ham, R. G. Methods for reducing the serum requirement for growth in vitro of nontransformed diploid fibroblasts. Dev. Biol. Stand. 37:96–108; 1977.
McKeehan, W. L. McKeehan, K. A.; Hammond, S. L., et al. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13:399–416; 1977.
Peehl, D. M.; Ham, R. G. Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro 16:526–540; 1980.
Pitas, R. E.; Innerarity, T. L.; Weinstein, J. N., et al. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophagesin vitro by fluorescence microscopy. Arteriosclerosis 1:177–185; 1981.
Rubin, H.; Koide, T. Mutual potentiation by magnesium and calcium of growth in animal cells. Proc. Natl. Acad. Sci. USA 73:168–172; 1976.
Rubin, A. H.; Terasaki, M.; Sanui, H. Magnesium reverses inhibitory effects of calcium deprivation on coordinate response of 3T3 cells to serum. Proc. Natl. Acad. Sci. USA 75:4379–4383; 1978.
Rubin, A. H.; Terasaki, M.; Sanui, H. Major intracellular cations and growth control: Correspondence among magnesium content, protein synthesis, and the onset of DNA synthesis in BALB/c 3T3 cells. Proc. Natl. Acad. Sci. USA 76:3917–3921; 1979.
Sherer, G. K.; Fitzharris, T. P.; Faulk, W. P., et al. Cultivation of microvascular endothelial cells from human preputial skin. In Vitro 16:675–684; 1980.
Shipley, G. D.; Ham, R. G. Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells. In Vitro 17:656–670; 1981.
Tsao, M. C.; Walthall, B. J.; Ham, R. G. Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell Physiol. 110:219–229; 1982.
Underwood, E. J. Trace elements in human and animal nutrition, 4th ed. New York: Academic Press; 1977.
Voyta, J. C.; Via, D. P.; Butterfield, C. E., et al. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J. Cell Biol. 99:2034–2040; 1984.
Wagner, R. C.; Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10:286–297; 1975.
Wechezak, A. R.; Mansfield, P. B. Isolation and growth characteristics of cell lines from bovine venous endothelium. In Vitro 9:39–45; 1973.
Weinstein, R.; Stemerman, M. B.; Maciag, T. Hormonal requirements for growth of arterial smooth muscle cells in vitro: an endocrine approach to atherosclerosis. Science 212:818–820; 1981.
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This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD.
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Knedler, A., Ham, R.G. Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro Cell Dev Biol 23, 481–491 (1987). https://doi.org/10.1007/BF02628418
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DOI: https://doi.org/10.1007/BF02628418