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

Abstract

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 and densely packed cells in the spheroids limit oxygen supply to the core region of the spheroids, which 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 react with water and generate O2 for 3 days. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. HepG2 spheroids were then prepared using either conventional PDMS microwells or O2-generating microwells. Using the conventional PDMS microwells, the O2 concentration in the culture medium reduced to ~67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells were greater in size and 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 in the core region of the spheroids. 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 functional activity.