Optimization of culture conditions for endothelial progenitor cells from porcine bone marrow in vitro.

OBJECTIVES: The aim of this study was to determine an optimal culture method for porcine bone marrow-derived endothelial progenitor cells (EPCs). MATERIALS AND METHODS: Mononuclear cells (MNCs) were isolated by density centrifugation and differentiated into EPCs in in vitro. At first-passage, EPCs were cultured at different cell densities (5 x 10(3), 1 x 10(4), 2 x 10(4) or 5 x 10(4)/cm(2)) and in basic medium (EGM, medium 199, DMEM or 1640) supplemented with FBS (2%, 5%, 10% or 20%) and different combinations of cytokines (VEGF, VEGF + bFGF, VEGF + bFGF + EGF, or VEGF + bFGF + EGF + IGF), the experiment being based on L(64) (4(21)) orthogonal design. RESULTS AND CONCLUSIONS: This demonstrated that the optimal culture method for our EPCs displayed higher expansion and migration rates as compared to other groups, by analysis of variance; that is, cultured at 1 x 10(4)/cm(2) in M199 supplemented with 10% FBS and VEGF + bFGF + IGF + EGF. Furthermore, percentage of positive cells stained by Dil-ac-LDL and FITC-UEA-1 was more than 65%, and as shown by immunohistochemistry, these cells also stained positively for CD133, CD34 and KDR. The present study indicates that the number and function of porcine EPCs significantly increased when using our optimized culture parameters.