基于微载体的犬脂肪间充质干细胞三维悬浮培养体系的优化

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中图分类号： Q813.1+1 文献标识码：A 文章编码：1005-8567（2025）05-0045-07

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

WANGXin 1 ，LIDongsheng²，WANGBingyun²，CHENShengfengl （1.Foshan University，Foshan Guangdong 528225; 2.VetCell BiotechnologyCo.，FoshanGuangdong528225）

Abstract：To enhance the expansion eficiency of canine adipose-derived mesenchymal stemcels（cADMSCs） and address the limitations of traditional two-dimensional culture systems in large-scale production，we developeda three-dimensional suspension culture system based on Synthemax II microcariers was employed.Key parameters， including seeding density，agitation method，andculture 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 eficiencyandcell viability，with viable cels counted using trypan blue exclusion.Dynamic culture evaluated the impactof intermitent versus intermitent-continuous agitation modes oncellproliferation using a spinner flask bioreactor，supplemented with glucose metabolism monitoring.Fluorescent staining（Calcein-AM/PI and TRITC-phalloidin/DAPI）wasapplied toassess cellviability，morphology，and cytoskeletal integrity，whileculture duration optimization 24-120h ）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 bycontinuous agitation（72 h）yielded 4.1×104 （20 cells on 3O0 cm² microcarriers，corresponding to an expansion fold of（6.7O±0.20 -fold） and 94.0% viability. Optimal culture duration was identified as 96h ，with intact cell morphology，no aggregation，and stable viability（ 94.0% ）. These findings indicate that the optimized microcarrier-based three-dimensional suspensionculture system， incorporating staged agitation strategiesandcontrolled parameters，significantly enhancescADMSC expansion efficiencyandquality，offringa viable solution forscalable production in veterinary regenerative medicine.

Keywords：Mesenchymal stem cells；Microcarriers；Three-dimensional culture；Dynamicagitation；Cell expansion

间充质干细胞（mesenchymal stem cells，MSCs）因具有多向分化潜能、免疫调节功能和旁分泌特性，已成为再生医学领域的核心研究对象。（剩余10703字）