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.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Isolation and culture of MSCs derived from human andcanine fat tissues
    Preparation of allogenic serum
    The measurement of population doubling time (PDT)
    Senescence associated β-galactosidase assay
    The analysis of cell apoptosis
    Quantitative real time-PCR (qRT-PCR) analysis
    Statistical analysis
RESULTS
    Differential in vitro growth capacity in human andcanine mesenchymal stem cells
    Effect of allogeneic serum on in vitro expansion ofcanine MSCs
    Allogeneic serum reduces cellular senescence ofcanine MSCs in vitro culture conditions
    Expression of pluripotency-related transcriptionfactors in canine MSCs cultured with FBS versusallogeneic serum
DISCUSSION
CONCLUSION
REFERENCES

• Yong-ho Choe(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Sang-Yun Lee(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Young-Bum Son(Department of Obstetrics, College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Korea)
• Won-Jae Lee(College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea)
• Hyeonjeong Lee(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Chan-Hee Jo(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Seong-Ju Oh(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Tae-Seok Kim(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Chae-Yeon Hong(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea)
• Sung-Lim Lee(College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea, Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea) Corresponding author