无血清培养基对深低温冻存复苏后的间充质干细胞免疫功能的研究

目的:探讨无血清培养基对冻存后的间充质干细胞（HUC-MSCs）免疫调节功能的影响，明确其成脂成骨生物学特性，为临床应用奠定基础。方法:（1）分离脐带中的HUC-MSCs，传代纯化后深低温冻存。（2）冻融后的HUC-MSCs分别以含10%胎牛血清的培养基（SCM），无血清培养基两种培养条件培养。（3）观察两组细胞生长状态，革兰染色计数，计算细胞活率。（4）流式细胞术检测两组细胞的表面标志物。（5）成脂成骨诱导，茜红素染色和油红染色观察诱导情况。结果:（1）两组细胞均以漩涡状贴壁，无血清培养基培养的细胞体积略小。（2）在细胞总数及活性方面，两组细胞有些微差异，可忽略不计。（3）流式细胞术显示其对PE-CD34不表达，对PE-CD105、PE-CD90、PE-CD73、PE-CD44和PE-CD29高表达。（4）对无血清培养基（SFM）所培养得到的HUC-MSCs历经约21 d的成骨细胞和脂肪细胞专向分化诱导，发现其具有可媲美SCM培养的HUC-MSCs的分化能力。结论:无血清培养基同有血清培养基相比，对深低温冻存复苏后的间充质干细胞免疫调节功能无明显差异，可替代有血清培养基进行细胞培养。

Study on the immune function of mesenchymal stem cells in serum-free medium after cryopreservation and resuscitation

Objective: To investigate the effect of serum-free medium on immune regulation of cryopreserved mesenchymal stem cells (HUC-MSCs), and to clarify the biological characteristics of adipogenic osteogenesis for clinical application. Methods: (1) Mesenchymal stem cells(HUC-MSC s) were isolated from the umbilical cord and subcultured for cryopreservation. (2) HUC-MSCs were cultured in two culture conditions: 10% fetal bovine serum medium and serum-free medium. (3) Cell growth was observed, and cell viability was calculated by Gram staining. (4) Flow cytometry was used to detect the surface markers of the two groups of cells. (5) adipogenic osteogenesis induction, alizarin staining and oil red staining were used to observe the induction. Results: (1) The cells of both groups adhered to each other in a whirlpool shape, and the cells cultured in serum-free medium were slightly smaller. (2) In terms of the total number and activity of cells, there were slight differences between the two groups. (3) Flow cytometry showed no expression of HLA-DR, PE-CD86, PE-CD80, PE-CD45, PE-CD11b, PE-CD34, PE-CD31, and PE-CD14, but low expression of PE-CD106, high expression of PE-CD105, PE-CD90, PE-CD73, PE-CD54, PE-CD44 and PE-CD29. (4) HUC-MSCs cultured in serum-free medium(SFM) were induced to differentiate into osteoblasts and adipocytes for about 21 days. It was found that the differentiation ability of HUC-MSCs cultured in SFM was close to that of HUC-MSCs cultured in SCM. Conclusion:Compared with the conventional medium, the serum-free medium shows no significant difference in the immune regulation function of mesenchymal stem cells after cryopreservation and resuscitation, that being said,the serum-free medium may be used asa substitution product used in MSCs culture.