Oil-sealed RGD-modified alginate hydrogel microwell array for analysis of single-cell-derived extracellular vesicles...

We propose an oil-sealed, arginine-glycine-aspartic (RGD) -modified alginate hydrogel microwell array for the analysis of single-cell-derived extracellular vesicles and particles (EVPs) secreted by adherent cells cultured in enclosed spaces. Taking advantage of the mesh structure of alginate hydrogel, we developed a microwell array with size-selective permeability that allows nutrients to pass through the hydrogel while preventing EVPs from doing so. Continuous single-cell culture in sealed microwells (>19 days) has been achieved, while retaining the EVPs inside the microwells. The single-cell-derived EVPs were collected from sealed microwells using a glass capillary to analyze surface membrane proteins. We believe that our oil-sealed RGD-modified alginate hydrogel microwell array will contribute to revealing the heterogeneity of cells, thereby advancing our understanding of the mechanisms of various diseases.