PDF(Pubmed)
Abstract：
BACKGROUND: While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified.
METHODS: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture.
RESULTS: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology.
CONCLUSIONS: This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.
METHODS: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture.
RESULTS: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology.
CONCLUSIONS: This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.
摘要：
背景：尽管多能干细胞（PSC）疗法以快速的速度向临床和商业应用发展，制造再现性和稳健性是监管批准的显著瓶颈。PSC的治疗应用需要在高度稳健的情况下产生大量细胞，定义明确，和经济上可行的条件。小规模和短期工艺优化，然而,通常以线性方式进行,其不考虑验证所使用的生物过程方案和分析方法所需的时间。可重复和强大的生物过程的设计应该是动态的，并且包括持续的努力，以了解该过程在过渡到应力将被放大的大规模生产之前，随着时间的推移和对不同应力的反应。
方法：本研究采用基线方案，为在Vertical-Wheel®生物反应器中短期培养PSC聚集体而开发，通过长期(连续传代)悬浮培养评估关键过程属性。这样做是为了在用各种市售的培养基和细胞系进行时获得整体过程的稳健性。过程输出变量，包括生长动力学，聚集体形态,收获效率，基因组稳定性，功能多能性通过短期和长期培养进行评估。
结果:在六天培养期内证明了扩增方案的稳健性质,其中对于测试的所有五种商业培养基,观察到球形聚集体形成和扩增具有高倍扩增。只有通过长期连续扩增和血管内解离操作才能揭示细胞生长和质量的深刻差异。测试的一些商业培养基制剂证明了细胞生长速率的维持，聚集体形态,通过使用多个PSC系的三个生物反应器串联通道和高收获回收效率。在10个连续的生物反应器通道中，持续的生长和质量维护甚至证明了出色的生物过程稳健性。然而,一些测试的商业培养基被证明不太适合在生物反应器中连续传代培养，因为培养物在解离过程中导致细胞裂解，增长率降低，和聚集体形态的损失。
结论：本研究证明了系统选择和测试生物过程输入变量的重要性，与多个生物过程输出变量通过连续通道创建一个真正的可重复和强大的方案临床和商业PSC生产使用可扩展的生物反应器系统。
方法：本研究采用基线方案，为在Vertical-Wheel®生物反应器中短期培养PSC聚集体而开发，通过长期(连续传代)悬浮培养评估关键过程属性。这样做是为了在用各种市售的培养基和细胞系进行时获得整体过程的稳健性。过程输出变量，包括生长动力学，聚集体形态,收获效率，基因组稳定性，功能多能性通过短期和长期培养进行评估。
结果:在六天培养期内证明了扩增方案的稳健性质,其中对于测试的所有五种商业培养基,观察到球形聚集体形成和扩增具有高倍扩增。只有通过长期连续扩增和血管内解离操作才能揭示细胞生长和质量的深刻差异。测试的一些商业培养基制剂证明了细胞生长速率的维持，聚集体形态,通过使用多个PSC系的三个生物反应器串联通道和高收获回收效率。在10个连续的生物反应器通道中，持续的生长和质量维护甚至证明了出色的生物过程稳健性。然而,一些测试的商业培养基被证明不太适合在生物反应器中连续传代培养，因为培养物在解离过程中导致细胞裂解，增长率降低，和聚集体形态的损失。
结论：本研究证明了系统选择和测试生物过程输入变量的重要性，与多个生物过程输出变量通过连续通道创建一个真正的可重复和强大的方案临床和商业PSC生产使用可扩展的生物反应器系统。
