Effect of different concentrations of fetal bovine serum on bone marrow mesenchymal stem cell purity and cycle / 安徽医科大学学报

ABSTRACT

Objective To compare the influence of three kinds of complete media with 0. 10 ,0. 15 ,0. 20 fetal bo-vine serum( FBS) on purity and cycle of rat bone marrow mesenchymal stem cells ( BMSCs) cultured in vitro and to seek suitable FBS concentration for the cultivation of the stem cells. Methods SD rats were executed by cervical dislocation method and used whole bone marrow adherence to isolate rat BMSCs. Experiment was divided into A, B,C,3 groups. Compared the expression of CD45, CD29, CD90, CD44 in the three groups in 2,3,4,5 passages ( P2 , P3 , P4 , P5 );the cells of P3 in group A were digested and cultured in three different concentration of com-plete culture media for four days, measuring cell cycle in 24,48,72,96 h by flow cytometry instrument. BMSCs of P3 were collected and inoculated to 6 pieces of 96-well plates, then vaccinated with complete media with 0. 10, 0. 15,0. 20 FBS in every plate, one culture plate was taken out for optical density(OD) meaturement every day with CCK-8. Results CD45 was negative, CD29, CD90, CD44 were positive. The difference of BMSCs surface markers cultured in the three kinds of complete media was bigger in the first two passages, but the difference was less in P3 , P4 , all could obtain pure BMSCs in P4 relatively;according to the results of the cell cycle at the same time, G0/G1 phasewith the increase of concentration of fetal bovine, G0/G1 phase reduced and had no diference in A, B, C groups;G2/M phasethere was difference between them after 24 h(P<0. 05,F=12. 412), but with the extension of time, the differences disappeared;S phasethere was no difference in the three groups;S+G2/M phase increased with the concentration of FBS. According to the result of cell vitality, the OD of ABC three groups increased in turn in 24 h, and there was difference(P<0. 05,F=5. 002), but with the extension of time, there was no obvious difference between them. Conclusion The three kinds of culture media in P4 can obtain pure BM-SCs, cell cycle and vitality show three complete media can promote the growth of BMSCs. There is no difference between them. Culture media with 0. 10 FBS can satisfy the isolation and amplification of BMSCs, in order to ob-tain pure BMSCs in the short term, using culture media with 0. 15 and 0. 10 FBS in the primary culture and subcul-ture respectively.