Summary
The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.
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References
Blake, D. A.; Yu, H.; Young, D. L., et al. Matrix stimulates the proliferation of human corneal endothelial cells in culture. Invest. Ophthalmol. Vis. Sci. 38(6):1119–1129; 1997.
Chappey, O.; Wautier, M. P.; Boval, B., et al., Endothelial cells in culture: an experimental model for the study of vascular dysfunctions. Cell. Biol. Toxicol. 12(4–6):199–205; 1996.
Cines, D. B.; Pollak, E. S.; Buck, C. A., et al., Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91(10):3527–3561; 1998.
Dichek, D. A.; Anderson, J.; Kelly, A. B., et al., Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors. Circulation 93(2):301–309; 1996.
Dichek, D. A.; Nussbaum, O.; Degen, S. J., et al. Enhancement of the fibrinolytic activity of sheep endothelial cells in retroviral vector-mediated gene transfer. Blood 77(3):533–541; 1991.
Engelmann, K.; Friedl, P.. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell. Dev. Biol. Anim. 25(11):1065–1072; 1989.
Geary, R. L.; Clowes, A. W.; Lau, S., et al. Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally transduced smooth muscle cells: a model for local and systemic gene therapy. Hum. Gene Ther. 5(10):1211–1216; 1994.
Gordon, E. L.; Danielsson, P. E.; Nguyen, T. S., et al. A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells. In Vitro Cell. Dev. Biol. Anim. 27A(4):312–326; 1991.
Gospodarowicz, D.; Ill, C.. Extracellular matrix and control of proliferation of vascular endothelial cells. J. Clin. Invest. 65(6):1351–1364; 1980.
Griendling, K. K.; Alexander, R. W.. Endothelial control of the cardiovascular system: recent advances. FASEB J. 10(2):283–292; 1996.
Hassell, T.; Gleave, S.; Butler, M.. Growth inhibition in animal cell culture: the effect of lactate and ammonia. Appl. Biochem. Biotechnol. 30(1): 29–41; 1991.
Henderson, A. H.. Endothelium in control. Br. Heart J. 65(3):116–125; 1991.
Herring, M. B.; Gardener, A.; Glover, J.. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery 84:498–504; 1978.
Hewett, P. W.; Murray, J. C.. Human microvessel endothelial cells: isolation, culture and characterization. In Vitro Cell. Dev. Biol. Anim. 29A(11):823–830; 1993.
Huber, T. S.; Welling, T. H.; Sarkar, R., et al. Effects of retroviral-mediated tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cells seeded onto expanded polytetrafluoroethylene. J. Vasc. Surg. 22(6):795–803; 1995.
Ingber, D. E.; Madri, J. A.; Folkman, J. Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro Cell. Dev. Biol. Anim. 23(5):387–394; 1987.
Jaffe, E. A.; Nachman, R. L.; Becker, C. G., et al., Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52(11):2745–2756; 1973.
Jarrell, B.; Levine, E.; Shapiro, S. et al. Human adult endothelial cell growth in culture. J. Vasc. Surg. 1(6):757–764; 1984.
Karasek, M. A.. Microvascular endothelial cell culture. J. Invest. Dermatol. 93(Suppl. 2):33S-38S; 1989.
Knedler, A.; Ham, R. G.. Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro Cell. Dev. Biol. Anim. 23(7):481–491; 1987.
Lewis, W. H.. Endothelium in tissue culture. Am. J. Anat. 30:39–59; 1922.
Nabel, E. G.; Plautz, G.; Boyce, F. M., et al., Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 244(4910):1342–1344; 1989.
Schulick, A. H.; Dong, G.; Newman, K. D., et al. Endothelium-specific in vivo gene transfer. Circ. Res. 77(3):475–485; 1995.
Scott, P. A.; Bicknell, R.. The isolation and culture of microvascular endothelium. J. Cell. Sci. 105(pt. 2):269–273; 1993.
Shireman, P. K.; Pearce, W. H.. Endothelial cell function: biologic and physiologic functions in health and disease. Am. J. Roentgenol. 166(1):7–13; 1996.
Stanley, J. C.; Burkel, W. E.; Graham, L. M., et al. Endothelial cell seeding of synthetic vascular prostheses. Acta Chir. Scand. Suppl. 529:17–27; 1985.
Thornton, S. C.; Mueller, S. N.; Levine, E. M.. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 222(4624):623–625; 1983.
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(6):2034–2040; 1984.
Walker, G. M.. The roles of magnesium in biotechnology. Crit. Rev. Biotechnol. 14(4):311–354; 1994.
Watkins, M. T.; Sharefkin, J. B.; Zajtchuk, R., et al. Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses. J. Surg Res. 36(6): 588–596; 1984.
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Terramani, T.T., Eton, D., Bui, P.A. et al. Human macrovascular endothelial cells: Optimization of culture conditions. In Vitro Cell.Dev.Biol.-Animal 36, 125–132 (2000). https://doi.org/10.1290/1071-2690(2000)036<0125:HMECOO>2.0.CO;2
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DOI: https://doi.org/10.1290/1071-2690(2000)036<0125:HMECOO>2.0.CO;2