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目的探讨人脐带源间充质干细胞(HUC-MSCs)临床输注前制备成细胞悬液,细胞活性和细胞表面抗原的变化。方法第3～5代人脐带源间充质干细胞用0.25%胰蛋白酶消化处理,PBS洗涤5次,分别重悬于磷酸盐缓冲液(PBS)或含有50 U/ml肝素-PBS、100 U/ml肝素-PBS、200 U/ml肝素-PBS中,放置于4℃、22℃或37℃条件下,分别延长至1 h、2 h、3 h,检测细胞存活率和细胞表面抗原的变化。结果 HUC-MSCs收集后重悬于PBS中,在4℃条件下,随着在体外放置时间的延长,细胞的存活率在随之下降,但细胞存活率明显高于放置22℃、37℃条件下的细胞。将细胞收集后重悬于含有肝素的PBS溶液中,细胞既无聚集现象,而且随着肝素浓度的增加,细胞的存活率相对较高,体外短暂保存HUC-MSCs的效果较好。在含200 U/ml肝素的PBS溶液中,4℃条件下细胞放置3 h后经苔盼兰染色镜下计数可知,HUC-MSCs存活率可维持在(91.3±3.08)%,而且HUC-MSCs表面仍高表达CD105、CD73、CD44、CD90。结论临床输注前制备HUC-MSCs重悬于含200 U/ml肝素-PBS中在4℃条件下3 h内可供临床有效输注。
Objective To investigate the changes of cell viability and cell surface antigen prepared from human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) before clinical transfusion. Methods The umbilical cord-derived mesenchymal stem cells (MNCs) from passage 3 to passage 5 were digested with 0.25% trypsin and washed five times with PBS, resuspended in phosphate buffered saline (PBS) or containing 50 U / ml heparin- ml Heparin-PBS and 200 U / ml Heparin-PBS for 1 h, 2 h and 3 h at 4 ℃, 22 ℃ or 37 ℃ respectively. The cell viability and cell surface antigen were detected. Results After resuspended in PBS, HUC-MSCs were collected and resuspended in PBS at 4 ℃. The survival rate of HUC-MSCs decreased with the prolonging of time in vitro. However, the viability of HUC-MSCs was significantly higher than that at 22 ℃ and 37 ℃ Under the cell. The cells were collected and resuspended in PBS solution containing heparin. The cells were neither aggregated nor aggregated. With the increase of heparin concentration, the cell survival rate was relatively high, and the effect of transiently storing HUC-MSCs in vitro was good. The cell viability was maintained at (91.3 ± 3.08)% in HUC-MSCs treated with 200 U / ml heparin in PBS at 4 ℃ for 3 h, The surface is still high expression of CD105, CD73, CD44, CD90. Conclusions The HUC-MSCs prepared before clinical infusion are resuspended in 200 U / ml heparin-PBS for effective clinical infusion within 3 h at 4 ℃.