PDF(Pubmed)
Abstract：
BACKGROUND: Stem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability.
METHODS: Utilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.
RESULTS: Expansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1-1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.
CONCLUSIONS: This study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.
METHODS: Utilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.
RESULTS: Expansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1-1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.
CONCLUSIONS: This study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.
摘要：
背景：干细胞衍生疗法具有治疗再生临床适应症的潜力。静态培养具有有限的放大能力,因此限制了其使用。悬浮培养可用于大量生产靶细胞，但也提出了与压力和聚集稳定性相关的挑战。
方法：在垂直轮式生物反应器中利用实验设计（DoE）方法，我们评估了具有多种特性的介质添加剂。评估的添加剂是肝素钠盐（HS），聚乙二醇（PEG），聚乙烯醇(PVA)，PluronicF68和硫酸葡聚糖(DS)。选择多个响应变量来评估细胞生长，响应于添加剂输入的多能性维持和聚集体稳定性，和数学模型被生成和调整为最大预测能力。
结果：在19种不同的培养基组合上使用100ml立式轮生物反应器测定4天扩增iPSC，从而产生可以优化多能性的模型，稳定性,和扩张。扩展优化导致了PA的组合，PVA和PEG与E8。该混合物导致比单独的E8短40%的膨胀倍增时间。多能性优化器强调了向E8培养基中添加1%PEG的重要性。使3D培养中的聚集体融合最小化的聚集体稳定性优化表明肝素和PEG两者的相互作用可以限制聚集以及增加hiPSC的维持能力和扩增。表明控制融合是扩展和维护的关键参数。在生物反应器中以40RPM的速度降低的两种细胞系上验证优化的解决方案，显示了对具有高频率的OCT4和S0X2的多能性标记物(>90%)的聚集体的一致性和延长的控制。在优化培养基中作为团块传代后,维持约1-1.4天的倍增时间。控制聚集体融合允许生物反应器速度降低，因此在大规模扩增中施加在细胞上的剪切应力降低。
结论：本研究控制了悬浮培养物中的聚集体大小，同时告知iPSC状态的伴随状态控制。这种方法的更广泛的应用可以解决培养基优化复杂性和生物反应器放大的挑战。
方法：在垂直轮式生物反应器中利用实验设计（DoE）方法，我们评估了具有多种特性的介质添加剂。评估的添加剂是肝素钠盐（HS），聚乙二醇（PEG），聚乙烯醇(PVA)，PluronicF68和硫酸葡聚糖(DS)。选择多个响应变量来评估细胞生长，响应于添加剂输入的多能性维持和聚集体稳定性，和数学模型被生成和调整为最大预测能力。
结果：在19种不同的培养基组合上使用100ml立式轮生物反应器测定4天扩增iPSC，从而产生可以优化多能性的模型，稳定性,和扩张。扩展优化导致了PA的组合，PVA和PEG与E8。该混合物导致比单独的E8短40%的膨胀倍增时间。多能性优化器强调了向E8培养基中添加1%PEG的重要性。使3D培养中的聚集体融合最小化的聚集体稳定性优化表明肝素和PEG两者的相互作用可以限制聚集以及增加hiPSC的维持能力和扩增。表明控制融合是扩展和维护的关键参数。在生物反应器中以40RPM的速度降低的两种细胞系上验证优化的解决方案，显示了对具有高频率的OCT4和S0X2的多能性标记物(>90%)的聚集体的一致性和延长的控制。在优化培养基中作为团块传代后,维持约1-1.4天的倍增时间。控制聚集体融合允许生物反应器速度降低，因此在大规模扩增中施加在细胞上的剪切应力降低。
结论：本研究控制了悬浮培养物中的聚集体大小，同时告知iPSC状态的伴随状态控制。这种方法的更广泛的应用可以解决培养基优化复杂性和生物反应器放大的挑战。
