We investigated the efficacy of three-dimensional porous ferric-ion-cross-linked alginate (Fe-alginate) gels as cell scaffolds, in comparison with calcium-ion-cross-linked alginate (Ca-alginate) gels. In a previous study, we had demonstrated that two-dimensional Fe-alginate film was an efficient material for use as a scaffold, allowing good cell adhesion and proliferation, unlike Ca-alginate film. In the present study, we fabricated three-dimensional porous Fe- and Ca-alginate gels by freeze-drying and evaluated their effects on cultured cells. The Fe-alginate gels showed higher protein adsorption ability than Ca-alginate gels. Cells formed multicellular spheroids in both types of alginate scaffold, but the number of cultured cells increased with culture time on Fe-alginate porous gels, whereas those on Ca-alginate gels did not. Moreover, it was revealed that the cells on Fe-alginate scaffolds were still viable inside the multicellular spheroids even after cultivation for 14 days. These results suggest that Fe-alginate provides a superior porous scaffold suitable for three-dimensional culture of cells. Our findings may be useful for extending the application of Fe-alginate to diverse biomedical fields. 1. Introduction For both research and therapeutic applications, fabrication of effective cell culture substrates is desirable. A suitable three-dimensional environment for cells is considered to be an important factor for in vitro cell cultivation, since cells within living organisms exist under such conditions. Two-dimensional cell culture is frequently used because it is convenient and manageable for the maintenance of cells and also for biological research. However, it has been reported that various cells lose their functions when cultured as a monolayer under two-dimensional conditions [1–5]. Thus, two-dimensional culture is considered to be an unnatural condition for many cell types. In order to overcome the shortcomings of two-dimensional culture, three-dimensional culture systems, such as multicellular spheroids, cellular multilayers, and matrix-embedded culture, have been devised [6]. Therefore, studies focusing on the optimal three-dimensional culture environment for cells have important implications. In a previous study, we produced two-dimensional ferric-ion-cross-linked alginate (Fe-alginate) films and demonstrated that they supported good cell adhesion and proliferation [7]. Alginates are composed of 1,4-linked -D-mannuronic acid (M) and -L-guluronic acid (G) residues, forming gels with certain multivalent metal ions [8, 9]. By exploiting
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