[Comparison of whole bone marrow culture method and density gradient centrifugation method of isolating hBMSCs].
摘要
OBJECTIVE: To make a comparative study on the effects of whole bone marrow culture method and density gradient centrifugation method in isolating hBMSCs. METHODS: hBMSCs were obtained from healthy adult volunteers and isolated by whole bone marrow culture method and density gradient centrifugation method. Primary cell morphology was observed using inverted phase contrast microscope and the cells in the 2nd passage were stained with HE after being cultured for 7 days. And then, the generation time of the primary, 2nd and 3rd passage hBMSCs was compared between two methods and the surface markers were detected by flow cytometer. In addition, the ALP expression in osteoinductive hBMSCs were evaluated by ALP activity kit at 3, 6 and 9 days and ALP staining was used for osteoinductive hBMSCs with Kaplow method at 9 days. RESULTS: Primary cells isolated with whole bone marrow culture method showed aggregation growth while cells isolated with density gradient centrifugation method showed diffusion growth. HE staining showed no significant difference in the morphology of the 2nd passage cells between these two methods. The generation time of primary cells isolated by whole bone marrow culture method (15.36 +/- 1.67) days was significantly shorter than that of cells isolated by density gradient centrifugation method [(18.57 +/- 1.05) days] (P < 0.01), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P > 0.05). The consent of positive surface markers (CD29, CD44, CD71, CD105, CD166) and negative surface marker CD34 in the 2nd cells showed no significant difference between these two isolation methods (P > 0.05); however, negative markers CD14 and CD45 showed significant difference (P < 0.01). The ALP expression in osteoinductive cells showed no statistical significant (P > 0.05) at 3, 6 and 9 days; and the ALP staining positive cell ratio of whole bone marrow culture method was basically in accord