In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

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Here we propose a novel electrochemical lithography methodology for fabricating calcium-alginate hydrogels having controlled shapes. We separated the chambers for Ca2+ production and gel formation with alginate with a semipermeable membrane. Ca2+ formed in the production chamber permeated through the membrane to fabricate a gel structure on the membrane in the gel formation chamber. When the calcium-alginate hydrogels were modified with collagen, HepG2 cells proliferated on the hydrogels. These results show that electrochemical hydrogel lithography is useful for cell culture.