Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.
Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.