COCULTURE OF ENDOTHELIAL-CELLS AND SMOOTH-MUSCLE CELLS IN BILAYER ANDCONDITIONED MEDIA MODELS

Smooth muscle cell (SMC) growth characteristics are affected by endoth elial cells (ECs) in vivo and in vitro. In this study, we compare a bi layer EC/SMC coculture model that allows cell contact with a model of SMCs growing in media continuously conditioned by ECs, but without phy sical contact. Bovine aortic SMCs were plated on one side of a 13-mu m -thick, semipermeable membrane. Three models were compared: (1) SMCs c ultured alone (with no cells on the opposite side of the membrane, O/S MC); (2) SMCs cultured with ECs on the opposite side of the membrane i n a bilayer coculture system that allows physical contact between ECs and SMCs (EC/SMC); and (3) SMCs cultured in media continuously conditi oned by adjacent ECs, without contact (conditioned media, CM). After c ultures were established, SMCs were harvested at 7 and 14 days after p lating (n = 5 cultures/day/group). SMC DNA and protein content and [H- 3]thymidine incorporation were measured in each group. On Days 7 and 1 4 after plating, ECs in both the EC/SMC and CM models stimulated SMC p roliferation >50% compared to O/SMC controls (P < 0.05). SMC density w as similar for the EC/SMC and CM models at Day 7, but SMC density was higher in the EC/SMC group at Day 14 in culture (P < 0.05). At Day 7, protein synthesis was similar in the three groups, but by Day 14, SMCs in the EC/SMC group had produced significantly less cellular protein/ DNA than SMCs in the CM group (P < 0.05), which in turn had less prote in/DNA than the control (O/SMC) group (P < 0.05). SMCs in the EC/SMC a nd CM groups retained a thin, spindle shape with filamentous projectio ns, compared to the hypertrophic appearance of SMCs in the absence of ECs. Electron microscopy revealed projections from SMCs which traverse d the pores in the coculture membrane and made intimate contact with E Cs. The degree of EC/SMC contact increased from 7 to 14 days (P < 0.05 ). Compared to SMCs alone, ECs in bilayer coculture or conditioned med ia altered SMCs growth characteristics similarly after 7 days in cultu re. By 14 days, however, the bilayer coculture had a significantly gre ater effect on SMC density and protein synthesis. The bilayer model is unique in terms of luminal/abluminal orientation of the cells, the pr oximity of the cell layers, and the presence of physical cell contact. Since the bilayer model amplifies the effect of ECs on SMCs, it may b e more useful than conditioned media to study EC-SMC interactions. (C) 1997 Academic Press.