## 4 Discussion
This mild condition required to form these gels as well as its high biocompatibility are some reasons for its superiority as a material for cell capsulation. Further development of cell encapsulation using alignates that can be applied to cell-based therapies is expected. However, calcium chloride, which has been commonly used as a crosslinker, forms alginate gel very rapidly, leading to an increased non-uniformity, permeability, and instability of the structure [34]. Therefore, the preparation protocol of these techniques should be optimized. During the alginate capsule preparation in this study, differences in the concentrations of each solution significantly impacted the morphology and strength of the prepared alginate capsules (Figure 1). On altering the concentration of the alginate solution and maintaining a fixed concentration of the calcium chloride solution, the capsules failed to retain their shape with decreasing the concentration of the alginate solution. However, on altering the concentration of the calcium chloride solution while maintaining the concentration of the alginate solution, the changes in their morphology were relatively small. Accordingly, the concentration of the alginate solution had a greater impact on the physical properties of the capsules than the concentration of the calcium chloride solution during the preparation of the alginate capsules. These results were in good agreement with previously reported results [36]. Data regarding the effect of capsule size on the viability of encapsulated cells remain limited. Herein, we demonstrated that the viability of cells encapsulated in large alginate capsules (50 uL) decreased in a long-term culture (Figure 2b). When the capsules become larger, the efficiency of substance exchange across the capsule decreases [37], suggesting that the viability of cells in the center of the capsules may decrease. Herein, the viability of the cells encapsulated in small alginate capsules (2.5 uL) was reduced, which could be attributed to the infiltration of the calcium chloride solution into the capsule during immersion, thus damaging the encapsulated cells. The cell damage can be explained by the cytotoxic effect of the 10 mM calcium chloride solution toward the encapsulated cells owing to its high ion concentration. Given that the relative light units decreased depending on the immersion time, the reaction time of calcium chloride with sodium alginate on the capsule surface was critical for the cell viability within the capsule. The cell density in alginate capsules has been shown to markedly impact cell viability [38]. Consistently, we revealed the effect of the cell density on cell viability in the capsules (Figure 4). The air exchange, liquid exchange, and nutrients required for cells in alginate capsules increase at a high cell density. In contrast, the surface areas of the capsules remain constant. Therefore, it is possible that cells in the center of the capsule experience a shortage of required substances and may die during long-term culture. Based on these results, we optimized the protocol for alginate capsule preparation with respect to the hardness, size, and cell density of the alginate capsule and the immersion time in calcium chloride solution. To evaluate the usefulness of the optimized preparation protocol for alginate capsules, we transplanted alginate capsules containing C3H10T1/2/Nluc or MIN6 cells into mice and demonstrated the long-term survival of the cells and a high cellular function post-transplantation (Figure 5). However, 15 capsules encapsulating MIN6 cells were transplanted for the evaluation of hypoglycemic effects, suggesting that a large number of capsules are required to exert therapeutic efficacy in clinical studies. Further investigations regarding the number of capsules for transplantation are needed for clinical studies. Considering these results, an optimal preparation protocol for alginate capsules is mandatory for effective cell-based therapy using encapsulated cells.