(J-405) Effects of Surface Coating Composition on Extracellular Vesicle Secretion are Cell Specific

Introduction: Over the past decade, research on the therapeutic applications of cell-secreted extracellular vesicles (EVs) has rapidly advanced, including several ongoing clinical trials. Differences in cell culture conditions, such as media composition, oxygen tension, cell density, 2D vs. 3D culture systems, and static vs. dynamic culture, have been demonstrated to significantly impact EV yield, content, and therapeutic potential. While culture substrate mechanical properties have recently been shown to modify EV secretion and cargo content, the effects of substrate composition on EV production remain underexplored. This project aims to study the impact of extracellular matrix (ECM) protein and ECM-mimetic polymer coatings on EV secretion and therapeutic potential in primary mesenchymal stromal cells (MSCs) and several common cell lines.

Materials and Methods: Surface coatings composed of collagen I (COLI), fibronectin (FN) and poly-l-lysine (PLL) were formed in tissue culture plates at a concentration of 5 g/cm2 and seeded with either HEK293 cells, NIH3T3 cells, RAW264.7 cells, primary bone marrow-derived mesenchymal stromal cells (BMSCs), or primary adipose-derived mesenchymal stromal cells (ASCs). Uncoated tissue culture plastic (TCP) was used as a control. EVs secreted from each cell type on each coating were isolated from the supernatant via ultracentrifugation and characterized using Nanoparticle Tracking Analysis. EV protein content was measured via BCA kit. Cell viability on each coating was measured via Live/Dead staining. Cell morphology on each coating was examined via phalloidin staining and fluorescence microscopy. Expression levels of EV-biogenesis related genes (Alix, HRS, TSG101, SMPD2/3, Rab27a/b, CD63) and cellular adhesion genes (ITGA2, ITGA5, ITGAV, ITGB1, ITGB3, CTGF) on each coating type were also analyzed. Analysis of the effects of EVs on the proliferation of primary bone marrow-derived macrophages is currently being performed.

Results, Conclusions, and Discussions: Preliminary data indicate that, while coating type had little effect on total EV yield in HEK293 cells and BMSCs, NIH3T3 cells produced significantly more EVs on FN coatings (p = 0.0029; n = 5), ASCs produced significantly more EVs on PLL coatings (p < 0.0001; n = 5), and RAW264.7 cells produced significantly less EVs on COLI coatings (p < 0.0001; n = 5), than on TCP controls. RAW264.7 cells on COLI generated EVs with significantly larger mode size than FN (p < 0.05; n = 5). Coatings did not appear to have significant effects on cell viability in most cell types, however, COLI coatings significantly decreased the number of viable RAW264.7 cells (p < 0.05; n = 3). Notable reorganization of actin structures can be observed in COLI and FN for NIH3T3 cells, and all coatings for ASCs. Gene expression analysis of NIH3T3s did not show a clear relationship between EV yield and expression of EV biogenesis related genes or cell adhesion genes (n =3). Most biogenesis gene expression changes were found on PLL at 24 h and COLI at day 4, which did not significantly change EV yield. NIH3T3 expression of cell adhesion genes changed more significantly at 24 h than at day 4. While COLI stimulated the strongest elevation in EV biogenesis gene expression in RAW264.7 cells at 24 h, it also yielded the lowest biogenesis gene expression at day 4 (n =3). Interestingly, RAW264.7 cells also showed significantly lower adhesion gene expression levels on COLI coatings at day 4, which correlated with the decreased number of viable RAW264.7 cells and lowest EV yield among all conditions. More gene expression analysis is currently underway. Future studies will examine whether EVs secreted from cells grown on different coatings show differences in in vitro functional assays.