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Abstract

An in vitro angiogenesis system was designed for screening angiogenic agonists and antagonists. In order to obtain large quantities of cells and reproducibility, human endothelial cells with extended life spans were developed by retroviral transfection. The resulting cells grown in a serum-free medium containing endothelial cell growth supplement (ECGS) have a telomerase activity, extended life spans of at least 21 passages, and an endothelial cell phenotype (diI-acetylated-LDL upake, factor VIII-related antigen, VEGFR-1 and R-2, and tissue-type plasminogen activator (tPA)) that resembled that of unaltered primary endothelial cells. Exceptions were (i) a higher expression of tPA, and (ii) a non-significant growth response to FGF-2 or VEGF stimulation. Within three-dimensional fibrin gels, specific cell clones rapidly formed tubular structures in a more reproducible manner than those observed with low-passage primary cells. Tube formation by primary endothelial cells and those with extended life spans was dependent upon FGF-2 and ECGS, respectively. Both cell types produced FGF-2 and VEGF cytokines. Increasing doses of suramin significantly decreased the size of microvessels formed by both cell lines. These functional results indicate that a vascular matrix system containing human cells with extended life spans can be successfully utilized as an in vitro assay for antiangiogenic compounds.

Controlled Delivery of Human Cells by Temperature Responsive Microcapsules

“…Human umbilical vein endothelial cells (HUVECs) were cultured on gelatin-coated cell culture flask in M199-medium supplemented with 50 µg·mL −1 ECGS, GlutaMAX™ 1X, heparin 50 µg·mL −1 , 5% fetal bovine serum, 100 U mL −1 penicillin and 100 µg·mL −1 streptomycin. HUVECs were immortalized using the human papilloma virus E6E7 proteins, which are believed to restore telomere length to primary human cells as previously published [ 28 , 29 ]. For this study, they were transfected with lentiviral vectors containing eGFP (LV/eGFP) to produce cells that fluoresced green for easy tracking after encapsulation.…”

Modeling human endothelial cell transformation in vascular neoplasias

“…However, a growth crisis prior to immortalization was noted in several studies (Ades et al, 1992; Fontijn et al, 1995; Rood et al, 2000; MacKenzie et al, 2002) and two studies showed that telomerase was spontaneously activated in the immortalized ECs (Rhim et al, 1998; Gagnon et al, 2002). Ectopic activation of telomerase was shown to cooperate with SV40 transformation to overcome crisis and efficiently promote immortalization of bone-marrow- and mammary-derived ECs (O’Hare et al, 2001; MacKenzie et al, 2002).…”

Development of immortalized mouse aortic endothelial cell lines

“…Moreover, characterization of primary endothelial cell cultures should be conducted regularly to confirm the lack of contamination or transdifferentiation. Previous reports have shown phenotypic change in cultured endothelial cells as a result of passaging [ 43 ]. It is difficult to maintain the phenotype of primary MAEC in sufficient amounts for a series of experiments without excessive time and effort.…”