Centre for Tissue Engineering and Regenerative Medicine, Universiti Kebangsaan Malaysia Medical Centre; My CytoHealth Sdn. Bhd.
School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University.
J Vis Exp. 2022 Jun 23(184). doi: 10.3791/64106.
The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs' size was 30-200 nm, with a particle concentration of 7.75 × 10 particles/mL and a protein concentration of 80 µg/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).
基于超速离心的方法被认为是分离小细胞外囊泡（sEVs）的常用方法。然而，这种分离方法的产量相对较低，并且这些方法在分离 sEV 亚型方面效率不高。本研究展示了一种简单的台式过滤方法来分离人脐带间充质干细胞来源的 MSC 小细胞外囊泡（hUC-MSC-sEVs），该方法成功地从小鼠 hUC-MSCs 的条件培养基中通过超滤分离出来。通过纳米颗粒跟踪分析、BCA 蛋白测定、western blot 和透射电子显微镜分别对分离的 hUC-MSC-sEVs 的大小分布、蛋白浓度、外泌体标志物（CD9、CD81、TSG101）和形态进行了表征。分离的 hUC-MSC-sEVs 的大小为 30-200nm，颗粒浓度为 7.75×10^9 个/mL，蛋白浓度为 80µg/mL。观察到外泌体标志物 CD9、CD81 和 TSG101 的阳性条带。本研究表明，hUC-MSC-sEVs 已成功地从小鼠 hUC-MSCs 的条件培养基中分离出来，并且表征结果表明，分离产物符合 2018 年细胞外囊泡研究最小信息（MISEV 2018）的要求。
