53 Molecular and Cellular Biochemistry 176: 53–59, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands. Introduction The normal arterial vessel wall consists of a regular arrange- ment of endothelial, smooth muscle and fibroblast cells, present in three distinct layers of endothelium, media and adventitia. A single layer of endothelial cells forms the luminal barrier to blood-borne signals that modulate vascu- lar function. The adventitia, which forms the outer layer around the artery, consists primarily of extracellular matrix as well as some fibroblasts, nerve fibres and microvessels. The media consists of numerous layers of smooth muscle cells (SMCs) intermixed with extracellular matrix that is bound by the internal and external elastic lamina. In some species such as swine and humans, an intima forms with maturity that consists of SMCs located on the luminal side of the internal elastic lamina. The response to injury or other stress stimuli (e.g. stretch) varies between the different cellular components of the vessel. Endothelial cells are capable of proliferation and migration, properties that permit re-endothelialization of the vessel after denudation or injury [1]. Medial SMCs are also able to reversibly modulate their phenotype which allows for their proliferation and/or migration into the intima at the site of injury [2]. It is these characteristics that lead to the adap- tive and pathogenic growth of SMCs which is key to vascu- lar remodelling and lesion formation. An emerging concept is that the medial SMC population consists of heterogeneous subpopulations of SMCs that differ in their growth and/or Address for offprints: P. Zahradka, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6, Canada Coronary artery smooth muscle in culture: Migration of heterogeneous cell populations from vessel wall Laura Saward and Peter Zahradka Department of Physiology, University of Manitoba; Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, R2H 2A6, Canada Abstract A method for establishing primary cultures of smooth muscle cells (SMCs) from the porcine coronary artery without either microdissection and/or enzymatic dispersion was developed using selective migration of cells from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and adherence ability to sepa- rate SMC from contaminating fibroblasts or endothelial cells. The cell type was determined by immunohistochemical staining with monoclonal antibodies to SM α-actin, SM myosin, h-caldesmon and von Willebrand factor. The first wave of migration (1–7 days) consisted of a mixture of fibroblasts and SMCs. Only SMCs were present in the second wave of migration (7–14 days). Endothelial cells, which exhibited a lower capacity for migration and adherence, were restricted to the third wave of migration (14–21 days). Cells obtained from the second wave of migration exhibited the characteristic single-layered, aligned, hill-and-valley pattern of SMCs when confluent. Quiescence was attained 4–5 days after removal of serum, as established by [ 3 H]-thymidine incorporation. Stimulation of the quiescent SMCs with 20% FBS 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 exhibit the structural markers and capacity to undergo phenotypic modulation that are characteristic of SMCs in vivo. This approach to establishing primary cultures of SMCs offers the advantage of selecting for the subpopulation of cells capable of migration in response to injury or growth factor stimulation. (Mol Cell Biochem 176: 53–59, 1997) Key words: smooth muscle, coronary artery, porcine, immunofluorescent microscopy