Scalable Cell Culture For Production Of Small Extracellular Vesicles For Regenerative Medicine Applications

Abstract

Mesenchymal stromal cells (MSCs) have evidenced to be a promising option for cellular therapy but often fail to be accepted at the clinical trial phase. Fortunately, there has been ongoing interest into MSC secreted small EVs (sEVs) due to their ability to transport cargo around the body, whilst evading the immune system and permeating hard to breach barriers. The generation of abundant small EVs for analysis is most commonly achieved by growing cells in multiple planar flasks in static culture, which can be both resource and time consuming, with relatively low yield of EVs. Alternatively, bioreactor based culture platforms have been proven to increase MSC and EV yields and hence provide a solution for scaling up. However this needs to be more thoroughly investigated in relation to the effects agitated suspension culture has on sEV profile. The aim of this project was therefore to investigate the effects of bioprocess parameters on small EV production by MSCs, both in terms of their characterisation and functional capacity. In order to do so, bone marrow Mesenchymal Stem Cells (bmMSCs) were expanded on microcarriers (MCs) in stirred spinner flasks (SFs) for 10 days. bmMSCs were then harvested, characterised for multipotency and EVs then collected, concentrated using Tangential Flow Filtration (TFF) and isolated using Size Exclusion Chromatography (SEC). MSC-EVs populations were characterised using a number of classic and novel methodologies and cargos revealed using mass spectrometry. Potency was demonstrated through changes in T cell immunosuppression and in vitro proliferation and wound healing assays. Optimising culture parameters led to changes in MSC metabolism and expansion, whilst cell stemness and differentiation potential were maintained. Bioreactor cultures produced greater quantities of EVs in comparison to standard planar cultures, and SEC isolated MSC-sEVs demonstrated immonopriviledge and regenerative potential in culture. Importantly, we demonstrate consistent, reproducible harvesting of MSC-sEVs from multiple batches from the same donor. Therefore, this research describes a new, consistent and reliable bioprocessing pipeline for the manufacture of therapeutically functional MSC-EVs at scale. This study is the first to investigate the number, potency and cargos of MSC-EVS from multiple time points during cell expansion in the small scale stirred tank bioreactor as well as the consistency of MSC-sEV product from multiple batches and donors.