Erna Jónsdóttir, Ragnar Axel Adolfsson, Jens Guðmundur Hjörleifsson, Berglind Benediktsdóttir
Extracellular vesicles (EVs) are cell-derived membranous nanoparticles that participate in cell-to-cell communication leading to functional changes in recipient cells, besides being implicated in various disease progressions. Various methods are used to isolate EVs from cell media such as size-exclusion-chromatography (SEC) and Strep-Tactin immuno-affinity chromatography (IAC) depending on the origin of EVs and downstream applications. There is limited knowledge of the applicability of SEC or IAC in the isolation of EVs from suspension-based cell lines such as MEXi-293. Therefore, the aim of this study was to compare SEC and IAC as EV isolation methods for MEXi-293-based cell lines.
Conditioned culture media from two cell lines, MEXi-HEK293 and MEXi-HEK293Ligand+/GFP+, was collected and EVs isolated using either SEC, with a Frac30 Åkta and an S-500HR column, or Strep-Tactin CD81 Fab-TACS® affinity chromatographic EV Isolation (Iba-LifeScience). After concentration with ultrafiltration, EV characterization was done using nanoparticle-tracking-analysis (NanoSight-NS300), and simple western blot (WB, ProteinSimple JESSTM), for EV protein markers ALIX and CD9.
Different size ranges of the isolated particles could be observed when using the two isolation processes or 83.68±7.89nm for IAC and 118±4.90nm particles for SEC. Significantly higher number of particles were isolated from IAC vs. SEC or 1.45±0.05x1011particles/mL vs 4.58±0.09x109particles/mL (t(12)=40.4890,p<0.001). EV isolations using IAC resulted in positive signals in WB for ALIX at around 100kDa and CD9 at 30kDa. However, no signal could be detected for SEC samples. IAC using Strep-Tactin is the better-suited method to isolate the EVs for this cell line with high particle numbers and positive EV WB bands.