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bioRxiv · 10.1101/2024.03.07.584007

Scalable Isolation of Surface-Engineered Extracellular Vesicles and Separation of Free Proteins via Tangential Flow Filtration and Size Exclusion Chromatography (TFF-SEC)

Kawai-Harada, Y.·Nimmagadda, V.·Harada, M.
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Abstract

BackgroundExtracellular vesicles (EVs) represent small lipid bilayer structures pivotal in mediating intercellular communication via biomolecular transfer. Their inherent characteristics, including packaging, non-immunogenicity, and biofluid stability, position EVs as promising drug delivery vectors. However, developing clinical quality EVs requires multifaceted technological advancement. MethodsIn this study, a method is introduced for engineering extracellular vesicles (eEVs) from cultured cells and their subsequent isolation using lab-scale tangential flow filtration (TFF). This is the first study to evaluate DNA loading efficacy into EVs isolated by TFF, marking a significant milestone in the field of targeted drug delivery. Initially, cells are transfected with EV-display constructs to facilitate the secretion of eEVs bearing the desired coding molecules. Following brief centrifugation, the cell culture media undergoes filtration using hollow fiber filters. TFF, by applying a constant flow, effectively segregates molecules based on designated molecular weight cut-off (MWCO), enriching particles between 50 nm and 650 nm. ResultsCompared to conventional methods like ultracentrifugation, TFF demonstrates higher efficiency in removing undesired molecules/aggregates while exerting less stress on EVs. Characterization of eEVs through various assays confirms TFFs superiority in isolating pure EV populations. Additionally, the necessity of size-exclusion chromatography (SEC) after tangential flow filtration (TFF) becomes evident for effectively removing unbound protein contaminants. ConclusionIn conclusion, TFF-SEC emerges as a scalable and superior approach for eEV isolation, promising significant advancements in clinical applications.

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Kawai-Harada, Y., Nimmagadda, V., Harada, M.. 2024-03-12. Scalable Isolation of Surface-Engineered Extracellular Vesicles and Separation of Free Proteins via Tangential Flow Filtration and Size Exclusion Chromatography (TFF-SEC). https://doi.org/10.1101/2024.03.07.584007

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