Monitoring HEK293 cell extracellular vesicle (EV) production via a polyester capillary-channeled polymer (C-CP) fiber column with HIC modality

Extracellular vesicles (EVs) are phospholipid bilayer-encased nanoparticles released from all living cells, with the potential to advance diagnostic and therapeutic applications. Human embryonic kidney (HEK293) cells, a widely-cultured cell type for recombinant protein and viral production, provide an abundant, scalable, and reproducible source of EVs. The monitoring of EVs released during cell culture growth provides insights into cell growth parameters, which could include the roles of culture medium composition, pH, glucose, serum presence, and cellular stress on the yields and quality. Therefore, a simple method for monitoring cell culture EV release is highly desirable for optimizing industrial cell culture growth and facilitating scalable EV collection. Potential cell culture EV isolation methods must address a number of primary metrics, including rapid separations, high throughput, pure and concentrated isolates, and high levels of reproducibility. Described here is a hydrophobic interaction chromatography (HIC) method using polyester (PET) capillary-channeled polymer (C-CP) fiber chromatography columns for the separation of EVs from HEK293 suspension cell culture supernatant. This method rapidly (<15 min) isolates EVs, enabling in-process concentration determination and subsequent collection for downstream characterization. The present protocol is applied to three parallel HEK293 cell cultures, monitoring the EV concentration as it relates to viable cell density (VCD) and cell viability. The efficacy of the HIC/C-CP fiber column for EV isolations is verified through multiple techniques, including a comparison of UV absorbance-based quantification, light scattering and immunofluorescence detection with nano-flow cytometry, and vesicle morphology confirmation with transmission electron microscopy (TEM). • EVs were isolated from HEK293 cell supernatant in <15 min per sample. • EV monitoring provides a powerful cell culture growth metric. • High concentrations of EVs were isolated, with a maximum of 1.48 × 10 11 EVs mL −1 . • Total EV concentration continues to increase during cell culture stationary phase. • EVs were characterized using TEM, UV scattering, and immuno-fluorescence.