## 4 Spheroid Culture of MSCs  
MSCs have been considered as the multifunctional agents for treating several types of degenerative diseases and immunological disorders [33]. Recently, the therapeutic efficacy of MSC spheroids has been tested in several animal disease models including wound healing as well as bone, cardiovascular, kidney, and liver disorders. Spheroid-forming MSCs secrete more bioactive factors such as fibroblast growth factor 1 (FGF1), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE2) to promote angiogenesis and reduce immune response, leading to the enhancement of wound regeneration [34; 35]. In a murine skin wound model, the injured site treated with MSC spheroids displayed higher regenerative and angiogenic rates [34]. In addition, the therapeutic efficiency of MSC spheroids was better than dispersed cells for the 5-fluorouracil-induced oral mucositis as well as weight loss [36], indicating the potential of MSC spheroids in alleviating the side effects for cancer patients after chemotherapy. MSC spheroids have been employed to in vivo cartilage and bone regeneration. Transplantation of MSC aggregates generated by hanging drop induced cartilage regeneration in the rabbit femur osteochondral defect model through the secretion of cartilage matrix [37]. On the other hand, MSC spheroids also promoted the osteogenesis and bone regeneration in the rat calvarial and femur defect models [38; 39]. Regarding the cardiovascular diseases, the therapeutic functions of MSC spheroids may be divided into cardiomyocyte regeneration and neovascularization. Adipose-derived MSC spheroids displayed higher cardiomyocyte potential as compared to 2D-cultured MSCs, and the functional recovery of impaired heart was demonstrated after injection of MSC spheroids in a chronic myocardial infarction rat model [40]. Moreover, the myocardial infarction of porcine can be obviously repaired by implantation of adipose-derived MSC spheroids [41]. Regarding the liver regeneration, 90% hepatectomized rats transplanted with the alginate hydrogel-encapsulated bone marrow cells displayed \(\sim\)5-fold survival rate and \(\sim\)1.3-fold liver weight, as compared to those transplanted with free bone marrow cells [42]. Another similar strategy to improve the survival rate of 90% hepatectomized rats was transplantation of hydrogel-embedded bone marrow MSCs into the spleen. In the latter case, the encapsulated MSCs rather than free MSCs continuously provided hepatotrophic factors with stable concentration, and resulted in \(\sim\)70% increase to the free MSC group for the survival rate of hepatectomized rats [43]. A more recent report demonstrated that the alginate-embedded MSCs differentiated into the liver cells and secreted IL-6 and other growth factors after transplantation into the spleen. Meanwhile, the secretion of the cytokines and growth factors for the encapsulated MSCs was \(\sim\)2-fold higher than free MSCs two days after surgery [44]. The above findings indicate that the therapeutic efficacy of MSCs can be obviously promoted when the MSCs are grown in an appropriate 3D environment. Furthermore, by the promotion of angiogenesis, paracrine secretion, cell differentiation, anti-inflammation, anti-apoptosis, or anti-oxidation, the greater therapeutic potential of MSC spheroids has also been demonstrated in kidney [23] and liver [45; 46] injuries, as compared with monolayer MSCs. These groups of evidence reveal the promising roles of spheroid-forming MSCs in future healthcare, especially in the functional recovery of damaged organs. Currently, the novel and advanced strategies used to generate MSC spheroids and to induce their differentiation toward specific cell types are being continuously developed, such as magnetic levitation and micro-fluidic system [47; 48]. Meanwhile, several novel events have been discovered in the MSC spheroids and co-spheroids. Rat adipose-derived MSCs were cultured on agarose plate and self-assembled into microspheres.