FMSG: Bio: End-to-End Continuous Manufacture of Cell Therapies Enabled by Robotics and Microfluidic Processing

该项目将寻求为基于微流体技术和机器人细胞处理的细胞治疗制造创建一个更连续，更集成的工作流程。从目前的批处理过渡到更连续的细胞治疗生产工作流程可以包括更高纯度和产量的有效细胞，这也有利于其他传统的生产工作流程，如化学生产。由于挽救生命的细胞疗法的成本为每剂10万美元，因此创新细胞疗法产品的新集成工艺和技术对于扩大可及性和提高创新率至关重要。本研究将解决细胞治疗制造的几个挑战。首先，细胞治疗产品目前使用由多步骤工艺组成的“批量”制造方法生产。因此，该工艺导致批次间差异，无法在生产中使用供体特异性变量，以及难以对关键质量属性进行系统控制。第二，用于细胞疗法制造的与细胞功能相关的传感器（例如，分子）不是实时的，并且不能用于灵活的过程控制。第三，由于自体疗法是从患者-供体中获得的，因此起始材料存在很大的可变性，但这种可变性并未纳入制造过程。该项目的更广泛影响包括对研究生和本科生进行细胞治疗制造微流控方法的培训，这可能会提高创新率并降低与临床细胞制造相关的成本，同时使小企业能够在治疗细胞的生产中进行创新。该项目将利用集成的微流控转染和分离操作在微观尺度上利用流体动力学，以及支持视觉和数据分析的机器人技术，以帮助自动化细胞培养过程。作为一个试验平台，该研究将把这些技术应用于诱导多能干细胞（iPSC）衍生的视网膜类器官制造过程。新技术和过程控制将用于产生功能性视网膜细胞移植物，即，来自iPSC衍生的视网膜类器官的3D工程化视网膜构建体。该项目可以通过更好地整合iPSC基因工程，基于机器人的培养和新的无标记细胞选择方法来改善再生策略，以纯化符合良好生产规范生产的所需细胞类型。该团队汇集了三个核心专业知识来完成转型：目前用于人类诱导多能干细胞培养的视网膜类器官制造的良好生产规范流程;微流体支持的细胞转染，表征和分离单元操作;以及机器人支持的细胞处理和图像分析能力。第一个目标是应用一种细胞微流体转染平台，该平台使用精心设计的机械变形来对流地将大型基因编辑CRISPR/Cas9和DNA货物递送到iPSC。此外，由于患者来源的标本因供体而异，该研究还将表征供体细胞的生物力学特性以优化转染。第二个目标是将联合收割机自动化细胞培养系统和机器学习相结合，以开发能够进行高质量自动化iPSC生成、CRISPR校正和视网膜分化的机器人平台。第三个目标是研究视网膜细胞的混合物如何使用无标记细胞分离微流体技术更好地产生功能性视网膜移植物。这个项目的智力价值将共同展示一种综合方法来生成3D工程视网膜结构，以解决遗传性失明问题。这个未来制造奖是由分子和细胞生物科学支持的。这个奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will seek to create a more continuous, integrated workflow for cell therapy manufacturing based upon microfluidic technology and robotic cell processing. The transition to a more continuous cell therapy manufacturing workflow from the current batch processing can include a higher purity and yield of potent cells, which has also benefited other conventional manufacturing workstreams such as chemical manufacturing. With the cost of life-saving cell therapies $100k per dose, innovating new integrated processes and technologies for cell therapy products is essential to expand access and increase the rate of innovation. There are several challenges to cell therapy manufacturing that this study will address. First, cell therapy products are currently produced using a “batch” manufacturing approach that consists of a multi-step process. As a result, the process results in batch-to-batch variation, an inability to use donor-specific variables in the manufacturing, and difficulty in system control of critical quality attributes. Second, the sensors that are used in cell therapy manufacturing that are related to cell function (e.g., molecular) are not real-time and cannot be used in nimble process control. Third, because autologous therapies are derived from a patient-donor, there is substantial variability of the starting material—yet this variability is not incorporated into the manufacturing process. The broader impacts of the project include the training of graduate and undergraduate students in microfluidic approaches to cell therapy manufacturing that may increase the innovation rate and decrease the costs associated with clinical cell manufacturing, while enabling small industry to innovate in the production of therapeutic cells.This project will harness fluid dynamics at the microscale using integrated microfluidic transfection and separation operations, as well as vision- and data analytics-enabled robotics to help automate the cell culture process. As a testbed, the study will apply the technologies to an induced pluripotent stem cell (iPSC) derived retinal organoid manufacturing process. The new technology and process control will be applied to generate functional retinal cell grafts, i.e., 3D engineered retinal constructs from iPSC-derived retinal organoids. The project can improve regenerative strategies through greater integration of iPSC genetic engineering, robotics-based culture, and new label-free cell selection methods to purify desired cell types that are consistent with Good Manufacturing Practices production. The team brings together three core expertises to accomplish the transformation: a current Good Manufacturing Practices process for retinal organoid manufacturing from human induced pluripotent stem cell culture; microfluidics-enabled cell transfection, characterization, and separation unit operations; and a capability for robotics-enabled cell processing and image analysis. The first objective is to apply a cell microfluidic transfection platform that uses choreographed mechanical deformations to convectively deliver large gene-editing CRISPR/Cas9 and DNA cargo to iPSCs. Moreover, because patient-derived specimens vary from donor to donor, the study will also characterize the biomechanical properties of donor cells to optimize the transfection. The second objective is to combine automated cell culture systems and machine learning to develop a robotic platform capable of performing high quality automated iPSC generation, CRISPR-correction, and retinal differentiation. The third objective examines how mixtures of retinal cells can better produce functional retinal grafts using a label-free cell separation microfluidic technology. Together the intellectual merits of this project will be to demonstrate an integrated approach to generate 3D engineered retinal constructs to address inherited blindness. This Future Manufacturing award was supported by Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.