Introduction: Construction of three-dimensional cellular aggregates in vitro is one of the most important issues for tissue engineering. Spherical cell aggregates known as spheroids have attracted much attention to build large cellular constructs. However, the most critical problem in the spheroid culture is a depletion of oxygen and cell necrosis in the core of large spheroids. In this study, we developed novel spheroid culture chip (Oxy chip) which enables direct oxygen supply to the cells because the chip is made with only gas-permeable polymer[1]. The aim of the present study was to investigate the influence of continuous oxygenation on 3D growth behavior, viability, functionality, differentiation, and hypoxia in the core of the spheroid from hepatocyte cells or mesenchymal stem cells using the Oxy chip, compared to both conventional non-gaspermeable chips and monolayer cultures.
Materials and Methods: We constructed spheroid culture chip to supply oxygen to the cells. The culture chip was fabricated using only polydimethylsiloxane. The Oxy chip and were designed to be comprised of multicavities (512 wells, 1.0 mm in diameter) in a triangular arrangement on a 25 x 25 mm section of the cell culture area. To test the ability of the Oxy chip, highly proliferative human hepatoma HepG2 cells were used because the cells exhibit a highly metabolic nature, high oxygen demand, and express some liver-specific functions HepG2 cells were inoculated into the culture chips. Furthermore, Mouse mesenchymal stem cell strain D1 cells were used to evaluate the effect of continuous oxygenation on osteoblastic differentiation of cells.
Results and Discussion: Both cells spontaneously dropped into the cavities, and a single spheroid was formed in each cavity within several hours. We found that the Oxy chip offered a high yield production of uniformly sized spheroids. The use of our chip dramatically prevented hypoxia and subsequent central necrosis of HepG2 cells, and enhanced the maintenance of metabolic functions compared to those in the conventional chip and monolayer culture. Moreover, we found that the chip promotes osteoblastic differentiation of D1 cells due to the formation of three-dimensional aggregates and oxygenation to the cells.
Conclusion: The Oxy chip dramatically prevented hypoxia in the core of the spheroids and subsequent central necrosis. This chip has advantages in the cell proliferation, viability, cellular function, and differentiation in comparison with the conventional non-oxygen-permeable chip and monolayer culture. The chip is useful for engineering 3D cellular constructs with high viability and functionality for tissue engineering and drug screening.
References:
[1] Takahisa Anada, Junji Fukuda, Yuko Sai, Osamu Suzuki, Biomaterials 33 (2012) 8430-8441