## 4 Discussion
However, because Stemfit 3D(r) can control the size of the spheroids, it shows improved stability over a longer period. Because of its economy, its ease of use, and the stability of the spheroids, Stemfit 3D(r) is commonly used in stem cell cytology and studies of other types of cells [3,4]. The diameter of the sPDLSC spheroids in the 3D culture decreased (75.3%) over the first 5 days, and decreased more slowly thereafter. The initial reduction in diameter was caused by cell aggregation and organization, but the subsequent reduction appears to have been a result of restricted nutrients and oxygen. Several models for the transport of nutrients, oxygen, and waste in spheroids have been verified [8]. When the diffusion of oxygen is limited, avascular tissue forms, and inefficient transport results in the accumulation of metabolic waste and the formation of a necrotic core at the center of the spheroid [10,12,14]. Several studies on MSC spheroids, including ones by [PERSON] et al. [14], who investigated human MSCs, and by [PERSON] et al. [18], who evaluated rat MSCs, have reported that spheroids form within 1 day and that their diameters decrease over time. However, [PERSON] et al. [19] reported that the diameter of human dental pulp stem cell spheroids, cultured on a six-well non-adhesive plate, increased over time. This discrepancy is due to the different culture methods used. [PERSON] et al. [14] and [PERSON] et al. [18] independently cultured MSCs in a small volume of medium, or used the scaffold method, whereas [PERSON] et al. [19] used a relatively large volume of medium. It is possible that spheroid-spheroid interactions may increase the diameter of the spheroids, but further study is needed. CCK-8 and live/dead assays were used to confirm the proliferative ability and viability of 3D-cultured sPDLSCs; these are frequently used in 2D culture experiments [22,23]. The CCK-8 assay revealed that the absorbance of the 2D culture increased over time, and that of 3D culture tended to decrease over time. This was probably due to continued proliferation of cells in the 2D environment, whereas in the 3D environment the differentiation rate decreased as a result of restricted nutrients and oxygen in the center of the spheroids [20]. In addition, in the 3D culture, the number of dead cells in the center of the spheroids increased slightly over time. Similarly, in one study, spheroids of human MSCs in pallet culture maintained a stable internal structure for one month. However, a necrotic area developed in the center of the spheroids, with a loss of proliferation, impaired structural stability, and decreased cell-to-cell contact at two months [20]. Although quantitatively evaluating 2D and 3D culture methods is difficult [24], these results support the notion that the characteristics of 3D-cultured cells are fundamentally different from those of 2D-cultured cells. Three-dimensional culture improves osteogenic differentiation compared to 2D culture. In addition, the intercellular interactions of embryonic stem cells are enhanced in a 3D environment [25]. For example, progenitor cells derived from the salivary gland can differentiate into hepatocyte and pancreatic islet cell lineages only when cultured in a 3D environment [26]. For this reason, 3D culture has attracted attention in stem cell biology and oncology, as well as in dentistry [12]. For example, 3D-cultured dental pulp stem cells or gingiva/papilla-derived stem cells show higher ALP activity than those grown in 2D culture [3,6,7]. In addition, the expression of genes associated with bone formation, such as BMP2, RUNX2, and dentine sialophosphoprotein, is increased in 3D-cultured cells [19]. The expression of BMP2 was upregulated 112.8-fold in 3D culture compared to in 2D culture. However, compared to our previous studies, the expression of osteogenic differentiation factors, such as RUNX2 and BMP2, tended to be lower in sPDLSCs than in pPDLSCs.