A Rapid, High-Resolution Chromatographic Method for Isolating Subpopulations of HEK293-derived Extracellular Vesicles.

Rationale: In recent years, significant advances have been made in understanding the basic underlying science of extracellular vesicles (EVs). This has opened multiple avenues for potential application, especially in areas such as biomarker discovery and drug delivery (vector) systems. Nonetheless, to achieve this, challenges such as the lack of reproducible isolation methods with high resolving power, especially for enriching subpopulations of EVs, need to be tackled. Here, we developed a hydrophobic interaction chromatography (HIC) method utilizing cost-effective polyester fiber columns for the isolation of EVs. Methods: Human embryonic kidney (HEK)-293 cells were cultured in shake flasks, centrifuged and filtered to remove cells and cell debris, respectively. The clarified media was injected onto capillary-channeled polymer (C-CP) fiber columns in HIC mode using step gradients. Alternative gradients that combine both positive and negative steps (termed a switchback gradient) during EV elution were scouted to improve the separation of partially resolved peaks. The optimized method was transferred to an analytical-scale column, and peaks were collected using an integrated fraction collector. For downstream characterization, fractionated peaks were buffer exchanged using Amicon filters with a 10 kDa molecular weight cut-off. Results: Polyester fiber columns, operated with both positive and negative step gradients during EV elution, yield baseline separation of three EV peaks within 12 minutes. Retention times show high repeatability (<0.33% RSD) and reproducibility (<1.3% RSD) across three column batches. Characterization of each peak fraction using nanoparticle tracking analysis (NTA) and nanoflow cytometry (nFCM) revealed similar trends in the size variation. The variation in the surface markers CD9 and CD81 among the collected fractions was confirmed by nFCM in the fluorescent detection mode, while intact double-layer and cup-shaped vesicles were observed in transmission electron microscopy (TEM) images. Conclusions: We demonstrated here, for the first time, a rapid chromatographic method for isolating and enriching EV subpopulations based on their chromatographic behavior which is reflective of their hydrophobicity (potentially a function of size or surface protein density) in a single unit operation. Three discrete size populations of EVs (based on NTA and nFCM sizing) were baseline separated within 12 minutes. Preliminary characterization of surface protein composition via nFCM showed significant differences among the isolated subpopulations.