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Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood-derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.

Comparison of Mixed and Lamellar Coculture Spatial Arrangements for Tissue Engineering Capillary NetworksIn Vitro