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2022, Vol. 26 ›› Issue (4): 553-559Method of constructing cell spheroids based on agarose and polyacrylic moldsHe Yunying1, Li Lingjie2, 3, 4, Zhang Shuqi2, Li Yuzhou2, 3, 4, Yang Sheng2, 3, 4, Ji Ping2, 3, 4
Abstract: BACKGROUND: The application prospect of cell spheroids is wide in tissue engineering, while the experimental cost of using commercial ultra-low adsorption culture plates to form spheroids is relatively high. Therefore, it is necessary to explore an alternative strategy for cell spheroids formation to promote the application of cell spheroids in related research fields. OBJECTIVE: Combined with agarose and polyacrylic mold, three-dimensional cell spheroids are established by layer-by-layer self-assembly technology, and so that the micro physiological environment of cells are partially restored. METHODS: In vitro, MC3T3-E1 cells of mice were coated with gelatin and sodium alginate respectively, through layer-by-layer self-assembly technology (LBL-MC3T3-E1). Gelatin-coated and sodium alginate-coated MC3T3-E1 cells were prepared as well. Agarose-coated plates and agarose microwell plates were prepared respectively. The cellular spheroids formation effect of the above treated cells in these culture plates was observed. The Live/Dead staining method was used to detect the viability of cell spheroids. The alkaline phosphatase staining and real-time PCR were used to confirm the osteogenesis of cell spheroids. RESULTS AND CONCLUSION: (1) As seen under the light microscope, in agarose-coated well plates, both untreated and treated MC3T3-E1 cells did not form into spheroids; and in agarose microwell plates, untreated and LBL-MC3T3-E1 cells formed ideal cell spheroids, in which the diameter of LBL-MC3T3-E1 cell spheroids was larger than that of untreated MC3T3-E1 cell spheroids (P < 0.05). (2) The results of Live/Dead staining showed that the cellular viability within LBL-MC3T3-E1 cell spheroids was better than that of MC3T3-E1 cell spheroids (P < 0.05). (3) The alkaline phosphatase activity of LBL-MC3T3-E1 cell spheroids was higher than that of MC3T3-E1 cell spheroids (P < 0.05). Collagen-I and Osterix gene expression levels were higher than those of MC3T3-E1 cell spheroids (P < 0.05), and the difference in Runt-related transcription factor 2 gene expression was not significant between the two groups (P > 0.05). (4) These results showed that a three-dimensional culture model of cell spheroids was successfully established by using agarose and polyacrylic acid molds combined with layer-by-layer self-assembly technology. This method is convenient, economical, efficient, and has good cell viability, preserves the osteogenic differentiation ability of cells, thus having potential applications in the field of bone tissue engineering and regenerative medicine. Key words:cell spheroid, three-dimensional culture, agarose, MC3T3-E1, layer-by-layer, cell viability, bone tissue engineering |