Co-Culturing Cells and Microcapsules in PDMS Microwell

To facilitate the observation of cellular distribution, Celltracker™ Green CMFDA and Red CMTPX (Invitrogen, c34552) were used to stain A549 cells and HUVECs, respectively, following the manufacturer's instructions. Before seeding, A549, HUVECs, and 3T3 cells were digested and resuspended to a cell density of 5 × 10⁵ cells/mL. The concentration of microcapsules was 5000/mL, and A549, HUVECs, and 3T3 cells were co-cultured in the ratio of 9:9:2 in a PDMS-based microwell device following previously established protocols [38 ,39 (link)]. Honeycomb microwell arrays of diameter 326 µm were prepared by casting PDMS on silicon wafers. The resulting PDMS membrane was peeled off, treated with hydrophobic agents, cut, and fixed in a 24-well culture plate for cell inoculation. The cells and microcapsules were cultured for 2 days before conducting the follow-up experiments.