Culturing cells as cell spheres results in a tissue-like environment that drives unique cell phenotypes, making it useful for generating cell populations intended for therapeutic use. Unfortunately, common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. Our team is developing an allogeneic cell therapy for the treatment of lumbar disc degeneration comprised of discogenic cells, which are progenitor cells expanded from human nucleus pulposus cells that are grown in a sphere configuration. We evaluate sphere production in Erlenmeyer, horizontal axis wheel, stirred tank bioreactor, and rocking bag format. We then explore the use of ramped agitation profiles and computational fluid dynamics to overcome obstacles related to cell settling and the undesired impact of mechanical forces on cell characteristics. Finally, we grow discogenic cells in stirred tank reactors (STRs) and test outcomes in vitro (potency via aggrecan production and identity) and in vivo (rabbit model of disc degeneration). Computation fluid dynamics were used to model hydrodynamic conditions in STR systems and develop statistically significant correlations to cell attributes including potency (measured by aggrecan production), cell doublings, cell settling, and sphere size. Subsequent model-based optimization and testing resulted in growth of cells with comparable attributes to the original static process, as measured using both in vitro and in vivo models. Maximum shear rate (1/s) was maintained between scales to demonstrate feasibility in a 50 L STR (200-fold scale-up). Transition of discogenic cell production from static culture to a stirred-tank bioreactor enables cell sphere production in a scalable format. This work shows significant progress towards establishing a large-scale bioprocess methodology for this novel cell therapy that can be used for other, similar cell therapies.

中文翻译：

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从静态培养过渡到搅拌罐生物反应器，用于异体生产治疗性椎间盘细胞球

将细胞培养为细胞球会产生类似组织的环境，驱动独特的细胞表型，使其可用于产生用于治疗用途的细胞群。不幸的是，利用静态悬浮培养的常见方法的可扩展性有限，使得此类细胞疗法的商业化具有挑战性。我们的团队正在开发一种用于治疗腰椎间盘退变的同种异体细胞疗法，该疗法由椎间盘细胞组成，这些细胞是从以球形结构生长的人髓核细胞扩增的祖细胞。我们评估了锥形、水平轴轮、搅拌罐生物反应器和摇袋形式的球体生产。然后，我们探索使用斜坡搅拌曲线和计算流体动力学来克服与细胞沉降相关的障碍以及机械力对细胞特性的不利影响。最后，我们在搅拌罐反应器 (STR) 中培养椎间盘细胞，并在体外（通过聚集蛋白聚糖产生和鉴定的效力）和体内（椎间盘退化的兔子模型）进行测试。计算流体动力学用于模拟 STR 系统中的流体动力学条件，并建立与细胞属性的统计学显着相关性，包括效力（通过聚集蛋白聚糖的生产量）、细胞倍增、细胞沉降和球体大小。随后的基于模型的优化和测试导致细胞的生长具有与原始静态过程相当的属性，如使用体外和体内模型测量的那样。最大剪切速率 (1/s) 在规模之间保持不变，以证明在 50 L STR 中的可行性（放大 200 倍）。从静态培养到搅拌罐生物反应器的盘状细胞生产的转变使细胞球生产能够以可扩展的形式进行。这项工作表明，在为这种可用于其他类似细胞疗法的新型细胞疗法建立大规模生物过程方法方面取得了重大进展。