Objective: To explore the appropriate procedures for preparing extracellular microvesicles (MV) derived from human mesenchymal stem cells (MSC).
Methods: Human MSCs from umbilical cords were cultured in a serum-free medium and maintained in a basal medium for 72 hours after the cell confluence reached to 80%. The supernatants of cultured cells were collected and MVs were enriched. MVs were identified by flow cytometry and electron microscopy. The total protein amount in MVs was used as a parameter for the content of MVs. The supernatants were adjusted to different pH values, and the output of MVs was detected. The supernatants were also collected for enriching the MV and detecting the protein content of MV after the cells were maintained in the basic medium for different time.
Results: Flow cytometric analysis showed that the MVs expressed CD9, CD63 and CD81, morphologically presented round under an electron microscope and the diameter of MV was around 100 nm. After enrichment of MV, the protein content of MVs in the supernatants was 416.8±128.1, 255.4±77.9 and 142.8±46.4 μg per 107 MSC，respectively at pH of supernatant 3, 7 and 9 (P＜0.05). The protein content of the supernatants per 107 MSC was 173.6±44.5, 262.4±49.6 and 364.2±37.8 μg respectively after starvation culture for 48, 72 and 96 hrs (P＜0.05).
Conclusion: MVs can be readily collected after MSCs were starved for 96 hours, and the pH of the supernatants is adjusted at 3.0.
题目: 制备人间充质干细胞源细胞外微囊条件探索.
目的: 探讨制备人间充质干细胞（mesenchymal stem cell, MSC）来源的细胞外微囊（extracellular microvesicle, MV）的适宜条件.
方法: 无血清条件下培养人脐带来源MSC，细胞融合达80%时,用基础培养液培养72 h后，收集培养细胞的上清，使用浓缩试剂浓缩细胞外微囊，经流式细胞术及电子显微镜鉴定，以蛋白总量为微囊含量指标。调整上清不同pH值，探测MV收获量。去营养后，收集不同培养时间的培养上清，富集微囊并测定蛋白含量，确定不同培养时间微囊产量.
结果: 富集的细胞外微囊表达CD9、CD63和CD81，电子显微镜下微囊呈圆形，直径在100 nm左右。使用微囊富集试剂盒收集、培养上清pH值分别为3,7和9时，每107个MSC收获的蛋白量分别为416.8±128.1、255.4±77.9和142.8±46.4 μg，3组比较有明显差异（P＜0.05）。饥饿培养48、72和96 h，每107个MSC收获的蛋白量分别为173.6±44.5、262.4±49.6和364.2±37.8 μg，3组存在显著差异（P＜0.05）.
结论: 去营养培养96 h后收集MSC培养上清，调整pH值至3.0，可获得较多的细胞微囊.