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

摘要： 为了提升犬脂肪间充质干细胞（cADMSCs）的扩增效率并解决传统二维培养体系规 模化生产的局限性，试验采用基于SynthemaxⅡ微载体的三维悬浮培养技术，通过静态与动态 培养策略优化接种密度、搅拌模式和培养周期等关键参数。研究通过静态培养对比不同接种 密度（1×104、2×104、3×104 cells/cm2）对扩增效率和细胞活率的影响，采用台盼蓝染色计数活细 胞并计算扩增倍数；动态培养中对比间歇搅拌与间歇?持续搅拌模式对细胞增殖的影响，利用 转瓶生物反应器结合葡萄糖代谢监测评估扩增效果；通过荧光染色（Calcein?AM/PI、TRITC?鬼 笔环肽/DAPI）检测细胞活性及形态特征，并分析不同培养周期（24-120 h）的细胞扩增趋势与 活率变化。结果表明：静态培养下，接种密度为2×104 cells/cm2时扩增倍数达（7.20±0.20）倍，活 细胞率为96.4%；动态培养采用间歇搅拌24 h后转为持续搅拌72 h的模式，在300 cm2微载体上 实现4.1×104个细胞扩增，扩增倍数达（6.70±0.20）倍，活细胞率为94.0%；培养周期优化显示， 96 h为最佳培养时间，此时细胞形态完整且未出现聚集脱落，活率稳定在94.0%。说明基于微 载体的三维悬浮培养体系通过优化接种密度、分阶段搅拌策略及培养周期，显著提升了cAD? MSCs的扩增效率与质量，为规模化生产提供了可行方案。

关键词: 间充质干细胞, 微载体, 三维培养, 动态搅拌, 细胞扩增