A Scalable Platform for Producing and Surface-Engineering Extracellular Vesicles with Enhanced Reparative Properties

Introduction

Extracellular vesicles (EVs) are increasingly recognized as bioactive components of cellular origin with potential utility across a range of military‑relevant injuries. However, translation of EV‑based approaches is limited by challenges in scalable manufacturing, cargo consistency, and the ability to enhance tissue targeting. Here, we describe a scalable fixed‑bed bioreactor platform for producing EVs from hTERT‑immortalized mesenchymal stromal cells (MSCs), coupled with post‑isolation surface engineering using the CD47 “don’t eat me” signal to modulate EV persistence and uptake.

Methods

Human MSCs were cultured in a fixed-bed bioreactor and EVs were isolated by Tangential Flow Filtration (TFF). EVs were characterized by nanoparticle tracking analysis, BCA, western blot, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multiplex immunoassays to assess yield, size distribution, and protein cargo. Comparative analyses were performed against EVs produced under conventional static culture conditions. For surface engineering, EVs were modified with CD47 via click chemistry. Functional evaluation was conducted in vitro using physiologically relevant human cell types, with assay endpoints including cell viability, inflammatory signaling, and expression of stress‑associated proteins.

Results

High yields of EVs (≥ 1.0 × 1010 EVs/mL) were recovered and EVs met all specifications for size distribution, protein expression, and sterility. Bioreactor‑derived EVs exhibited enhanced expression of proteins associated with immunomodulatory and reparative signaling compared to static culture EVs. CD47 surface modification significantly increased EV uptake and persistence across multiple human cell types in vitro and resulted in greater attenuation of inflammation‑ and stress‑associated markers relative to unmodified EVs.

Discussion

These data demonstrate a scalable platform for the manufacture and engineering of hTERT MSC‑derived EVs. Integration of fixed‑bed bioreactor production with surface protein modification enables generation of EVs with enhanced cargo and tissue‑targeting properties. This approach supports the development of standardized, EV-based products for pre-clinical research and provides a foundation for future evaluation of EV‑based strategies addressing diverse forms of military‑associated tissue injury.