Trailhead Biosystems, 23215 Commerce Park, Beachwood, OH, 44122, USA.
Cleveland State University, 2121 Euclid Ave, Cleveland, OH, 44115, USA.
Stem Cell Res Ther. 2024 Nov 9;15(1):408. doi: 10.1186/s13287-024-03973-0.
Type 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture.
We examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors.
A three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies.
This study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment.
1 型糖尿病是一种导致胰腺β细胞破坏的自身免疫性疾病，需要终身胰岛素治疗。胰岛移植提供了一种有前途的解决方案，但面临着供体有限和需要免疫抑制等挑战。诱导多能干细胞（iPSC）为功能性β细胞提供了潜在的替代来源，并且具有大规模生产的能力。然而，目前的分化方案主要在混合或 2D 环境中进行，缺乏可扩展性和悬浮培养的最佳条件。
我们研究了一系列可能影响分化过程的生物反应器放大过程参数和质量目标产品特性。这项研究使用了一种经过优化的高维设计实验（HD-DoE）方案，该方案旨在实现可扩展性，并在 0.5L（PBS-0.5 Mini）垂直轮生物反应器中实施。
开发了一种三阶段悬浮制造工艺，从贴壁培养过渡到悬浮培养，TB2 培养基在放大过程中支持 iPSC 的生长。采用分阶段优化方法和延长分化时间来增强 iPSC 衍生的胰岛样簇的标志物表达和成熟。连续生物反应器运行用于研究营养和生长限制及其对分化的影响。连续生物反应器与控制介质更换生物反应器进行比较，显示出代谢变化和更类似于β细胞的分化特征。从运行中收获的冷冻保存的聚集体在恢复后显示出保持活力和胰岛素分泌能力的能力，表明它们具有储存和未来移植治疗的潜力。
本研究表明，在大规模悬浮环境中培养时，增加阶段时间和有限的补料并积累乳酸可以提高产生胰岛素的细胞的分化能力。
