FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan
Abstract: We developed a reproducible method for affinity purification of EVs by using Tim4 protein, which specifically binds phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Here, we tested the profiles of the purified EVs, including biological activities of EVs, and compared with other commonly used methods for large-scale purification of EVs, such as tangential flow filtration (TFF) combined with size exclusion chromatography (SEC) or anion exchange chromatography (AEX) methods. Agarose resin immobilized with recombinant Tim4 protein was packed into a 1mL column. 200 mL of bone marrow-derived MSC culture supernatant was directly applied into the Tim4 column. After washing the column, 4 mL of EDTA solution was applied to elute the captured EVs. The resulted EVs were analyzed by using NTA, ELISA, BCA methods, and cell-based functional reporter assay. In parallel, EVs were also purified from the 200 mL of the same culture supernatant by using TFF, TFF + SEC, or TFF + AEX, and the resulted EVs were analyzed for comparison. It was confirmed that EVs can be purified with higher yield (recovery was 70 - 90%) and purity than the other methods. We also observed that the purified EVs by using the Tim4 column showed similar surface tetraspanin distributions to what observed with EVs purified with the Tim4-immobilized magnet beads-based small scale purification method, suggesting that this affinity-based method is scalable. Furthermore, the Tim4 column purified EVs showed higher anti-inflammatory effect than other conventional methods. Therefore, we propose that our affinity purification method has potential for use in manufacturing process of therapeutic EVs.
