Hollow-fibre biomanufacturing and cell-free engineering of HEK293 extracellular vesicles

Extracellular vesicles (EVs) are lipid-delimited particles produced by most cell types that can have therapeutic effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro, which can integrate with exogenously added EVs. To advance this type of cell-free EV engineering, we established an end-to-end cell-free EV engineering workflow. Firstly, human embryonic kidney (HEK293) cells were cultured within a hollow-fibre bioreactor to generate several batches of HEK293 EVs. Subsequently, these EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. Nano-flow cytometry analyses revealed that, under optimal conditions, up to 4.83 × 10¹¹ /ml of HEK293 EVs were successfully cell-free engineered to incorporate CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3)-monomeric green lantern membrane fusion proteins. Finally, we demonstrated that cell-free engineered EVs can be functionally assessed using nano-flow cytometry and cell-based assays. We envision that, in the future, cell-free EV engineering workflows could be used to help accelerate future EV discoveries and advancements in EV therapeutics.