## 4 Spheroid Culture of MSCs
After induction, the expression of endothelial and osteoblast markers were both detected in the MSC spheroids, suggesting that it is a potential and simple method to fabricate a vascularized bone tissue [49]. Activity of angiogenic promotion for MSCs was proven in another MSC and endothelial cell (EC) co-culture experiment. ECs organized into a network structure in the MSC/EC heterotypic co-spheroids formed by the non-adherent U-bottomed plates. Replacing the MSCs by fibroblasts decreased the length and width of EC networks as well as branch formation. By the use of different inhibitors, platelet-derived growth factor (PDGF)-involved signaling pathway has been found as the predominant regulator for the EC alignment in this model [50]. Positive effects for the cells co-cultured with MSCs in 3D co-spheroids have been mentioned in other literature. In the conventional culture, the islet cells are difficult to maintain. Although insulin secretion was not stimulated for islet cells, a current finding has demonstrated that the survival rates of islet cells were largely enhanced by forming the co-spheroids with MSCs on a special glass micromold [51]. Moreover, the proliferation of hematopoietic stem/progenitor cell (HSPC) was promoted by MSCs in a monolayer co-culture condition. However, this support is still too weak to produce sufficient HSPCs for clinical usage. Currently, co-culture of HSPCs and MSCs has been evaluated in a 3D spheroid by using non-adherent Pluronic-coated polydimethylsiloxane (PDMS) microwell platforms. The results demonstrated that the number of CD34-positive cells was increased on PDMS microwells; nevertheless, the co-culture strategy and system still needed further improvement before being subjected to clinical applications [52]. For cell spheroid preparation, the major advantage of scaffold-free systems is the convenient collection of the formed spheroids. However, the drawbacks of these systems mentioned above, such as hanging-drop, low-adherent plate, micro-fluidic system, and PDMS microwells, are deficient in environmental interaction during and after the spheroid formation. CS-based substrates also belong to the scaffold-free platforms; however, the transient interaction of MSCs and chitosan surface has been observed. An illustration demonstrating the processes of MSC spheroid/co-spheroid formation on CS substrates and other common scaffold-free methods is shown in Figure 1. In a previous study, human adipose-derived MSCs self-organized into cell spheroids on the CS-coated surfaces. MSCs displayed better chondrogenic differentiation when cultured on CS substrates as compared to those on TCPS. During the spheroid formation, MSCs adhered on the CS substrates at initial culture period, and then gradually merged and self-assembled into the cell spheroids, revealing the existence of crosstalk between the cells and CS substrates [21]. Rho/ROCK signaling pathway regulates the cytoskeleton rearrangement and cell adhesion/migration [53]. After the treatment of a ROCK inhibitor, Y-27632, MSCs still adhered and migrated on the CS substrates; however, MSC spheroids were not further organized. Meanwhile, CD44, a stemness marker of MSCs, was required for the movement and spheroid formation of MSCs on CS substrates. As blocking the formation of MSC spheroids by ROCK inhibitor or CD44 antibody, the overall stemness of MSCs was reduced [21], suggesting the positive correlation between the spheroid formation and stemness maintenance for MSCs cultured on CS-based substrates. Furthermore, based on the observation on the relationship between MSC spheroid formation and their stemness, a further study has demonstrated that CS substrates can be employed as a rapid platform for selecting MSCs from adipose tissues. Adipose-derived MSCs isolated by CS substrates displayed higher proliferation rates and stemness, as compared to those collected by conventional plastic adherence approach.