Characteristics and Function of Cryopreserved Bone Marrow–Derived Endothelial Progenitor Cells

Abstract

This study examined ex vivo expansion of bone marrow-derived endothelial progenitor cell (EPC) from cryopreserved bone marrow-derived mononuclear cells, and evaluated proliferation and migration function of the cryopreserved EPC (Cryo-EPC). Bone marrow samples were taken from swine iliac bone (n = 6). Isolated bone marrow-derived mononuclear cells were cultured or cryopreserved at -80 degrees C for 2 to 3 months. After cell culture for 4 days, attached cells, EPCs with or without cryopreservation, were collected. Direct fluorescent staining by acetylated low-density lipoprotein, isolectin B4, and 4',6-diamidino-2-phenylindole were performed to confirm the attached cells as EPC. Endothelial progenitor cell proliferation by vascular endothelial growth factor was evaluated by the tetrazolium method. Endothelial progenitor cell migration in response to stromal-derived factor-1alpha was also evaluated by using a Boyden chamber assay. The percentage of cells positively stained by direct fluorescent staining by acetylated low-density lipoprotein and isolectin B4 was similar between fresh and Cryo-EPC (EPC = 96.0 +/- 0.42 versus Cryo-EPC = 95.2 +/- 1.2; p = 0.21). Vascular endothelial growth factor increased proliferation activity in fresh and Cryo-EPC (p < 0.01). Stromal-derived factor-1alpha increased migration activity in fresh and Cryo-EPC (p < 0.01). There was no difference in proliferation and migration activity between fresh and Cryo-EPC. Ex vivo expansion by cell culture was a useful method for collection of bone marrow-derived EPC from cryopreserved mononuclear cells. Proliferation and migration function of EPC is preserved after cryopreservation.