Isolation, culture and identification of endothelial progenitor cells from porcine peripheral blood in vitro

Background: Endothelial progenitor cells (EPCs) are precursor cells that can directly differentiate into vascular endothelial cells. Researches have suggested that the porcine cardiovascular anatomic structure is similar to human compared with other inferior mammal. It is of great clinical significances to establish cardiovascular disease models using porcine cardiovascular system. Objective: To explore the method to isolate and culture EPCs from porcine peripheral blood in vitro. Design, time and setting: The cytology in vitro study was performed at the Centor for Research in Cardiovascular Diseases, University at Buffalo, State University of New York and Department of Pathophysiology, Capital Medical University from September 2007 to May 2008. Materials: Six 12-week-old healthy adult pigs were supplied by Animal Experimental Center of University at Buffalo. Methods: The mononuclear cells (MNCs) of porcine peripheral blood were isolated by the density gradient centrifugation and incubated into 6-well tissue culture plates coated with type I rat tail collagen at 3x10⁷ cells per well in 2 mL of endothelial growth medium-2 (EGM-2). Cells were purified and amplified by the adherence method. Cells at the second passage were used for determination. Main outcome measures: The expression of EPC surface markers was detected by immunofluorescence and flow cytometer. The biological function of EPCs was examined by the adsorption of Dil-Ac-LDL and Matrigel. For colony-forming assay, EPCs at passages 2 to 10 were incubated at a low cell density (500 cells per well). Results: After 7-10 days culture, cobblestone-appearing cells colonies emerged, which expressed CD9, CD31, CD105, VE-Cadherin, VEGFR2 and endothelial nitric oxide synthase. Some cells also expressed c-Kit, but did not express CD1 33 and CD45. These cells could uptake Dil-acLDL and formed capillary-like structure in Matrigel in vitro, and had strong clone forming ability. Cell colonies at passages 2, 4, 6, 8 and 10 were identical to the total number of EPCs (F=0.167, F=0.195, P > 0.05). Conclusion: EPCs can be successfully isolated from porcine peripheral blood by the density gradient centrifugation and the adherence method. Cell proliferation ability does not change following in vitro amplification.