Mesenchymal stem cells (MSCs) are primary candidates for cell therapy and tissue engineering applications. A two-dimensional (2D) culture system is typically used for cell growth, but that method affects the characteristics of stem cells. The physiological cell environment connects cells not only to each other, but also to the extracellular matrix providing mechanical support, exposing the entire cell surface, and opening signaling pathways. The hanging drop method is the most widely used 3D culture method for spheroid formation. In this study, we investigated the relationship between spheroid size and changes in gene expression to determine the optimum spheroid size for use in tissue engineering. The expression levels of stemness factors such as NANOG, OCT4, and SOX2, angiogenic factors such as VEGF and IL-8, and osteogenic factors such as COX2 and TGF-β1 increased with spheroid size in the respective spheroid formation groups unlike the responses in their monolayer groups. Therefore, our results indicate that spheroid formation through the hanging drop method can increase the efficiency of MSCs-based tissue engineering over that obtained via traditional 2D cell culture systems.

Mesenchymal stem cells (MSCs) have been researched for use in biomedical applications, particularly for cell-based therapies and regenerative medicine due to their self-renewing capacity and ability to differentiate into multiple cell types such as adipose, bone, and tendon tissues. Cryopreservation of MSCs is a common preservation method that is advantageous for cellular therapies in human and veterinary medicine. Adipose tissue-derived cells have been shown to maintain their properties after cryopreservation. In this study, we investigated the morphology, proliferation (cumulative population doubling level and doubling time), cell surface markers (CD34, CD90, and CD105), and ability to differentiate into adipose, bone, and cartilage tissues in vitro of equine adipose tissue-derived MSCs (eAD-MSCs) and miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with and without long-term cryopreservation. The eAD-MSCs and mpAD-MSCs were analyzed immediately and after being frozen in liquid nitrogen for 3 years and 2 years, respectively. Cryopreserved eAD-MSCs maintained their morphology, proliferation rate, and cell surface markers compared with fresh cells. With the exception of proliferation rate, cryopreserved mpAD-MSCs also maintained their fresh cell characteristics. The proliferation rate of cryopreserved mpAD-MSCs was higher than that for fresh cells. Cryopreservation did not change the adipogenic, chondrogenic, or osteogenic differentiation potentials of eAD-MSCs and mpAD-MSCs. In summary, long-term cryopreservation maintains the cell phenotype and differentiation ability of eAD-MSCs and mpAD-MSCs. These results might be useful when developing veterinary medicine and clinical applications.

Bone fractures are most often seen in racetrack horses because of the high level of intensity in racing. These issues are the main cause of decreased performance in racehorses. Mesenchymal stem cells (MSCs) have been explored to improve intra-articular therapy in racehorses. MSCs are essential for the repair and regeneration of damaged tissues. In this study, the effect of intra-articular injection of MSCs in racehorses was investigated. Before accessing the MSC therapy, synovial fluids were obtained from the fracture site of racehorses, and adipose tissue was collected for MSC isolation. Using the MSC specific marker, adipose tissue-derived MSCs were identified. The racehorses received intra-articular injection of autologous MSCs (or allogeneic) (3 × 107 cells/3 mL). After 1 or 2 weeks, synovial fluids were collected from racehorses. To test the effect of MSC injection using ELISA, we analyzed inflammatory factors from the untreated samples compared to MSC-treated samples of racehorses. The level of pro-inflammatory factors (interleukin-1β and prostaglandin E2) was significantly decreased in synovial fluids of MSC-injected racehorses, compared to before accessing the MSC therapy, whereas, the level of anti-inflammatory factor (interleukin-10) was higher than prior to accessing the MSC therapy. Further studies are needed to investigate the anti-inflammatory mechanism of MSC in racehorses.