Data-Driven Model Development for Cost-Effective, Reliable Cardiac Tissue Manufacturing

美国的心脏病死亡率正在上升，尤其是年轻人的死亡。天然人类心脏组织的有限可用性阻碍了研究、药物发现和临床心脏再生的努力。用干细胞衍生的心脏组织（由功能性收缩的心脏细胞称为心肌细胞）进行治疗，具有很高的实现临床有意义结果的潜力。然而，治疗效果所需的细胞数量估计为每位患者10 - 100亿个。目前制造工程心脏组织的技术挑战和有限的可扩展性阻碍了临床应用的进展。该研究项目的主要目标是设计可靠且具有成本效益的制造工艺来生产工程心脏组织。该项目采用了一种新颖的可扩展的一步式心脏组织生产平台，结合了实验和建模研究，并利用它们的集成来了解不同操作条件对心脏组织制造过程的影响。这项研究有可能改变制造用于药物测试和心脏再生应用的工程心脏组织的能力。该项目的劳动力发展方面包括通过联合高级课程开发，让本科生、研究生和实习工程师参与进来，与持续努力的团队一起支持当地产业，包括为新的跨学科先进制造工程专业建立生物制造轨道。并与阿拉巴马州立大学生命科学系合作，培养学生对生物制造、组织工程和生物打印至关重要的基础知识。为了支持长期的劳动力发展，这项跨学科的研究正在被纳入多个K-12外展工作。本科生研究人员和阿拉巴马州立大学暑期实习生将是研究团队的主要成员。目前制造工程心脏组织的技术挑战和有限的可扩展性，包括普遍使用的预分化心肌细胞（CMs），阻碍了临床应用的进展。在可扩展的搅拌烧瓶生物反应器中缺乏CM成熟和可变结果是治疗性CM生产的关键障碍。Lipke教授最近建立了一个新的平台，直接将水凝胶封装的人类诱导多能干细胞（hiPSCs）分化为工程心脏组织。该研究采用定制的微流体系统将hiPSCs快速封装在高度均匀的微球体中，微球体具有可控的大小和形状。通过改变组织轴比，分化cm在自发收缩过程中会受到各向异性的机械刺激；这在可扩展的悬浮培养中通常是不可能实现的，并且具有推动CM成熟的独特潜力。为了利用这种潜在的变革性方法进行单阶段心脏组织生产，需要一个强大的制造工艺。因此，该项目的首要目标是设计可靠且经济高效的制造工艺，以实现可重复、经济高效和高质量的心脏组织生产。为了实现这一目标，该项目追求3个目标：(1)研究利用人类诱导多能干细胞（hiPSC）水凝胶微球包封和悬浮培养直接分化的单阶段过程形成的工程心脏组织的特征；(2)利用数据驱动模型确定在悬浮培养和旋转瓶生物反应器系统中可靠地制造工程心脏组织的最佳或接近最佳参数范围区域；(3)测试水凝胶微球在更容易扩展的旋转瓶生物反应器系统中支持单阶段工程心脏组织制造的能力。在数据驱动模型的指导下，该项目研究了水凝胶微球形状和大小、聚合物前体浓度、交联时间和光照强度以及施加的剪切应力对单阶段制造心脏组织的安全性和有效性属性的影响。正在进行实时功能监测和深入的初始和终点表征，以评估初始和最终的细胞表型。该方法整合了实验研究和数据驱动模型，通过设计适应性实验活动，直接从水凝胶封装的hipsc中单阶段制造心脏组织。通过对数据驱动模型的逆分析，确定了工艺参数的范围，以最大限度地减少工程心脏组织的电生理和机械收缩属性的可变性，这对其可靠制造至关重要。组织工程、聚合物科学和微流体学的基础知识将被用来直接从hipsc中制造心脏组织微球，而不是使用预分化的心脏细胞。这种方法是独特的，通过消除多个细胞处理步骤和使用聚乙烯乙二醇纤维蛋白原光交联基质，它有可能在制造用于药物测试和心脏再生应用的工程心脏组织的能力方面提供革命性的进步。

U.S. mortality rates from heart disease are increasing, driven particularly by deaths in younger adults. Limited availability of native human heart tissue impedes research, drug discovery, and clinical cardiac regeneration efforts. Treatment with stem cell-derived heart tissues, composed of functional contracting heart cells called cardiomyocytes, has high potential to achieve clinically meaningful outcomes. However, the number of cells required for therapeutic benefit has been estimated to be 1-10 billion per patient. Technical challenges and limited scalability of current processes for manufacturing engineered heart tissues hinder progress toward clinical use. The primary goal of this research project is to design reliable and cost-effective manufacturing processes for producing engineered heart tissues. The project employs a novel scalable, one-step heart tissue production platform, combines experimental and modeling studies, and leverages their integration to understand how different operating conditions impact the heart tissue manufacturing process. The research has the potential to transform the ability to manufacture engineered heart tissue for drug testing and heart regeneration applications. Workforce development aspects of the project include engagement of undergraduate and graduate students and practicing engineers through joint upper level curriculum development, teaming with ongoing efforts to support local industry, including establishing a biomanufacturing track for the new cross-disciplinary Advanced Manufacturing engineering specialization, and collaborating with Alabama State University's Life Sciences Department to train students in fundamentals important to biomanufacturing, tissue engineering and bioprinting. To support long-term workforce development, this interdisciplinary research is being incorporated into multiple K-12 outreach efforts. Undergraduate researchers and Alabama State University summer interns will be key members of the research team. Technical challenges and limited scalability of current processes for manufacturing engineered cardiac tissues, including the ubiquitous use of pre-differentiated cardiomyocytes (CMs), has hindered progress toward clinical use. Lack of CM maturation and variable outcomes in scalable stirred flask bioreactors are critical barriers in therapeutic CM production. Professor Lipke has recently established a novel platform to directly differentiate hydrogel-encapsulated human induced pluripotent stem cells (hiPSCs) into engineered cardiac tissues. The research employs a custom microfluidic system to rapidly encapsulate hiPSCs in highly uniform microspheroids with controllable size and shape. By modifying tissue axial ratio, differentiating CMs will experience anisotropic mechanical stimulation during spontaneous contraction; this has been typically impossible to achieve in scalable, suspension cultures and has the unique potential to drive CM maturation. To leverage this potentially transformative approach for single-stage cardiac tissue production, a robust manufacturing process is needed. Thus, the overarching goal of this project is to design reliable and cost-effective manufacturing processes for reproducible, cost-effective, and high-quality production of cardiac tissues. To achieve this goal, the project pursues 3 aims: (1) Investigate the characteristics of engineered cardiac tissues formed using a single-stage process of human induced pluripotent stem cell (hiPSC) hydrogel microspheroid encapsulation and direct differentiation in suspension culture, (2) Identify regions of optimal or near-optimal parameter ranges to manufacture engineered cardiac tissues reliably in suspension culture and spinner flask bioreactor systems using data-driven models, and (3) Test the ability of hydrogel microspheres to support single-stage engineered cardiac tissue manufacturing in a more readily scalable, spinner flask bioreactor system. Guided by data-driven models, the project investigates the effects of hydrogel microspheroid shape and size, polymer precursor concentration, crosslinking time and light intensity, and applied shear stress, on safety and efficacy attributes of single-stage manufactured cardiac tissues. Real-time functional monitoring and in-depth initial and end-point characterization are being performed to assess initial and resulting cell phenotypes. The approach integrates experimental studies and data-driven models through design of adaptive experimental campaigns for manufacturing cardiac tissue in a single-stage process directly from hydrogel encapsulated hiPSCs. Using inverse analysis on the data-driven models, the range of process parameters is being determined to minimize variability in electrophysiological and mechanical contraction attributes of engineered cardiac tissues, which is essential for their reliable manufacturing. Fundamentals of tissue engineering, polymer science, and microfluidics will be drawn upon to manufacture cardiac tissue microspheres directly from hiPSCs, rather than employing pre-differentiated cardiac cells. This approach is unique and, by eliminating multiple cell handling steps and employing a polyethyleneglycol-fibrinogen photocrosslinkable matrix, it has the potential to provide a transformative advance in the ability to manufacture engineered heart tissue for drug testing and cardiac regeneration applications.