Summary
A simple reproducible method is described for isolating pure clonal populations of bovine aortic endothelial cells in culture. Single cells exposed to conditioned medium grow rapidly (clonal cell doubling time approximately 20 h) until a confluent monolayer is reached. This method provides virtually 100% cloning efficiency. In addition, the cell population doubling level of endothelial cell cultures, when continuously exposed to conditioned medium, is increased 40% over controls in fresh medium.
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Gimbrone, M. A., Jr.; Cotran, R. S. Human vascular smooth muscle in culture. Lab. Invest. 33: 16–27; 1975.
Thorgeirsson, G.; Robertson, A. L., Jr. The vascular endothelium-pathobiologic significance. Am. J. Pathol. 93: 801–848; 1978.
Schwartz, S. M. Selection and characterization of bovine aortic endothelial cells. In Vitro 14: 966–980; 1978.
Folkman, J.; Haudenschild, C. C.; Zetter, B. R. Long-term culture of capillary endothelial cells. Proc. natl. Acad. Sci. USA 76: 5217–5221; 1979.
Olander, J. V.; Marasa, J. C.; Kimes, R. C.; Johnston, G. M.; Feder, J. An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells. In Vitro 18: 99–107; 1982.
Gospodarowicz, D.; Moran, J.; Braun, D.; Birdwell, C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc. Natl. Acad. Sci. USA 73: 4120–4124; 1976.
Puck, T. T.; Marcus, P. I.; Cieciura, S. J. Clonal growth of mammalian cells in vitro. J. Exp. Med. 103: 273–284; 1956.
Laug, W. E.; Tokes, Z. A.; Benedict, W. F.; Sorgente, N. Anchorage independent growth and plasminogen activator production by bovine endothelial cells. J. Cell Biol. 84: 281–293; 1980.
Smith, U.; Ryan, J. W. Electron microscopy of endothelial cells collected on cellulose acetate paper. Tissue Cell 5: 333–336; 1973.
Ryan, U. S.; Mortara, M.; Whitaker, C. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding use of enzymes. Tissue Cell 12: 619–635; 1980.
Gimbrone, M. A.; Cotran, R. S.; Folkman, J. Human vascular endothelial cells in culture. J. Cell Biol. 60: 673–684; 1974.
Gajdusek, C.; DiCorletto, P.; Ross, R.; Schwartz, S. M. An endothelial cell-derived growth factor. J. Cell Biol. 85: 467–472; 1980.
Gajdusek, C. M.; Schwartz, S. M. Ability of endothelial cells to condition culture medium. J. Cell Physiol. 110: 35–42; 1982.
Vogel, A.; Raines, E.; Kariya, B.; Rivest, M.-J.; Ross, R. Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components. Proc. Natl. Acad. Sci. USA 75: 2810–2814; 1978.
Schwartz, S. M.; Selden, S. C., III; Bowman, P. Growth control in aortic endothelium at wound edges. Sato, G.: Ross, R. eds. Hormones and cell culture. Vol. 6. Cold Spring Harbor, New York: Cold Spring Harbor 3rd conference on cell proliferation; 1979: 593–610.
Cotta-Pereira, G.; Sage, H.; Bornstein, P.; Ross, R.; Schwartz, S. M. Studies of morphologically atypical (“sprouting”) cultures of bovine endothelial cells. Growth characteristics and connective tissue protein synthesis. J. Cell. Physiol. 102: 183–191; 1980.
Mueller, S. N.; Rosen, E. M.; Levine, E. M. Cellular senescence in a cloned strain of bovine fetal aortic endothelial cells. Science 207: 889–891; 1979.
Rosen, E. M.; Mueller, S. N.; Noveral, J. P.; Levine, E. M. Proliferative characteristics of clonal endothelial cell strains. J. Cell. Physiol. 107: 123–137; 1981.
Martin, G. M. Cellular aging- clonal senescence. A review (Part I). Am. J. Pathol. 89: 484–511; 1977.
Greenberg, S. B.; Grove, G. L.; Cristofalo, V. J. Cell size in aging monolayer cultures. In Vitro 13: 297–300; 1977.
Duthu, G. S.; Smith, J. R. In vitro proliferation and lifespan of bovine aorta endothelial cells: effects of culture conditions and fibroblast growth factor. J. Cell. Physiol. 103: 385–392; 1980.
Greenburg, G.; Vlodavsky, I.; Foidart, J. M.; Gospodarowicz, D. Conditioned medium from endothelial cell cultures can restore the normal phenotypic expression of vascular endothelium maintained in vitro in the absence of fibroblast growth factor. J. Cell. Physiol. 103: 333–347; 1980.
Cristofalo, V. J. Metabolic aspects of aging in diploid human cells. Holeckova, E.; Cristofalo, V. J. eds. Aging in cell and tissue culture. New York: Plenum Press; 1970: 83–119.
Macieira-Coelho, A. Action of cortisone on human fibroblasts in vitro. Experientia 22: 390–391; 1966.
Gospodarowicz, D.; Brown, K. D.; Birdwell, C. R.; Zetter, B. R. Control of proliferation of human 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: 774–788; 1978.
Gospodarowicz, D.; Hirabayashi, K.; Giguere, L.; Tauber, J.-P. Factors controlling the proliferative rate, final cell density, and lifespan of bovine vascular smooth muscle cells in culture. J. Cell Biol. 89: 568–578; 1981.
Heldin, C.-H.; Westermark, B.; Wasteson, A. Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc. Natl. Acad. Sci. USA 78: 3664–3668; 1981.
Levin, E. G.; Loskutoff, D. J. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J. Cell Biol. 94: 631–636; 1982.
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This work was supported in part by grants HL-18645 and HL-03174 from the National Institutes of Health, Bethesda, MD, and a grant from R. J. Reynolds Industries. Dr. Schwartz is an Established Investigator for the American Heart Association.
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Gajdusek, C.M., Schwartz, S.M. Technique for cloning bovine aortic endothelial cells. In Vitro 19, 394–402 (1983). https://doi.org/10.1007/BF02619556
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DOI: https://doi.org/10.1007/BF02619556