Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in 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 leads to accelerated cell death. In this study, we developed O 2 -generating microwells by incorporating calcium peroxide (CaO 2 ) into polydimethylsiloxane (PDMS)-based microwells. The CaO 2 -containing PDMS was shown to generate O 2 for 3 d. Then, CaO 2 -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 2generating 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 (i.e., 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.

3D Stem Cell Spheroids with 2D Hetero‐Nanostructures for In Vivo Osteogenic and Immunologic Modulated Bone Repair

“…The first problem is the delivery of adequate oxygen and nutrients to cells embedded in the middle of the spheroids. [20,21] When forming the 3D spheroids, the cells adhere to each other, and the cells that are trapped in the interior part of the spheroids may experience limited access to essential oxygen and nutrients due to diffusion limitations. As spheroids experience longer culture time and grow larger, the cells in the inner core become even more distant from the external medium or blood, leading to inadequate diffusion of oxygen and nutrients.…”

“…With sufficient oxygen and nutrient supply to all cells within the spheroids, there may be more optimal cell growth and differentiation, thus facilitating new tissue formation. [20,21] The second problem is the nonspecific differentiation of stem cells after transplantation. [22] After implantation, the stem cells were immersed in a surrounding environment full of a large variety of bio-factors, which may result in nonspecific and uncontrolled differentiation.…”

Controlled Release of Oxygen from Calcium Peroxide in a Weak Acidic Condition by Stabilized Amorphous Calcium Carbonate Nanocoating for Biomedical Applications

“…In the tissue engineering field, cell death due to an oxygen shortage is still a major issue for the construction and transplantation of three-dimensional (3D) tissues. − To address this problem, oxygen-releasing materials have attracted much attention in recent years. − Calcium peroxide (CaO 2 ) is one of the most common oxygen sources for these types of materials because it generates oxygen, hydrogen peroxide, and calcium hydroxide (Ca­(OH) 2 ) by the reaction with water . Many oxygen-releasing materials have been reported by incorporating CaO 2 in various substrates such as hydrogels − and polymer materials − in a variety of structures.…”

Hydroxyapatite Nanocoating on Calcium Peroxide Microparticles for Sustained Oxygen Release

“…1,2 Moreover, in vitro construction of thick three-dimensional (3D) tissues is still a major challenge because limited diffusion of oxygen inside these tissues causes cell death. 3,4 Solving this oxygen shortage problem is therefore considered important for the development of regenerative medicine and tissue engineering.…”

“…Regenerative medicine is a new medical technique for recovering tissue function by transplanting cells and tissues to the disease sites. However, before the angiogenesis to the transplanted tissues, insufficient oxygen supply results in cell death, preventing efficient tissue regeneration in vivo . , Moreover, in vitro construction of thick three-dimensional (3D) tissues is still a major challenge because limited diffusion of oxygen inside these tissues causes cell death. , Solving this oxygen shortage problem is therefore considered important for the development of regenerative medicine and tissue engineering.…”