兔骨髓间充质干细胞体外分离培养及冻存

对分离获得的兔骨髓间充质干细胞(BMSCs)进行低温冻存研究。用红细胞裂解法体外分离培养BMSCs,采用SABC法进行鉴定;冻存后复苏,台盼蓝染色检测细胞复苏率,观察冻存时间(15d,30d,90d)对各代(P1,P3,P9代)细胞复苏效果的影响;向脂肪细胞诱导分化验证其干细胞多潜能分化特性。结果表明:BMSCs呈长梭形,聚集成旋涡状均匀生长,SABC法鉴定其为BMSCs;冻存前后P1、P3代细胞活力好,增殖速度快;P9代细胞增殖缓慢;冻存时间对P1、P3代细胞复苏率的影响不显著(P0.05),复苏率较高;但是P9代细胞复苏率极低,并且随着冻存时间的延长,细胞复苏率逐渐降低;冻存复苏的细胞向脂肪细胞诱导分化,油红浸染红色。可见:红细胞裂解液法分离获得的BMSCs生长生物学性状稳定;P3代以前冻存增殖和分化特性不受影响,可作为种子细胞用于后续研究。

Cultivation and Cryopreservation of Rabbit BMSCs in Vitro

To study the cryopreservation of rabbit bone marrow mesenchymal stem cells (BMSCs) isolated by red blood cell lysis buffer. Rabbit BMSCs were cultured and identified in vitro, the thawed cells after 15d, 30d, and 90d frozen storage detected with trypan blue to evaluate the cell recovery rate. Adipogenic differentiation of BMSCs testified the characteristics of pluripotent differentiation. The result showed that primary and passage cells were spindle shaped gathering like eddy identified by immunocytochemistry. The cells of P1 and P3 generation grew rapidly and the expansion ability improved. Instead, the cells of 9 generation grew slowly. After a period of time, the frozen thawed BMSCs of P1 and P3 generation had the relative recovery ratio with non-significant difference (P>0.05); the cryopreservation recovery rate of P9 generation was extremely low as cellular senescence with increased numbers of passage. BMSCs differentiate into adipogenesis stained by oil red. It illustrated that BMSCs isolated by the red blood cell lysis method in vitro had better potentiality of proliferation and differentiation. Rabbit BMSCs were freeze-stored in liquid nitrogen during a certain period. P3 generation and the previous cells after cryopreservation, the proliferation and differentiation characteristics were not affected. They could be used as the seed cells of tissue engineering for subsequent research.