Background: The clinical application of canine mesenchymal stem cells (MSCs) necessitates efficient and safe culture methods to produce large quantities of cells. Traditionally, fetal bovine serum (FBS) has been used for MSC expansion, but it carries risks such as contamination and adverse immune responses. Methods: In this study, we investigate the efficacy and efficiency of canine allogeneic serum as an effective alternative to FBS for the in vitro culture of canine MSCs. We measured the population doubling time of canine MSCs in allogeneic serum conditions and utilized qRT-PCR, flowcytometric analysis, and cellular staining/color-metric assay for investigating its effects on cellular senescence during long-term culture and the expression of key pluripotency-related transcriptomes. Results: Our findings demonstrate that canine MSCs cultured with allogeneic serum exhibited enhanced proliferation rates, reduced cellular senescence, and lower apoptosis levels compared to those cultured with FBS. Additionally, the expression of key pluripotency-related transcription factors, including Oct4, Sox2, and Nanog, was increased in canine MSCs cultured with allogeneic serum. Conclusions: These results highlight the potential of canine allogeneic serum to provide a safer and more effective culture environment, supporting the large-scale expansion and maintenance of canine MSCs for clinical applications.

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/ drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and antiinflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient’s synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RAhMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.