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 viva-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 O-2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O-2 for 3d. Then, CaO2-containing PDMS was used to fabricate O-2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O-2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O-2 generating microwells maintained O-2 at constant levels. The HepG2 spheroids prepared using the O-2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O-2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O-2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (Le., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O-2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Automated summary

The study developed O-2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells, maintaining oxygen levels and improving cell viability in multicellular spheroids. This technique proved to be effective in preparing multicellular spheroids with high cell viability.