Comparison of Yield, Purity, and Functional Properties of Large-Volume Exosome Isolation Using Ultrafiltration and
Min Gao1, Junrong Cai1, Helen S Zitkovsky1
1From 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.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Clinical Relevance Statement:
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.
