## 3 Results  
MSC Spheroids Are Dense, Compact Cell Aggregates, Whereas C-MSC, That Are Composed of Cells and Extracellular Matrices, Can Be Fabricated to a Larger Size  
The size and morphology of the cell aggregates such as MSC spheroids and C-MSCs can be tightly controlled by the number of cells composing them. Thus, we initially fabricated MSC spheroids and C-MSCs using varying cell numbers. MSC spheroids could be generated from 25,000, 50,000, and 100,000 cells forming spherical structures (Figure 1B, upper panels). However, when using 200,000 cells with the hanging drop method, spherical morphogenesis was compromised, resulting in a destabilized, membrane-like structure. In contrast, C-MSCs consistently formed spherical structures, even at elevated cell numbers of 200,000, in addition to 50,000 and 100,000 cells (Figure 1B, lower panels). To generate smaller C-MSCs with fewer than 50,000 cells, it would be necessary to generate cell sheets from a 384-well culture plate. However, this method was technically challenging, leading us to forgo this approach in our study. The size of the cell aggregates increased in a cell-number-dependent manner for both MSC spheroids and C-MSCs (Figure 1C). Interestingly, when fabricated from equivalent cell counts, C-MSCs exhibited a markedly larger diameter compared to MSC spheroids. The C-MSCs derived from 200,000 cells were conspicuously larger compared to other cell aggregates (Figure 1C). Given the variations in size and morphology between MSC spheroids and C-MSCs, it was speculated that C-MSCs might incorporate non-cellular elements, possibly matrix proteins, that contribute to their enlarged structure. Moreover, it is well reported that enlarged cell spheroids can experience central nutrient and oxygen deprivation, potentially leading to cellular apoptosis [25]. Accordingly, we next conducted a histological examination of MSC spheroids and C-MSCs. HE staining demonstrated that MSC spheroids generated from 25,000 or 50,000 cells exhibited a dense cellular organization (Figure 2A,C). However, the spheroids from 100,000 cells, although maintaining an external spherical appearance, exhibited disrupted internal architectures. This structural anomaly was accompanied by increased cell apoptosis in the 100,000-cell MSC spheroids, as demonstrated by TUNEL staining (Figure 2A,D). In contrast, all tested C-MSCs retained their 3D spherical integrity evidenced by HE staining. In addition to cellular components, non-cellular ECM elements were apparently observed within C-MSCs, substantiating their unique composition (Figure 2B). These observations are congruent with the semi-quantitative analysis of the cell numbers that the cellular density of C-MSCs was significantly lower than that of MSC spheroids (Figure 2C). Additionally, C-MSCs did not exhibit an augmented apoptotic cell number even at larger sizes, underscoring their structural and compositional stability (Figure 2B,D). These findings suggested that MSC spheroids, formed by densely aggregated cells, are stable when their size is small, whereas C-MSCs can be larger constructs consisting of both cells and the non-cellular ECM component. Accordingly, MSC spheroids derived from 25,000 cells and C-MSCs generated from 200,000 cells were selected for further analysis. This selection allows for a direct comparison under conditions where each type of 3D cellular construct exhibits its most stable and representative structural characteristics. MSC Spheroids Primarily Rely on Cell-Cell Contacts Mediated by N-Cadherin, Whereas C-MSCs Are Predominantly Structured by the Extracellular Matrix, COL1  
To investigate the key molecules that compose each cell aggregate, immunofluorescence staining for COL1, F-actin, and N-cadherin was conducted. The expression level of COL1, a primary ECM protein for stromal cells, was notably low in MSC spheroids (Figure 3A,C). Additionally, F-actin integrity appeared compromised in the inner cells of the spheroids, unlike the outer cells, suggesting limited matrices for cell attachment.