Microvesicles preparation from mesenchymal stem cells

Abstract

Background: 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, we isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation.

Methods: We 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.

Results: 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.

Conclusion: The choice of exact speed greatly influences the purity of MVs and their yield. Our findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.

Keywords: DLS method differential centrifugation; exosomes; extracellular vesicles; microvesicles.

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Fig. 3a

A. Representative dot plots from the flow cytometer. R1 is the gate for MVs and R2 is the gate for beads. The size of MVs in R1 was less than 1μm.

Fig. 3b

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).

Fig. 4a

Dynamic Light Scattering (DLS) results for the size distribution of MVs obtained at the centrifugation rate of 10,000× g. From the relative intensities of the size distribution peaks, appears that there is likely some apoptotic debris within the sample.

Fig. 4b

Dynamic Light Scattering (DLS) results for the size distribution of MVs obtained at the centrifugation rate of 20,000× g.

Fig. 4c

Dynamic Light Scattering (DLS) results for the size distribution of MVs obtained at the centrifugation rate of 40,000× g. The contamination possibility with exosomes at 40,000× g is less than at 60,000× g.

Fig. 4d

Dynamic Light Scattering (DLS) results for the size distribution of MVs obtained at the centrifugation rate of 60,000× g.