Rapid fabrication of cell spheroids by using MEMS chamber

Objectives: One of the effective uses of cell spheroids in the biomedical field is to engineer biological tissues. However, when the cell spheroid is excessively large, the lack of nutrients and oxygen causes necrosis of cells in the internal region of the spheroid. In addition, to obtain reproducible results, the number of cells in a spheroid should remain constant. Therefore, the aim of this study was to establish a simple method for fabricating spheroids, using which it would be possible to control the overall size of a spheroid and the number of cells contained in it. Methods: We used microchambers fabricated by a microelectromechanical systems (MEMS) technique. Mouse bone marrow stromal cells (BMSCs) were seeded in quadrangular microchambers (small, 100×100µm; large, 200×200µm), and were cultured in alpha-MEM containing 20% FBS. Spheroids formed in the chamber were collected and observed under an optical microscope. Spheroid sizes and numbers were measured using image analysis software (Image J and Lumina Vision). Results: There were 10000 small and 2401 large microchambers in each 35-mm tissue culture dish. When 1×106 cells were seeded in the dish, the microchambers contained 11±3.4 cells per small chamber and 124±7.3 cells per large chamber. The size of the spheroids increased from 50±12µm (estimated diameter) at 10 h to 61±16µm at 12h. The spheroid number also increased from 7900 at 10h to 9800 at 12h. The spheroid size obtained from the large chamber showed significantly large compared to the spheroid obtained from small chamber at 10h (p<0.05). Conclusion: Spheroids are obtained in a simple manner in the microchambers fabricated by the MEMS technique. The spheroid size and the cell number per spheroid were regulated by varying the cell culturing conditions.