冻存复苏对脐带间充质干细胞生物学特性的影响

Effects of cryopreservation and resuscitation on the biological characteristics of umbilical cord mesenchymal stem cells

目的观察脐带间充质干细胞经冻存复苏后的生物学特性,并鉴定其是否具有间充质干细胞的特征。方法采用组织块贴壁法从人脐带组织中分离培养间充质干细胞,将传至第3代的细胞进行冻存6个月后复苏,观察冻存前和冻存复苏后细胞的形态学变化,比较冻存前后细胞的存活率,测定细胞生长曲线,并用流式细胞仪检测冻存后细胞表面抗原的表达情况。结果组织块贴壁法可以成功地分离培养脐带间充质干细胞;经低温冻存复苏后细胞仍保持贴壁生长的特性,形态未发生明显变化;复苏后细胞存活率与未冻存细胞相比差异无统计学意义;冻存前后细胞的生长曲线相似;流式细胞仪分析冻存后细胞表面高表达间充质干细胞抗原CD29、CD90、CD44、CD105,低表达或不表达造血干细胞抗原CD34、CD45和白细胞相关抗原HLA-DR。结论冻存复苏对脐带间充质干细胞的生物学特性无明显影响,冻存后细胞符合间充质干细胞的基本要求。

Objective It is to observe the biological characteristics of umbilical cord mesenchymal stem cells after cryopr -eservation, and identify whether it has the characteristic of mesenchymal stem cells .Methods By tissue explants adherent method, the MSCs were isolated and cultured from umbilical cord .And the third passage cells were frozen and thawed after six month.Then the morphological changes before freezing and frozen thawed cells were observed , the survival rate was com-pared, the cell growth curve was determined , and the expression of cell surface antigen was detected by flow cytometry .Re-sults MSCs were successfully obtained from Wharton ’ s jelly of human umbilical cord via the proposed approach of tissue ad-herence.After cryopreservation, the cells still maintained the characteristics of adherent growth , and the morphology did not change significantly The survival rate of frozen thawed cells had no significant difference compared with that of the non frozen cells.Cell growth curve was similar before and after cryopreservation .The flow cytometry analysis showed that frozen thawed cells highly expressed CD29, CD90, CD44, CD105 of stromal cell antigen, but low expression of hematopoietic stem cell as-sociated antigens CD34, CD45 and leukocyte antigen HLA -DR.Conclusion The biological characteristics of human umbili-cal cord mesenchymal stem cells were not significantly changed after cryopreservation , and the cells were in accordance with the basic requirements of mesenchymal stem cells after cryopreservation.