High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Abstract

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function can be mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include priming MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/567730v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1fca79forg.highwire.dtl.DTLVardef@60f543org.highwire.dtl.DTLVardef@186394borg.highwire.dtl.DTLVardef@11b94bd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMSCs morphologically respond to inflammatory priming conditions. C_LIO_LIPriming hits identified from morphological screen increase MSC-EV production. C_LIO_LIPriming MSCs in bioreactors enhances MSC-EV modulation of microglia. C_LIO_LIChanges in MSC-EV production and potency reflected by lipid content. C_LIO_LIFirst demonstration of effects of priming on MSC production of EVs in a bioreactor. C_LI