## 3 Cell-Material Interface and Interaction in Scaffold-Free Stem Cell 3D Culture Systems  
Scaffold-free culture platforms are simple, rapid, and low-cost methods to culture stem cells in a 3D environment, and thus are suitable for biomedical applications requiring high throughput property. In these systems, stem cells are cultured in a non- or low-adherent environment, and then are compelled to organize into the suspended cell spheroids. Hanging drop is the easiest approach to generate cell spheroids and does not require any additional or specific culture materials. Spheroids formed by hanging drop are aggregated by gravitational force and maintained in a liquid environment [22]. Due to the lack of direct contact with solid supporting interfaces, the cell-environment interaction only exists within the interior of cell spheroids. A few other similar approaches, such as pellet culture and low-attachment culture also demonstrate the spontaneous secretion of ECM proteins from the spheroid-forming cells [23; 24; 25], indicating that the composition of ECM proteins is the crucial environmental factor for the regulation of cell properties in these types of 3D culture systems. Biomaterial-based planar substrates are major systems to generate cellular spheroids as well, such as agarose and CS-based substrates [26; 27]. Agar/gaarose system belongs to the non-adherent spheroid-forming approach. Therefore, the cell-substrate interaction is also limited in the agar/gaarose culture system. In contrast, the carbohydrate structure of chitosan is similar to the glycosaminoglycans (GAGs) in the ECM [27]. Meanwhile, the cell-chitosan contact has been demonstrated by experiments [21]. Therefore, the cell-environment interactions in the CS-coated system are more complicated than those of hanging drop and low-attachment culture approaches. Several kinds of stem cells are assembled into cellular spheroids or co-spheroids and then used to demonstrate various medical applications. The physiological features of cell spheroids may be different due to the approaches of spheroid formation. Some distinct effects are found in MSC spheroids formed by hanging drop, low-adherent culture, or CS-based substrates, as compared to the planar-cultured MSCs. Hanging-drop-derived MSC spheroids displayed over 100-fold increase in the expression of anti-inflammatory [TNF\(\alpha\) stimulated gene/protein 6 (TSG-6)] and anti-cancer [IL-24 and TNF\(\alpha\)-related apoptosis inducing ligand (TRAIL)] genes, compared with the adherent MSCs on TCPS [28]. The stemness of MSCs was well maintained in the spheroids generated by the hanging drop approach, and the expression levels of stemness markers including Oct4, Sox, and Nanog were higher than those of 2D-cultured MSCs [29]. On the other hand, MSC spheroids formed by low-adherent plates secreted more angiogenic factors, such as vascular endothelial growth factor (VEGF) as well as stromal cell derived factor (SDF), and the cell proliferation of MSC spheroids displayed \(\sim\)2-fold increase as compared to 2D-cultured MSCs in vivo after transplantation to the mice ischemic hindlimb [30]. Moreover, MSCs derived from human adipose and placenta tissues displayed increased stemness and chondrogenic differentiation potential after culture on CS and CS-based substratum [21]. Meanwhile, by the microarray technique, the changes of expression levels of \(\sim\)300 genes were discovered for the MSC spheroids on CS substrates compared with the MSC grown on TCPS, indicating the substantial alternation for the MSC behavior after the spheroid formation [31]. Based on the findings mentioned above, the cell behavior of MSCs is dramatically changed as the culture environment shifts from 2D to 3D. Furthermore, the cell properties of MSCs may be different in the spheroids generated by the distinct forming approaches.