Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed
Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed
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DOI：
10.1016/j.jcyt.2014.08.011
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发表时间：
2015-02-01
期刊：
影响因子：
4.5
通讯作者：
Oh, Steve Kah Weng
中科院分区：
文献类型：
--
作者：
Chen, Allen Kuan-Liang;Chew, Yi Kong;Oh, Steve Kah Weng
背景目标。临床细胞治疗需要大量的人间充质基质细胞（MSCs）。在以前的出版物中，我们描述了一种基于微载体的MSC扩增方法。本研究通过选择合适的基础培养基、微载体浓度和补料方式对该工艺进行了优化，以获得更高的细胞产量和更有效的培养基利用率。方法. MSC在Cytodex 3微载体上用含有10%胎牛血清的培养基在搅拌培养物中扩增。在转瓶中进行工艺优化。使用带有自动补料系统的2 L生物反应器验证转瓶培养中探索的优化参数。结果最低必需培养基-基于α的培养基支持MSC在微载体上比Dulbecco改良的Eagle培养基更快的生长（倍增时间，31.6 +/-1.4 vs 42 +/-1.7 h），并缩短了处理时间。在8 mg/mL的微载体浓度下，实现了1.08 x 10（6）个细胞/mL的高细胞浓度，融合细胞浓度为4.7 x 10（4）个细胞/cm（2）。我们设计了基于葡萄糖消耗速率的全培养基补料，而不是每2天更换50%培养基。由1.5g/L葡萄糖组成的最佳培养基补料支持MSC生长至完全汇合，同时实现3.29mL/10（6）个细胞的低培养基使用效率。最后，具有优化参数的受控生物反应器实现了最大汇合细胞浓度，扩增16倍，并且进一步提高了1.68 mL/10（6）个细胞的培养基使用效率。结论.我们已经优化了基于微载体的MSC扩增平台，以更有效和更具成本效益的方式产生高细胞产量。本研究强调了MSC生产工艺优化中的关键参数。
Background aims. Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. Methods. MSCs were expanded in stirred cultures on Cytodex 3 microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. Results. Minimum essential medium-alpha-based medium supported faster MSC growth on microcarriers than did Dulbecco's modified.Eagle's medium (doubling time, 31.6 +/- 1.4 vs 42 +/- 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 x 10(6) cells/mL with confluent cell concentration of 4.7 x 10(4)cells/cm(2) was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10(6)cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10(6)cells. Conclusions. We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.