Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product.

Researchers from the University of North Carolina present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From these results, the researchers propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.

Diagram of cross-flow filtration

(a) A cross-flow chamber containing the input fluid (tissue culture supernatant, human plasma, tumour fluid, etc.) is pumped into a molecular weight cut-off filter. Molecules smaller than the size exclusion of the MW filter are sent to a waste chamber, whereas molecules larger than the size exclusion MW filter (in this case, EV) are returned to the cross-flow chamber. As the volume in the cross-flow chamber decreases, equilibration buffer is pumped in from the equilibration chamber. After equilibration buffer has been depleted, a final clarified product of concentrated EV can be used. (b) Particle size distribution (from cultured BCBL-1 cells) for Input 300 g and 50 g “Post-Clean” samples, with the modes arbitrarily standardized to 1 (N = 6). (c) Particle sizes were determined by NTA at progressive time points during the CFF concentration step. (d) Concentration of particles measured in each fraction during the CFF concentration step (N = 6).