The endothelial cell participates in numerous functions of vascular physiology. Many factors, such as cytokines, can alter the surface of the endothelial cell and thereby modulate the role of the endothelium in coagulation, inflammation, vaso-regulation, and adhesion. See, for example, R. P. Hebbel et al., J. Lab. Clin. Med., 129, 288 (1997); J. S. Pober, Am. J. Path., 133, 426 (1988); E. J. Favaloro, Inmmunol. Cell. Biol., 71, 571 (1993). The endothelial cell may also have a key role in the vascular pathology of sickle cell anemia, including the vaso-occlusions that cause acute painful crises. However, research in this area has been hindered by the inaccessibility of vascular endothelium in patients. For example, E. M. Levine et al. (U.S. Pat. No. 5,132,223) disclosed cloning and serial cultivation of adult human endothelial cells derived from brain-dead, but heart-beating cadaver organs. K. Gupta et al., Exp. Cell. Res., 230, 244 (1997) reported the culture of microvascular endothelial cells derived from newborn human foreskin. Thus, circulating endothelial cells might provide useful material for the study of vascular pathologies, for gene therapy, and for biomedical engineering applications. In previous investigations increased numbers of circulating endothelial cells have been found in sickle cell anemia and other conditions associated with vascular injury, such as that due to cytomegalovirus infection, rickettsial infection, myocardial infarction, intravascular instrumentation, and endotoxinemia. See, for example, F. George et al., Blood, 80, Suppl: 12a, abstract (1992); E. Percivalle et al., J. Clin. Invest., 92, 663 (1993), F. George et al., Blood, 82, 2109 (1993); C. A. Bouvier et al., Thomb. Diath. Haemorrh. Suppl., 40, 163 (1970); F. George et al., J. Immunol. Meth., 139, 65 (1991) and R. G. Gerrity et al., Exp. Mol. Pathol., 24, 59 (1976).
However, in normal donors, there are only about 2-3 circulating endothelial cells per ml of blood; they have a quiescent phenotype, and about 50% of them are microvascular as evidenced by CD36 positivity. See, A. Solovey et al., New Engl. J. Med., 337, 1584 (1997), who reported using the methodology of Gupta et al., cited above, to coculture viable circulating endothelial cells identified in the blood of patients with sickle cell anemia with primary microvascular endothelial cells (MVEC). T. Asahara et al., Science, 275, 964 (1997) isolated putative endothelial cell (EC) progenitors from human peripheral blood after CD34+ enrichment by magnetic bead selection on the basis of cell surface antigen expression. The cells were cultured on fibronectin-coated wells in modified M-199 medium containing bovine brain extract and 20% fetal bovine serum. Q. Shi et al., Blood, 92, 362 (1998) characterized bone marrow-derived precursor endothelial cells by isolating CD34+ cells derived from peripheral blood using murine anti-CD34+ antibody binding followed by exposure to anti-mouse immunomagnetic beads. The cells were cultured in gelatin or fibronectin-coated plastic wells in M199 medium containing VEGF, FBS, bFGF and IGF.
However, due to the low concentration of CEC in blood, a need exists for a culture method and medium that will permit the rapid expansion of CEC from blood, without the attendant difficulties of isolation discussed above.