Isolation, culture and identification of rabbit bone marrow-derived vascular endothelial progenitor cells / 中国实验血液学杂志

ABSTRACT

The aim of study was to set up a suitable method of isolation, culture and identification of endothelial progenitor cells (EPC) derived from rabbit bone marrow. Density gradient centrifugation was used to isolate mononuclear cells from bone marrow, the isolated mononuclear cells were cultured with specific culture medium for EPCs. EPCs were identified by cellular morphologic observation, immunohistochemistry testing, flow cytometry and the function test of taking up Dil-ac-LDL and FITC-UEA-1. The results indicated that the newly isolated bone marrow-derived mononuclear cells exhibited a round appearance, following culture for 48 hours, adherent cells grew in colony cluster, presenting with round or irregular appearance, and nuclear division was obvious. On day 7, flaky cell colonies mutually connected together, presenting with spindle-shaped cells. Immunohistochemistry testing in the EPCs showed CD133(+), CD34(+), VIII factor(++), KDR(++); flow cytometry testing showed that the positive rate of CD133 was (18.23+/-7.12)%, the positive rate of CD34 was 47.71+/-14.85%, the positive rate of CD31 was (71.61+/-13.51)%, the positive rate of KDR was (87.24+/-11.40)%. And more than 80% EPC could take up both Dil-acLDL and FITC-UEA-1. It is concluded that the mononuclear cells isolated from bone marrow by density gradient centrifugation can differentiate into EPCs under special culture situation.