Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by anion-exchange chromatography

The development of a new large-scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, researchers from the Mie University Graduate School of Medicine have developed an effective method for preparing high-performance exosomes (EXOs) by using an anion-exchange method. Cytotoxic T-lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut-off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M-0.3 M) and high (0.3 M-0.5 M) NaCl concentrations (approximately 2 × 10¹² and 1.5 × 10¹² particles, respectively) through the anion-exchange column chromatography. The former are abundant in EXO proteins, including late endosome-associated proteins and rab-family and integrin-family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)-like particles including DNA, core histone and ribosomal proteins, and GC-rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor⁺ Kupffer cells. Thus, the anion-exchange method is suitable for the large-scale separation of bioactive EXOs and MV-like EVs as a cargo for dangerous nucleic acids at high-purity.

Elution of bioactive CTL EVs at low-salt concentrations by DEAE column chromatography

(a) Preparation workflow of CTL EVs by DEAE column chromatography after concentration and deproteinization of culture supernatant with 750 kDa MWCO UF. (b) A typical elution pattern by NaCl linear gradient (0.15 M–0.8 M) of CTL EVs from DEAE column chromatography based on particle number, protein concentration, and expressions of CD9 and CD90 (FACS analysis). (c) Western blot analysis of CTL EVs of the indicated fractions by CD9 and Tsg101 mAbs. (d) Fr. 20 and Fr. 26 CTL EVs, and UC CTL EVs (2 × 10⁹ EVs/tumour) were treated i.t. in subcutaneous CMS5a tumours (fibrosarcoma: 1 cm in diameter).