Optimization Of A Microcarrier-based Three-Dimensional Suspension Culture System For Canine Adipose-Derived Mesenchymal Stem Cells

WANG Xin; LI Dongsheng; WANG Bingyun; CHEN Shengfeng

doi:10.19978/j.cnki.xmsy.2025.05.07

Abstract：To enhance the expansion efficiency of canine adipose-derived mesenchymal stem cells(cADMSCs)and address the limitations of traditional two-dimensional culture systems in large-scale production,we developed a three-dimensional suspension culture system based on Synthemax Ⅱ microcarriers was employed.Key parameters,including seeding density,agitation method,and culture duration,were optimized through static and dynamic culture strategies.Static culture experiments compared the effects of different seeding densities(1×104,2×104,and 3×104 cells/cm2)on expansion efficiency and cell viability,with viable cells counted using trypan blue exclusion.Dynamic culture evaluated the impact of intermittent versus intermittent-continuous agitation modes on cell proliferation using a spinner flask bioreactor,supplemented with glucose metabolism monitoring.Fluorescent staining(Calcein-AM/PI and TRITC-phalloidin/DAPI)was applied to assess cell viability,morphology,and cytoskeletal integrity,while culture duration optimization(24-120 h)analyzed expansion kinetics and viability changes.Results showed that under static conditions,a seeding density of 2×104 cells/cm2 achieved the highest expansion fold(7.20±0.20-fold)with 96.4%viability.Dynamic culture with intermittent agitation(24 h)followed by continuous agitation(72 h)yielded 4.1×104 cells on 300 cm2 microcarriers,corresponding to an expansion fold of(6.70±0.20-fold)and 94.0%viability.Optimal culture duration was identified as 96 h,with intact cell morphology,no aggregation,and stable viability(94.0%).These findings indicate that the optimized microcarrier-based three-dimensional suspension culture system,incorporating staged agitation strategies and controlled parameters,significantly enhances cADMSC expansion efficiency and quality,offering a viable solution for scalable production in veterinary regenerative medicine.