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

Abstract Extracellular vesicles (EVs) are lipid-delineated nanoparticles that are produced by most cell types. EVs contain complex molecular cargoes that can have useful therapeutic or vaccine immunological effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro , that can co-localise and integrate with exogenously added EVs. To advance this type of cell-free EV engineering we manufactured, isolated and characterised HEK293 cell EVs. These EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. In our most optimal conditions, up to 4.83 ×10 11 /ml of HEK293 EVs were successfully cell-free engineered with a fusion membrane protein incorporating CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3) and monomeric green lantern (mGL). Finally, we also demonstrated that nano flow cytometry is a powerful tool for assessing cell-free EV engineering efficiency. In the future cell-free EV engineering could help accelerate future EV discoveries and the development of EV translational applications. Graphical abstract Technology readiness Cell-free systems are a well-established part of the engineering biology toolkit. Indeed, several applications are already at TRL9 stage, including commercially available cell-free protein synthesis kits, drug screening assays, field-tested medical biosensors, as well as therapeutic antibody manufacturing that has now reached cell-free reaction volume scales of up to 4500 L. However, cell-free extracellular vesicle (EV) engineering is currently at TRL3 / 4 stage. Whilst our study helps to further advance cell-free EV engineering, key challenges remain. To accelerate technology readiness, improvements in scalable EV isolation methods, increased eukaryotic cell-free protein synthesis yields, and assay automation will likely speed up cell-free EV engineering workflows. Furthermore, artificial intelligence-guided design workflows could be used to rapidly create community accessible libraries of EV membrane protein scaffolds, therapeutic, cell targeting and other modular elements for use in EV engineering studies. Finally, the development of EV potency assays for functional assessment of cell-free engineered EVs are needed to progress prototyped designs towards clinical translation. If these challenges are addressed, we envision that cell-free EV engineering will become an important approach in EV biological research and clinical applications. Highlights Cell-free extracellular vesicle (EV) engineering could be utilised to rapidly prototype and test novel biotechnological, vaccine, or therapeutic EVs for foundational or translational applications. Our data highlight some of the impacts that hollow fibre-based cell culture, EV isolation methods and different cell-free engineering approaches have on cell-free EV engineering workflows. We demonstrate several characterisation assays and technologies, including nanoflow cytometry, that can be used to assess cell-free EV engineering efficiency.