人脐带间充质干细胞的微生物学安全性评价

Microbiological safety evaluation of human umbilical cord mesenchymal stem cells

目的对按标准化工艺流程制备的人脐带间充质干细胞(hUC-MSCs)制剂进行微生物学安全性评价。方法人脐带华通氏胶分离的间充质干细胞培养扩增至P5代。显微镜下观察细胞的形态特征,流式细胞仪检测细胞表面标志物的表达;培养法检测细菌、真菌污染,指示剂培养显色法和快速聚合酶链反应(PCR)法检测支原体污染,鲎试剂凝胶法检测细菌内毒素;胶体金法检验特定人源病毒、梅毒螺旋体;荧光定量PCR法检测人乳头瘤病毒(HPV)。结果按照标准化流程分离培养hUC-MSCs传代至P5代呈均一的长梭型成纤维细胞样贴壁生长,细胞表面标志物CD73、CD90和CD105阳性率大于98%,CD14、CD19、CD34、CD45和HLA-DR<2%;受检人脐带间充质干细胞鲎试剂凝胶法检测内毒素水平小于0.25 EU/mL,培养检测未见细菌、真菌菌落生长,指示剂培养显色法和快速PCR检测均显示无支原体污染;胶体金法和荧光定量PCR法检测特定人源病毒、梅毒螺旋体和HPV(包含18种高危亚型)全部为阴性结果。所有检测方法的阳性对照标本均呈阳性结果,证实检测方法稳定可靠。结论本研究采用的标准化干细胞制备工艺流程稳定可靠,采用该工艺制备人脐带间充质干细胞符合临床应用的微生物学安全性要求。

Objective To conduct the safety evaluation of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)prepared by the standardized technological process.Methods hUC-MSCs isolated by Wharton′s Jelly were cultured and expanded to passage 5(P5).The morphological characteristics of the resulting cells were observed by microscope,and the expression of cellular surface markers was detected by flow cytometry.The bacterial and fungal contamination was detected by using the culture method,the mycoplasma contamination was detected by using the indicator culture chromogenic assay and rapid PCR method,and the bacterial endotoxin was detected by the limulus reagent gel method;the specific human virus and treponema pallidum were detected by using the colloidal gold method and HPV was detected by adopting the fluorescence quantitative PCR.Results hUC-MSCs isolated by standardized technological process showed the homogeneous long shuttle like fibroblast like adherent growth until p5,and the positive rate of cellular surface markers CD73,CD90 and CD105 was more than 98%,which of CD14,CD19,CD34,CD45 and HLA-DR was less than 2%;the endotoxin content of hUC-MSCs in the subjects detected by the limulus reagent gel method was less than 0.25 EU/mL,no growth of bacterial and fungal colonies was found by the culture,the indicator culture chromogenic assay and rapid PCR method all showed no mycoplasma contamination;the specific human virus,treponema pallidum and HPV(containing 18 high-risk subtypes)detected by the colloidal gold method and the fluorescence quantitative PCR all had the negative results.All positive control samples of all detection methodsshowed the positive results,verifying that the detection method was stable and reliable.Conclusion The standardized stem cells preparing technological process adopted by this study is stable and reliable,adopting the technological process to prepare hUC-MSCs conforms to the requirements of clinical application microbiological safety.