Cultivation and characterization of coronary microvascular endothelial cells: A novel porcine model using micropigs

Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO4, and 500 muM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelialspecific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for a-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern. (C) 2002 Elsevier Science (USA).