## 4 Discussion  
The biological properties of MSC spheroids, emblematic of 3D cell aggregates, have been well delineated in comparison to conventional 2D MSC culture. Herein, this present study investigated the attributes of C-MSCs, an alternative 3D cell aggregate that we have previously developed [18], contrasting them with the 3D MSC spheroids. We revealed that whereas MSC spheroids are dense cell clusters constructed primarily from Cadherin-mediated cell-cell contacts, C-MSCs are cell aggregates based on the ECM component COL1, allowing them to form larger aggregates than MSC spheroids. This study represents the first attempt to simultaneously create and compare MSC spheroids and C-MSCs across a range of cell numbers, providing novel insights into their structural and functional differences. More specifically, this approach allowed us to hypothesize that the abundance of ECM in C-MSCs contributes to their reduced cell death compared to MSC spheroids as discussed below. Anyway, this simultaneous comparison led to the discovery of N-Cadherin-expressing cells within C-MSCs, though its expression level was obviously lower than that of MSC spheroids. Importantly, our transcriptome analysis comparing these 3D cell aggregates with distinct structures demonstrated that C-MSCs have enhanced capacities for producing immunomodulatory and cytoprotective factors, a hallmark biological feature of MSCs. These findings contribute significantly to the understanding of MSCs in 3D cultures and address the gap identified in previous research. Previous research has established that, compared to 2D-MSCs, MSC spheroids cultured in a 3D suspension have a heightened production of immunomodulatory and cytoprotective factors [14; 26; 27]. Intriguingly, our study found that C-MSCs, also cultured in three dimensions, further augmented this paracrine property than MSC spheroids (Figure 4), suggesting that the other factors, beyond simply being cultured in 3D, may contribute to this elevation. One potential candidate for this enhancement is the abundant ECM in C-MSCs. Given that stromal cells inherently function within an ECM environment in vivo [28; 29], it is plausible that C-MSCs, being rich in ECM components, could more readily manifest MSCs' paracrine effects. Supporting this hypothesis, [PERSON] et al. reported that ECM conditions affected the inflammatory response in MSCs through NF-kB signaling in response to TNF stimulation [30]. Further studies utilizing C-MSCs, which explore protein production levels and include functional experiments such as T cell/macrophage co-culture systems or in vivo transplantation studies using an immune-disorder animal model, may provide new insights into the mechanisms underlying paracrine effects driven by ECM-cell interactions. As illustrated in Figures 1 and 2, C-MSCs were enlarged more than MSC spheroids, maintaining their 3D structure without cell death even at diameters around 1 mm. In spheroid culture, it is well reported that enlargement beyond approximately 500 \(\upmu\)m is challenging, often attributed to dense cell aggregation leading to internal nutrient and oxygen deprivation [25]. Given the abundant ECM within C-MSCs and their tissue-like structure with a lower cell density than spheroids, it is conceivable that they facilitate gas and fluid exchange. This ECM-rich configuration might be the key to the fabrication of larger 3D cell aggregates. Conversely, the MSC spheroids produced in this study using the hanging drop method were, consistent with previous reports, predominantly formed through Cadherin-mediated cell-cell contacts. Notably, cell-cell interactions facilitated by N-Cadherin are reported to play a role in the osteogenic, chondrogenic, or myogenic differentiation of MSCs [31; 32; 33], suggesting possible differences in bone induction capabilities between MSC spheroids and C-MSCs. Although no differences were observed in the expression of trilineage differentiation master genes under standard culture conditions (Figure 4B), distinctions might emerge under induction mediums.