## 4 Discussion
Furthermore, it is of note that the diverse MSC spheroid fabricating techniques improving the biological properties of the MSCs, such as forced aggregation technique, low-attachment surfaces, magnetic levitation, and a spinner flask bioreactor system, have been developed [27; 34]. Additional comparative analysis between C-MSCs and those varied MSC spheroid types that possess better biologicalproperties than the conventional spheroids will provide deeper insights into 3D MSCs aggregate. Anyway, given the objectives and context of the research, discerning whether to employ MSC spheroids, which mainly rely on cell-cell contact, or C-MSCs, enriched in ECM components, is essential for the rigorous execution of foundational studies. It is intriguing to consider whether the distinct properties observed in 3D cell constructs made from other cell types also manifest in MSC spheroids and C-MSCs, potentially enhancing our understanding of 3D cell culture. Notably, 3D culture systems are known to better preserve the biological properties of original tumors, such as epithelial-mesenchymal transition (EMT), compared to 2D cultures. For example, a recent study demonstrated that 3D spheroids generated with cancer stem cells exhibited heightened expression of ECM-related transcription factors, mesenchymal markers, and TGF-\(\beta\) signaling-related genes in comparison to 2D cultured cells [35]. In light of this, we revisited our RNA-seq data to see if similar cellular properties are observed in MSC 3D cell aggregates (Supplementary Figure S3). As a result, the expression levels of the ECM-related transcription factor Twist (but not Snail) and the stromal cell marker (also an MSC marker) CD44 were increased in MSC spheroids and C-MSCs. Additionally, TGF-\(\beta\) signaling-related genes, such as SMADs and MMPs, were also elevated in MSC 3D cell aggregates compared to 2D cultured cells. These findings, observed both in cancer stem cells and MSC 3D cell aggregates, indicate that 3D cell culture systems could potentially steer cell fate toward mesenchymal lineages. Regarding medical application, distinctions in usage and indications can be envisioned for these 3D cell aggregates. For instance, C-MSCs, primarily composed of ECM and fabricable up to approximately 1 mm in diameter, could serve as an effective scaffold-free cell transplantation therapy for substantial tissue defects. In fact, C-MSCs demonstrated good handleability, allowing for direct transplantation in bone or periodontal tissue defects. Notably, C-MSC transplantation without an artificial scaffold into the defects induced successful tissue regeneration via grafted cell differentiation at the defect sites [36; 37], suggesting the stability of the grafted cells. Attempting such procedures using MSC spheroids of a diameter less than 500 \(\upmu\)m might present challenges. On the other hand, MSC spheroids, characterized mainly by cell-cell interactions, have unique medical applications. Although cleanly dispersing cells from ECM-based C-MSCs is challenging, MSC spheroids can be relatively easily dissociated using trypsin. This relative ease of dissociation has a huge advantage for the cell-priming approach. Briefly, by forming MSCs into spheroids, their paracrine function is elevated. Subsequently, the \"primed\" cells are dispersed and injected intravenously to treat the disease in animal models. This priming strategy has been well tested and indicated reliable therapeutic effects on immunological disorders or ischemic diseases [38; 39]. In addition, recent preclinical studies in cynomolgus monkeys have demonstrated the possibility of intravenously delivering MSC spheroids with diameters under 450\(\upmu\)m. Remarkably, the intravenously injected MSC spheroids did not induce adverse reactions, such as embolism in the small vessels, and the grafted cells were distributed and stable at the various organs for 21 days [40]. This represents a clear advantage of spheroids, which can be fabricated in a smaller size than C-MSCs.