关键词: 间充质干细胞, 微载体, 三维培养, 动态搅拌, 细胞扩增

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 SynthemaxⅡ 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.

Key words: Mesenchymal stem cells, Microcarriers, Three ? dimensional culture, Dynamic agitation, Cell expansion