## 4 Discussion  
In the physiological state, the DPCs do not proliferate [9; 29]. At the same time, they are highly functional, so they can interact with epithelial cells, which are mainly composed of matrix cells [29]. DPC is a permanent part of the hair follicle [20; 30]. DPCs do not go into apoptosis so that they can re-enter the hair cycle though in the anagen-catagen transition there is a decrease in the number of cells in the DP, with migration to the DS [8; 12]. Different intrinsic and extrinsic factors keep some of DPCs in dormancy, which helps to preserve stemness of these cells. Upon activation, the DPC exists from dormancy and/or quiescence to enter new cycle [31; 32; 33; 34]. Compared with the in vivo state, once DPCs are cultivated, they are highly proliferating [35]. However, many studies have reported that DPCs lose their trichogenetic ability as the number of passages increases [20; 35; 36]. Many hair biologists  
Figure 5: The gene expression level of dermal papilla alginate spheres by qPCR. The human dermal papilla cell marker genes expressed in different passage state and alginate spheres, cultivate in different days. Note. P1, P3, P10: passage state 1, 3 and 10 respectively. D10, D50, D80: post-spherical formation day 10, 50 and 80 respectively. have attempted to find a way to maintain the trichogenicity and the functional properties of DPCs after cultivation. To recover the trichogenicity of human DPCs, we created alginate spheres containing human DPCs. To apply them in cell therapy for androgenetic alopecia, we investigated their cellular proliferation over the short-term and long-term. We concluded that the alginate spherical formation can regulate the DPCs so that they do not proliferate, as in physiological in vivo conditions. However, unlike in in vivo conditions, in 3D sphere formation, the number of cells is not maintained but decreases. Those short-term cellular number decreasing might be due to the condition of making alginate sphere such as voltage, 1% of CaCl\({}_{2}\), or lysis caused by washing procedures of distilled water. However, its decreasing rate is consistent even after day 30 in alginate sphere, necessitating other reasons for explanation. If there is little secretory factor from the epithelial component, there is not sufficient proliferative potential in dermal papilla [20; 37; 38]. We recently reported that factors from the epithelial cells could induce and maintain DPCs' trichogenicity as well as modulate the regulation of DPC gene expression [39]. In further study, it would be worth to investigate the interactional effect of the secretory factors of epithelial cellular to the alginate sphere dormancy of dermal papilla cells. Recently, our group has developed a method to recover trichogenicity of epithelial cells in 3D cultivation using a biodegradable scaffold [24; 40]. We used sponge-type, fiber sheet and newborn epithelial skin scaffold (NESS) to restore the hair follicle formation properties of the cells. That study mainly focused on the epithelial compartment, not the mesenchymal compartment, although we investigated the nature of the epithelial-mesenchymal interaction. On the other hand, this study was carried out using DPCs. We investigated the effect of the cellular density as well as the size of the alginate sphere containing human DPCs. Intriguingly, though the number of live cells decreased, it was stabilized after 70 days of cultivation. We found that the cellular convergence number per alginate volume, the optimal cellular density in alginate, was not dependent on the number of cells we input but on the volume of the alginate sphere. Moreover, the average number of cells divided by the volume was similar after 70 days of cultivation, range between 2235.05 cells/mm\({}^{3}\) and 2628.80 cells/mm\({}^{3}\). We concluded that the remaining cells had to occupy a certain volumetric space or optimal density to maintain their properties.