人骨髓间充质干细胞原位冷冻保存方法的改进

本研究的目的是分离纯化、大量扩增人骨髓间充质干细胞(mesenchymal stem cells，MSC)。在细胞原住冻存后通过鉴定其生物学特性有无变化。以探讨这种原位冻存方法的可行性。采用常规方法体外分离培养并扩增骨髓MSC，将培养扩增的细胞用含10％二甲亚砜、30％胎牛血清、DMEM—LG的细胞冻存体系，以不同的细胞贴壁密度在细胞培养瓶内原住冻存于-70℃冰箱内。冻存后第4，8和12周在38℃至40℃的水浴中复苏冻存的贴壁细胞，通过细胞周期分析并检测其多向分化功能以鉴定该方法的可行性。结果表明，MSC不经消化，直接在培养瓶内原住冻存于-70℃低温冰箱，3个月内细胞生物学特性没有明显改变，细胞仍具有向成骨、脂肪和成软骨细胞多向分化的能力。结论：体外培养的骨髓MSC原住贴壁冻存于-70℃。至少在12周内不影响其生物学功能，此种短期冷冻保存是一种可行的方法。

Modification of in situ Cryopreservation of Human Bone Marrow Mesenchymal Stem Cells

The study was aimed to evaluate if the modified in situ cryopreservation could affect the biological function of mesenchymal stem cells (MSC) in vitro. Mesenchymal stem cells from human bone marrow were isolated by standard method and characterized with their morphology, cell-surface antigen profile and differentiation repertoire in vitro. The culture-expanded MSC were cryopreserved in situ with culture medium (DMEM-LG) containing 10% D MSO and 30% selected FCS in -70 degrees C. Following recovery of cryopreservation, differentiation to adipocytes, chondrocytes, and osteoblast in vitro and cell cycle analysis were performed to investigate whether the cryopreservation would change the differentiation potential of MSC. The results showed that after recovery of cryopreservation, there was no changes detected as compared with the culture-expanded MSC in both differentiation potency and growth pattern at 12 weeks. In conclusions: this optimized short term in situ cryopreservation at -70 degrees C could retain biological characteristics of human MSC for at least 3 months, and this method may be useful for cryopreservation of hum an bone marrow MSCs.