Coculture of Endothelial Cells and Smooth Muscle Cells in Bilayer and Conditioned Media Models

Smooth muscle cell (SMC) growth characteristics are affected by endothelial cells (ECs)in vivoandin vitro.In this study, we compare a bilayer EC/SMC coculture model that allows cell contact with a model of SMCs growing in media continuously conditioned by ECs, but without physical contact. Bovine aortic SMCs were plated on one side of a 13-μm-thick, semipermeable membrane. Three models were compared: (1) SMCs cultured alone (with no cells on the opposite side of the membrane, O/SMC); (2) SMCs cultured with ECs on the opposite side of the membrane in a bilayer coculture system that allows physical contact between ECs and SMCs (EC/SMC); and (3) SMCs cultured in media continuously conditioned by adjacent ECs, without contact (conditioned media, CM). After cultures were established, SMCs were harvested at 7 and 14 days after plating (n= 5 cultures/day/group). SMC DNA and protein content and [3H]thymidine incorporation were measured in each group. On Days 7 and 14 after plating, ECs in both the EC/SMC and CM models stimulated SMC proliferation >50% compared to O/SMC controls (P< 0.05). SMC density was 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 protein/DNA than the control (O/SMC) group (P< 0.05). SMCs in the EC/SMC and CM groups retained a thin, spindle shape with filamentous projections, compared to the hypertrophic appearance of SMCs in the absence of ECs. Electron microscopy revealed projections from SMCs which traversed the pores in the coculture membrane and made intimate contact with ECs. 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 media altered SMCs growth characteristics similarly after 7 days in culture. By 14 days, however, the bilayer coculture had a significantly greater effect on SMC density and protein synthesis. The bilayer model is unique in terms of luminal/abluminal orientation of the cells, the proximity of the cell layers, and the presence of physical cell contact. Since the bilayer model amplifies the effect of ECs on SMCs, it may be more useful than conditioned media to study EC–SMC interactions.