## 5 Physical and Chemical Factors Affecting Cell Behaviors in Scaffold-Free 3D Culture Systems  
In 2D culture systems, the predominant physical factors affecting cell behaviors are the stiffness and topology of culture materials [58]. For scaffold-based 3D culture platform, such as hydrogelencapsulated system, the differentiation potential of encapsulated MSCs is also directly affected by the stiffness of hydrogel. The hydrogels with higher and lower stiffness induced the differentiation of embedded MSCs toward myocytes/osteocytes and adipocytes/chondrocytes, respectively. Meanwhile, the differentiation tendencies of these MSCs were regulated by transforming growth factor \(\beta\) (TGF-\(\beta\))- and integrin-involved signaling, revealing the relationship between the cell-environment interaction and cell fate determination [59; 60]. For common scaffold-free 3D culture systems, the direct contact of cells and culture materials is very limited. In the hanging drop, cells are maintained and suspended in the culture medium, and no cell-substrate interaction exists. For the non- or low-adherent spheroid-forming system, cells \"touch\" rather than \"interact\" to the surfaces of culture materials. In a CS-based system, the cell adhesion is observed transiently. Therefore, the overall influences of surface stiffness and topology of culture materials in scaffold-free 3D culture systems are much slighter than those in 2D and scaffold-based 3D culture platforms. Based on the different approaches for spheroid formation, other extrinsic physical  
Figure 1: Formation of mesenchymal stem cells (MSC) spheroids/co-spheroids by chitosan (CS) substrates and other common scaffold-free methods. During the monotypic or heterotypic spheroid formation on CS substrates, cells transiently adhere and interact with CS substrates, and then merge and further organize into cell spheroids. On the contrary, cells directly assemble into cell spheroids without cell–substrate interaction in other scaffold-free methods (hanging drop and low-adhesive methods). Furthermore, for the heterotypic spheroid formation, cell–cell and cell-substrate interactions result in the self-assembly of heterotypic cells and specific cell arrangement in the co-spheroids on CS substrates. However, cells are forced to form co-spheroids with random organization by other scaffold-free methods. ECM: extracellular matrix. stimulation also affects the MSC behaviors. For instance, several evidences have demonstrated the functions of fluid flow in MSC differentiation. It has been reported that the osteodifferentiation of dexamethasone-treated MSCs was enhanced by the shear stress produced by a flow perfusion system during the period of culture [61]. A recent report also demonstrated that several indicators of osteodifferentiation of MSCs, including alkaline phosphatase (ALP) activity, type I collagen expression, and intracellular calcium ion concentration, were up-regulated in the MSCs after the stimulation of mechanical stress (4000 microstrain). Meanwhile, the mechanical stress-induced osteodifferentiation of MSCs was related to the activation of p38 mitogen-activated protein kinase (MAP kinase) [62]. Furthermore, the static or dynamic perfusion fluid also regulates the transportation of nutrients, waste, and gas in the culture systems. Thus, these factors have to be carefully concerned, especially in a micro-fluidic 3D system. Moreover, the hydrostatic pressure is also mentioned in the regulation of the cytoskeletal composition and MSC differentiation [63]. In scaffold-free 3D culture platforms, besides the gravity, the water pressure is the only resource of external pressure for the cultured cells. Therefore, it may be another choice to control the differentiation of MSCs by adjusting the hydrostatic pressure given to the spheroid-forming cells. Physical factors affecting the MSC behaviors within the spheroids are generally related to the diameter and cell density of spheroids.