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

人类多能干细胞(HPSCs)以及由其分化而来的细胞预计对基于细胞的治疗和工程化组织的需求很大。为了满足这一需求，需要提供足够数量的这些细胞来供应实验室和未来的治疗医生。因此，它们实际上需要制造。三维(3D)搅拌悬浮培养，由于其可伸缩性和易于自动化，是一个有希望的平台，以满足这种需求，同时潜在地显著降低生产成本。然而，使用该技术培养未分化的hPSCs是近几年来相对较新的发展，对流体环境和搅拌过程对细胞的作用知之甚少。各种特征的表现。这项研究将研究通过流体环境施加的剪切力对细胞的影响，包括它们的基因表达、分化和生存。预计这项研究的新发现和工具将适用于其他与医学相关的细胞类型的研究，如神经球、胰岛和成体干细胞，潜在地解决了降低其当前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推广和教育活动，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；在尖端研究方面招聘和指导本科生和研究生，特别是女性和代表性较低的少数族裔；通过扩展努力吸引当地K-12教师和学生。目前的研究旨在通过开展三项任务来解决与干细胞生物制造相关的关键知识缺口：1)确定流体剪切在干细胞聚集体生长中的作用；2)确定剪切诱导的机械转导的生物分子机制；以及3)设计和验证一种将对干细胞施加均匀剪切的悬浮培养。这项研究的预期成果包括：1)剪切力、集合体大小和细胞命运之间的定量关联；2)剪切力控制的机械转导的分子途径模型；3)对干细胞及其集合体施加和量化均匀剪切力输入的新方法。总而言之，这些结果有望实现一种新的方法，使用流体微环境(例如，流体剪切)作为关键输入参数来扩增和分化干细胞。

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.