CORONARY-ARTERY SMOOTH-MUSCLE IN CULTURE - MIGRATION OF HETEROGENEOUSCELL-POPULATIONS FROM VESSEL WALL

A method for establishing primary cultures of smooth muscle cells (SMC s) from the porcine coronary artery without either microdissection and /or enzymatic dispersion was developed using selective migration of ce lls from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and ad herence ability to separate SMC from contaminating fibroblasts or endo thelial cells. The cell type was determined by immunohistochemical sta ining with monoclonal antibodies to SM alpha-actin, SM myosin, h-calde smon and von Willebrand factor. The first wave of migration (1-7 days) consisted of a mixture of fibroblasts and SMCs. Only SMCs were presen t in the second wave of migration (7-14 days). Endothelial cells, whic h exhibited a lower capacity for migration and adherence, were restric ted to the third wave of migration (14-21 days). Cells obtained from t he second wave of migration exhibited the characteristic single-layere d, aligned, hill-and-valley pattern of SMCs when confluent, Quiescence was attained 4-5 days after removal of serum, as established by [H-3] -thymidine incorporation. Stimulation of the quiescent SMCs with 20% F BS resulted in a synchronous re-entry into the cell-cycle with S phase reached 15-18 h later. The SMCs prepared using this protocol thus exh ibit the structural markers and capacity to undergo phenotypic modulat ion that are characteristic of SMCs in vivo. This approach to establis hing primary cultures of SMCs offers the advantage of selecting for th e subpopulation of cells capable of migration in response to injury or growth factor stimulation.