Combinatorial effect of calcium chloride and polyethylene glycol on efficient isolation of small extracellular vesicles.

Small extracellular vesicles (sEVs) are cargo-carrying cellular nano-vesicles that have been explored for developing organic drug delivery modalities (DVM), an alternative to synthetic liposomes. However, scaled-up production of sEVs is a notable challenge in bringing sEV-based DVMs from the bench to the clinic. Ultracentrifugation is the most accepted isolation approach, but the cumbersome logistical issues and aftereffects of intense 'g' force hinder their applicability. In this study, we developed a new amenable isolation strategy for sEVs using a combinatorial treatment of calcium chloride and polyethylene glycol (PEG). An equivalent volume of cell culture medium from growing lung cancer A549 and H1299 cells was incubated overnight at 4 °C with different formulations (0.1 M CaCl2, 8% PEG, 12% PEG, 0.1 M CaCl2 + 8% PEG, and 0.1 M CaCl2 + 12% PEG) and centrifuged at 4000g to purify the precipitated sEVs as a pellet. Next, the extra CaCl2 was chelated out and the buffer was exchanged with PBS. The sEV number and protein content were assessed using the NTA (nanoparticle tracking analysis) and the BCA assay, respectively. Finally, transmission electron microscopy (TEM) was used to visualize the sEVs. The data from the present study demonstrated that the combination of 8% PEG and 0.1 M CaCl2 produced comparable numbers of sEVs with the ultracentrifugation technique. The sEV characteristics and structural integrity also remained intact, as evident from the TEM images and western blot assay. Thus, here we report an efficient technique for sEV isolation that can be easily scaled up.