Serial subculture and relative transport of human endothelial cells in serum-free, defined conditions.

Endothelial cells bind, process, and transport bioactive molecules and thus provide an interactive interface between the plasma and adjacent tissues. Various hormones and factors induce endothelial cells to synthesize and secrete interactive factors. However, study of endothelial cell synthesis, processing, and transport of these bioactive molecules has been impeded because of the serum requirement for cell growth. Many of these bioactive molecules are derived from or are modified by serum components. We have developed a short-term culture system that supports sequential subculturing of endothelial cells in a serum-free culture medium on a defined extracellular matrix. The cells have a doubling rate of 33 h and the total cell number can be expanded more than 800-fold. Expression of specific markers; factor VIII related antigen-von Willebrand factor, Wiebel-Palade bodies, a cobblestone appearance of confluent cell monolayers, and angiotensin-converting enzyme activity confirm the normal morphologic integrity and biochemical function of these cultures. Using this defined serum-free medium, we have grown human endothelial cell monolayers on porous polycarbonate membrane inserts, thereby creating an upper and a lower chamber that models the vascular architecture and demonstrates an inverse relationship between transport and molecular weight. By eliminating serum components, this model system should facilitate the study of endothelial cell binding, metabolism, and transport of bioactive molecules and may contribute to a better understanding of the blood-tissue interface.

Erythropoietin binding and induced differentiation of Rauscher erythroleukemia cell line red 5-1.5.

We isolated from the Rauscher erythroleukemia cell line (Red 5), a subclone (Red 5-1.5), which contains erythropoietin (epo) binding sites and demonstrates an epo-dependent erythroid differentiation. One class of high affinity binding sites was detected with a Kd (+/- S.D.) of 0.43 +/- 0.09 nM and a mean density/cell of 1200 +/- 311. The cell-associated 125I-epo was displaced by nonlabeled epo but not by other hormones or factors. The 125I-epo binding to Red 5-1.5 cells was maximal within 3 h at 15 degrees C and 1 h at 37 degrees C and proportional to cell number. The addition of epo increased [3H] uridine incorporation into RNA by 6 h and [3H]thymidine incorporation into DNA by 60 h followed by 59Fe incorporation into protein, cell proliferation, and formation of hemoglobin-containing colonies. The incorporation of 59Fe into protein demonstrated a linear dose response (from 0.002 to 1.5 units of epo/ml) beginning 60 h after addition of the hormone to the cultures, and there was a dose-dependent increase (from 0.1 to 1.0 unit of epo/ml) in the formation of hemoglobin-containing colonies. We concluded that the binding of 125I-epo to Red 5-1.5 suggests the presence of specific epo receptors. The sequence of the epo-induced proliferation and differentiation events is similar to primary erythroid cultures but requires longer epo exposure. Receptor occupancy correlates with the induced biological response.