Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O₂-generating microwells by incorporating calcium peroxide (CaO₂) into polydimethylsiloxane (PDMS)-based microwells. The CaO₂-containing PDMS was shown to generate O₂ for 3d. Then, CaO₂-containing PDMS was used to fabricate O₂-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O₂ concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O₂generating microwells maintained O₂ at constant levels. The HepG2 spheroids prepared using the O₂-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O₂-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O₂-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16–BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O₂-generating microwells are useful for preparing multicellular spheroids with high cell viability.