## 5 Physical and Chemical Factors Affecting Cell Behaviors in Scaffold-Free 3D Culture Systems
With the growth or aggregation of cells, the diameter or cell density of spheroids is gradually increased and internal acidity may be changed due to the metabolic status. The internal material transfer may be blocked or decelerated by the size expansion of spheroids. However, it has been demonstrated that the cell viability of MSC spheroids or co-spheroids was over 80% when the sizes of these spheroids were larger than 300 \(\upmu\)m [64; 65], suggesting that the tight interaction between the cells would establish a strong communication network to support the cell vitality in the spheroids. Oxygen gradient is also a common issue in the study of cell spheroids. It is generally considered that the cells within the core of spheroids have to face the hypoxic state [66]. Recently, an approach to estimate the oxygen tension in the MSC spheroids has been developed. By a simulation approach, the researchers concluded that the change of oxygen concentration from the center to the surface was lower than 10% when the spheroid diameter was \(\sim\)700 \(\upmu\)m. Meanwhile, the cell density and metabolism were both decreased by the increment of spheroid sizes, indicating that the cells would self-adjust the physiological status to avoid cell death through promoting mass transfer and reducing energy consumption [67]. It may be a mechanism for maintaining the cells with high viability in the spheroids. The change of signal transduction is the major responses to the environmental chemical factors. Certainly, a part of these chemical effects was also derived from the mechanical stimulation. Ion strength may be one of chemical factor regulating MSC behaviors. To date, ion concentration in the spheroids has not been explored. It has been reported that the mechanical stress affected the activation of calcium ion channels on the cell membrane of MSCs, and the calcium ion-involved signaling played important roles in the differentiation of MSCs [68; 69]. On the other hand, the cytoskeleton would be dramatically rearranged during the spheroid formation on CS substrates. Blocking the cytoskeleton rearrangement by the treatment of a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor interrupted the self-assembly of MSC spheroids [21]. Meanwhile, ROCK signaling was also found to regulate the calcium ion-involved actin remodeling [70]. Therefore, the activation of calcium channels and the change of calcium influx may be the key regulators for the spheroid formation and the subsequent MSC differentiation on CS substrates. Integrin-involved signaling may be a crucial factor for regulating the physiology of cell spheroids. Integrins are the important anchor proteins for cells bound to ECM. We have previously mentioned that ECM proteins are secreted by spheroid-forming cells. Thus, the combination of alpha and beta units of integrin on the membrane of MSCs as well as which ECM proteins they interact with may determine the stemness or differentiation potential of MSCs in the spheroids. For example, human placenta MSC spheroids displayed lower expression of integrin \(\beta 1\) compared to normal 2D culture, leading to a decrease of cell-ECM interaction and enhancement of MSC stemness. By other approaches to release cytoskeleton tension, the stemness of MSCs was promoted as well, revealing the negative relationship between stemness and cytoskeleton tension in MSCs [71]. In addition to cell-matrix interaction, the cadherin-mediated cell-cell interaction cannot be ignored. Cadherin is a kind of calcium-dependent adhesive molecule between neighboring cells. When calcium ions bind on the extracellular region of cadherin, the cadherin forms a homodimer and then binds to another cadherin homodimer on the neighboring cells. Intercellular cadherin interaction regulates the cell-cell adhesion, cell migration, and multiple types of cell signaling by association with the actin filaments [72].