Bioprocess Development for Mass Production of Size-Controlled Human Pluripotent Stem Cell Aggregates in Stirred Suspension Bioreactor
Bioprocess Development for Mass Production of Size-Controlled Human Pluripotent Stem Cell Aggregates in Stirred Suspension Bioreactor
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DOI：
10.1089/ten.tec.2012.0161
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发表时间：
2012-11-01
影响因子：
3
通讯作者：
Baharvand, Hossein
中科院分区：
文献类型：
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作者：
Abbasalizadeh, Saeed;Larijani, Mehran Rezaei;Baharvand, Hossein
目前用于人类多能干细胞(HPSCs)可伸缩悬浮培养的方案受到多种生物学和技术挑战的限制，在将其用于临床试验之前需要解决这些挑战。为了克服这些挑战，我们开发了一种新的生物处理平台，用于大规模扩增人类胚胎和诱导的多潜能干细胞系，作为三维大小可控的聚集体。这一新的生物工艺利用了静态和动态悬浮培养条件的逐步优化。在静态悬浮培养中筛选了8种无异种培养基并在动态条件下优化了单细胞传代后，在搅拌生物反应器中从静态悬浮培养扩大到动态悬浮培养，以细胞计数和代谢活性衡量，扩张率提高了两到三倍。我们通过使用不同搅拌速度和剪切保护剂浓度的组合，通过优化生物反应器的流体动力学条件，成功地生产了特定尺寸的聚集体。在常氧条件下通过控制氧浓度进一步提高了膨胀率，两种类型的hPSCs的扩张率都达到了最大8倍。随后，我们展示了一种简单而快速的放大策略，通过将“悬浮适应的冷冻细胞”直接转移到搅拌悬浮生物反应器中，在1个月内生产出临床相关数量的hPSCs(类似于2×10(9)细胞)。我们省略了静态悬浮培养中所需的预适应传代。在无异种的动态悬浮培养中，细胞经过多次传代增殖，同时保持其自我更新能力，这是由标志物的表达和体外自发分化决定的。总之，hPSCs的悬浮培养方案可以通过鉴定和优化关键的生物过程参数，并辅之以简单、快速的放大平台，来批量生产均质和多潜能的未分化细胞。
Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (similar to 2 x 10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells'' to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess parameters that are complemented by a simple and rapid scale-up platform.