Zhao Ashley G, Shah Kiran, Cromer Brett, Sumer Huseyin
Department of Chemistry and Biotechnology, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, John St, Hawthorn, VIC, 3122, Australia.
Department of Chemistry and Biotechnology, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, John St, Hawthorn, VIC, 3122, Australia; Australian Veterinary Stem Cells and Magellan Stem Cells P/L, 116-118 Thames St, Box Hill, VIC, 3129, Australia.
Exp Cell Res. 2022 May 15;414(2):113097. doi: 10.1016/j.yexcr.2022.113097. Epub 2022 Mar 9.
Various types of cells secrete extracellular vesicle (EVs) which contain proteins, lipids and nucleic acids and play important roles in inter-cellular signalling and pathological processes to impact the recipient cells. EVs have demonstrated their potential as biomarkers for disease and as therapeutic agents in regenerative medicine. In recent times, EVs derived from mesenchymal stem cells (MSCs), which are widely used as a promising medicinal product in many clinical applications, are being tested in many preclinical trials. However, the lack of standardization of MSC-derived EV isolation and analysis methods, restricts the utility of MSC-derived EVs in the clinical setting. Here, we focused on optimising the isolation method for EVs derived from MSCs. Four samples of EVs were isolated from human adipose derived MSC culture medium by differential ultracentrifugation with three different ultracentrifuge durations to investigate the influence of ultracentrifuge time on quality and quantity of MSC-derived EVs. Additionally, we used a commercial kit to extract EVs from MSC cultured medium and compared it with the ultracentrifugation method. The EV samples were then characterised for particle concentration, protein concentration, size distribution and the presence of known EV protein markers, by western blot and flow cytometry. A comparison of these results for the five samples demonstrated that 1 h of differential ultracentrifugation was optimal to isolate high quality and quantity of MSC-derived EVs from MSC cultured medium. Additionally, fluorescence imaging of the freshly isolated vs frozen EVs showed that freshly isolated EVs are taken up by cells more efficiently than frozen EVs. These finding establish a simple and reliable method of EV isolation from MSCs.
多种类型的细胞分泌细胞外囊泡（EVs），其包含蛋白质、脂质和核酸，并在细胞间信号传导和病理过程中发挥重要作用，从而影响受体细胞。EVs已展现出作为疾病生物标志物和再生医学治疗剂的潜力。近年来，源自间充质干细胞（MSCs）的EVs在许多临床应用中被广泛用作有前景的医药产品，并正在许多临床前试验中进行测试。然而，MSCs来源的EV分离和分析方法缺乏标准化，限制了其在临床环境中的应用。在此，我们专注于优化源自MSCs的EVs的分离方法。通过差速超速离心法，以三种不同的超速离心时间从人脂肪来源的MSCs培养基中分离出四个EVs样本，以研究超速离心时间对MSCs来源的EVs质量和数量的影响。此外，我们使用商业试剂盒从MSCs培养基中提取EVs，并将其与超速离心法进行比较。然后通过蛋白质免疫印迹和流式细胞术对EVs样本的颗粒浓度、蛋白质浓度、大小分布以及已知的EV蛋白标志物的存在情况进行表征。对这五个样本的这些结果进行比较表明，1小时的差速超速离心是从MSCs培养基中分离高质量和高数量的MSCs来源的EVs的最佳方法。此外，对新鲜分离的EVs与冷冻EVs的荧光成像显示，新鲜分离的EVs比冷冻EVs更能有效地被细胞摄取。这些发现建立了一种从MSCs中分离EVs的简单可靠方法。
