# Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study
## 4 Discussion
These results indicate that the initial reduction in diameter was due to cell aggregation and organization, but the subsequent reduction appears to be due to restricted nutrients and oxygen. Several models for the transport of nutrients, oxygen, and waste in spheroids have been verified [29; 30; 31]. 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 [14; 16; 30; 31]. Several studies on MSC spheroids, including [PERSON] et al. [15] who studied human MSCs and [PERSON] et al. [32] who studied rat MSCs, have reported that spheroids form within 1 day and their diameters decrease with time [22; 23; 28; 33; 34]. On the other hand, [PERSON] et al. reported that the diameter of human dental pulp stem cell spheroids cultured using a 6-well non-adhesive plate increase over time [21]. This discrepancy is due to the different culture methods used in the studies. [PERSON] et al. and [PERSON] et al. independently cultured MSCs in a small volume of media or used the scaffold method, whereas [PERSON] et al. [21] used a relatively large volume of media. It is possible that spheroid-spheroid interactions may increase the diameter of the spheroids, but further study is needed. Many previous studies on the morphology of spheroids have only made short-term observations of about 5-7 days [15; 22; 23; 28; 33; 34]. In 2016, [PERSON] et al. [35] used a pellet culture method to study the morphology of human MSC spheroids and found that the diameter of the spheroids decreased rapidly over a 14-day period, after which it remained constant for 2 months. In general, depending on the culture method, the morphology of the spheroids is maintained for 3-21 days and the internal structure is homogeneous and biologically stable for at least 1 month [35; 36]. In this study, the spheroids were observed for 20 days to confirm their long-term stability. In addition to the decrease in diameter, the surface integrity of the spheroids began to diminish on day 13 but the shape of the spheroids remained stable (Figure 1I-P). It has been found that 3D culture methods using low-adhesive plates have a lower long-term stability than other methods because of the difficulty limiting the size of the spheroids [36]. However, because Stemfit 3D can control the size of the spheroids, it shows improved stability over a longer period. Further research is needed to determine whether the internal structure and function of cells cultured using the Stemfit 3D system are equally stable in the long-term. CCK-8 and LIVE/DEAD assays were used to confirm the proliferation ability and viability of 3D-cultured PDLCs, which are frequently used methods in 2D culture experiments [37; 38; 39; 40]. The CCK-8 assay revealed that the absorbance in the 2D culture increased over time while in the 3D culture it decreased over time. This is probably due to the continued proliferation of cells in the 2D environment, while in the 3D environment, the differentiation rate decreased due to the inevitable restriction of nutrients and oxygen in the center of the spheroids (Figure 2) [14; 24; 25; 35]. In addition, in the 3D culture, the LIVE/DEAD assay results showed that the number of dead cells increased over time in the center of the spheroids (Figure 3). Similar results were seen in a previous study of human MSCs grown via pellet culture, where the spheroids maintained a stable internal structure for 1 month, however at 2 months, a necrotic area developed in the center of spheroid, together with a loss of proliferation, impaired structural stability, and decreased cell-to-cell contact [35]. Although quantitative evaluation of 2D- and 3D-culture methods is difficult [7], these results are significant because it supports the idea that the characteristics of the 3D-cultured cells are fundamentally different from the 2D-cultured cells. One of the benefits of 3D culture is it improves osteogenic differentiation compared to 2D culture.