Unnur Ástrós Magnúsdóttir, Erna María Jónsdóttir and Berglind Eva Benediktsdóttir
Extracellular vesicles (EVs) are released from human cells and have been investigated for their role as drug delivery nanocarriers. Common EV isolation techniques such as ultracentrifugation or size-exclusion chromatography, often accompanied with density gradient are not scalable and may cause agglomeration of EVs, hindering their applicability for isolation of EVs as drug nanocarriers. Immunoaffinity capture, is a viable alternative to isolate intact EVs. This project aimed to refine the current immunoaffinity isolation setup to increase EV yield.
Using the same volume (25ml) of culture medium from the HEK293E cell line, EVs were isolated using Strep-Tactin CD81 Fab-TACS® affinity chromatography, with variable agarose amount but constant CD81 Fab-Strep (25 µg/ml). EVs were eluted with 100 mM biotin and concentrated using 100kDa ultrafiltration, then characterized using NanoSight and capillary western blot (cWB) for EV protein marker ALIX.
Results showed that using 1 ml agarose resulted in 9.37±0.26*10ˆ10 particles/ml, confirmed to be EVs with ALIX cWB, with increased yield when using 1,5 ml (1.04±0.23*10ˆ11 particles/ml) and 2 ml (2.06±0.97*10ˆ11 particles/ml) agarose. Subsequent reduction was observed in the particle amount in the flowthrough (non-EVs) or 95,8% when moving from 1,5 ml to 2 ml agarose. Contribution from the pure cell culture media or the Fab-strep itself towards the measured particle amount was minimal, as determined with NanoSight and cWB.
Increasing the agarose amount in the isolation process improved EV yield, with minimal influence from isolation materials, demonstrating the efficacy of immunoaffinity capture in enhancing EV isolation for future drug nanocarrier formulation.
