Extracellular vesicles are particles ranged from 30 nm to 5μm and subcategorized into three groups; exosomes, microvesicles and apoptotic bodies, each of which have different biological impact. Lack of a standard method for the detection and isolation of MVs has led to a challenging issue that is a worth considering. In this study, researchers from the Yasuje University of Medical Sciences isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation.
The researchers examined the efficacy of differential centrifugation ranging from 10,000×g to 60,000×g on UCMSCs- derived microvesicles yield and purity. The fractions were evaluated by Dynamic Light Scattering (DLS) method, total protein quantification and flow cytometry.
UC-MSCs were spindle cells that adhered to plastic culture flasks. These cells expressed MSC markers such as CD44 and CD73, whereas were negative for hematopoietic markers CD45 and CD34. UC-MSCparticles were successfully isolated. Particles were heterogeneous vesicles of approximately 50 to 1250 nm in diameter that bear the surface-expressed molecules UC-MSCs such as; CD90, CD106, CD166 and CD44, and negative for CD34, CD63, and CD9. According to the results of DLS method, centrifugation at 10,000, 20,000, 40,000 and 60,000 ×g, all gave MVs of less than 1000 nm. It is of notion that only at the centrifugation rates of 40,000 and 60,000×g, particles of less than 100 nm in diameter were also obtained.
The isolated microvesicles were stained with fluorochrome–conjugated antibodies; CD90-FITC, CD105-PE, CD166-PE, CD44-FITC, CD63-PE, CD9PE, and CD34-FITC plus reagent. They were positive for CD90 (36.96%), CD105 (39.47%), CD166 (17.41%), CD44 (55.33%) and negative for CD34 (1.48%), CD63 (0.4) and CD9 (0.45).
The choice of exact speed greatly influences the purity of MVs and their yield. These findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.