Understanding the Role of Fluidic Microenvironment in Stem Cell Suspension Culture toward Scalable Biomanufacturing

人类多能干细胞（hPSCs）和从这些细胞分化出来的细胞预计在基于细胞的治疗和工程组织中有很大的需求。为了满足这一需求，这些细胞需要有足够的数量来供应实验室和将来的治疗医生。因此，它们实际上需要被制造出来。三维（3D）搅拌悬浮培养由于其可扩展性和易于自动化，是满足这种需求的一个有前途的平台，同时有可能显著降低生产成本。然而，使用这种技术培养未分化的人造血干细胞在过去几年中是一个相对较新的发展，并且对流体环境和搅拌过程对细胞的作用知之甚少。各种特征的表达本研究将探讨通过流体环境施加剪切力对细胞的影响，包括细胞的基因表达、分化和存活。预计这项研究的新发现和工具将适用于其他医学相关细胞类型的研究，如神经球、胰岛和成体干细胞，有可能解决当前降低其生产效率的现有瓶颈。结合概述的研究活动，该项目将开发和整合几项外展和教育活动，包括：为干细胞生物学和机械工程课程开发新的交叉课程材料；招收和指导本科生和研究生，特别是女性和少数族裔，从事前沿研究；并通过外展活动吸引当地K-12教师和学生。目前的研究旨在解决与干细胞生物制造相关的关键知识缺口，通过承担三个任务：1)确定流体剪切在干细胞聚集体生长中的作用；2)确定剪切诱导的力学转导的生物分子机制；3)设计和验证悬浮培养，使干细胞受到均匀剪切。本研究工作的预期成果包括：1)剪切应力、聚集体大小和细胞命运之间的定量相关性；2)剪切控制的机械转导的分子途径模型；3)应用和量化均匀剪切应力输入干细胞及其聚集体的新方法。总的来说，这些结果有望实现一种利用流体微环境（例如流体剪切）作为关键输入参数来扩展和分化干细胞的新方法。

Human pluripotent stem cells (hPSCs) and cells that differentiate from these are anticipated to be in great demand for cell-based therapies and engineered tissues. In order to meet this demand, these cells need to be made available in sufficient number to supply both laboratories and, in the future, treating physicians. Therefore, they actually need to be manufactured. Three-dimensional (3D) stirred suspension culture, due to its scalability and ease of automation, is a promising platform for meeting such a demand, while potentially reducing the cost of production significantly. However, culturing undifferentiated hPSCs using this technique is a relatively new development in the past several years, and little is known about the role of the fluid environment and stirring process on the cells? expression of various traits. This research will investigate the effect of shear forces applied through the fluid environment on the cells, including their gene expression, differentiation, and survival. The new discoveries and tools anticipated from this study are expected to be applicable to studies of other medically relevant cell types, such as neurospheres, pancreatic islets, and adult stem cells, potentially addressing the existing bottlenecks that reduce the efficiency of their current production. In conjunction with the outlined research activities, the project will develop and integrate several outreach and educational activities, including: developing new crosscutting course materials for stem cell biology and mechanical engineering courses; recruiting and mentoring both undergraduate and graduate students, particularly women and underrepresented minorities, in cutting-edge research; and engaging local K-12 teachers and students through outreach efforts.The current research aims to address a critical knowledge gap relevant to stem cell biomanufacturing, by undertaking three tasks: 1) determining the roles of fluidic shear on growing stem cell aggregates; 2) identifying biomolecular mechanisms of shear-induced mechanotransduction; and 3) designing and verifying a suspension culture that will impose uniform shear on the stem cells. Expected outcomes of this research effort include 1) quantitative correlations between shear stress, aggregate size, and cell fates, 2) molecular pathway models for shear-controlled mechanotransduction, and 3) new means to apply and quantify homogeneous shear stress inputs to the stem cells and their aggregates. Collectively, these outcomes are expected to enable a novel approach to expand and differentiate stem cells using the fluidic microenvironment (e.g., fluid shear) as a critical input parameter.