From the Laboratory of Tissue Regeneration, Division of Plastic Surgery, Lahey Hospital & Medical Center; Department of Burns and Plastic Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine; and Department of Plastic Surgery, Nanfang Hospital, Southern Medical University.
Plast Reconstr Surg. 2022 Mar 1;149(3):638-649. doi: 10.1097/PRS.0000000000008830.
Mesenchymal stem cell-derived exosomes are known to produce effects similar to those of source cells and therefore represent a new approach in cell-free regenerative medicine. Their potential clinical application demands efficient isolation of stable and functional exosomes from a large volume of biological fluid.
Exosomes from adipose-tissue conditioned medium of the same volume were isolated using either (1) ultrafiltration with size exclusion or (2) ExoQuick-TC. The isolated exosomes were characterized by protein concentration, particle size, exosomal marker expression, RNA expression profiles, and roles in dermal fibroblast proliferation and migration.
Both isolation methods produced exosomes within the size range defined for exosomes (50 to 200 nm) and common markers were enriched. Compared to the ExoQuick-TC precipitation method, the ultrafiltration method produced a significantly higher protein yield (p < 0.001) but a lower particle-to-protein ratio (p < 0.05); it also yielded higher RNA contents from the same fat tissue indicated by housekeeping genes, but with overall lower purity. The expression of several mRNAs and miRNAs related to tissue regeneration showed that there was no statistical difference between both methods, except miR-155 and miR-223 (p < 0.05). However, there was no difference in overall fibroblast proliferation and migration between exosomes isolated by these two methods.
Ultrafiltration with size exclusion demonstrated higher yields, acceptable purity, and comparable biophysical properties and biological functions to the more expensive commercial precipitation method. Therefore, it may conceivably be translated into yield-efficient and cost-effective modalities for therapeutic purposes.
Ultrafiltration with size exclusion may be amenable for exosome isolation from large-volume complex fluids such as tissue conditioned media for clinical application in future regenerative medicine.
间充质干细胞衍生的外泌体被认为能产生与源细胞相似的作用，因此代表了无细胞再生医学的一种新方法。它们的潜在临床应用要求从大量生物液中有效分离稳定且功能正常的外泌体。
使用（1）超滤法（大小排阻）或（2）ExoQuick-TC 从相同体积的脂肪组织条件培养基中分离外泌体。通过蛋白浓度、粒径、外泌体标志物表达、RNA 表达谱以及对真皮成纤维细胞增殖和迁移的作用来对分离的外泌体进行表征。
两种分离方法均产生了大小在定义的外泌体范围内（50 至 200nm）的外泌体，且富集了常见的标志物。与 ExoQuick-TC 沉淀法相比，超滤法产生的蛋白产量显著更高（p<0.001），但颗粒与蛋白的比值更低（p<0.05）；它还从相同的脂肪组织中产生了更高的 RNA 含量，这由管家基因表明，但总体纯度较低。与组织再生相关的几种 mRNA 和 miRNA 的表达表明，两种方法之间没有统计学差异，除了 miR-155 和 miR-223（p<0.05）。然而，这两种方法分离的外泌体对整体成纤维细胞增殖和迁移没有影响。
超滤法（大小排阻）显示出更高的产量、可接受的纯度，以及与更昂贵的商业沉淀法相当的生物物理特性和生物学功能。因此，它可能被转化为用于治疗目的的高效且具有成本效益的方法。
超滤法（大小排阻）可能适用于从组织条件培养基等大容量复杂流体中分离外泌体，以便在未来的再生医学中进行临床应用。
