3D Culture of mES Cells in Four-Compartment Bioreactors

四室生物反应器中 mES 细胞的 3D 培养

• 批准号: 7021496
• 负责人: JOERG C. GERLACH
• 金额: $ 20.34万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-06 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021496
• 关键词: animal tissue; bioreactors; biotechnology; cell differentiation; cell proliferation; embryonic stem cell; mixed tissue /cell culture; pluripotent stem cells; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): Embryonic stem (ES) cell research and scale-up for development of possible clinical therapies is limited by the existing 2D dish culture methods. Our proposed studies present a new approach, in which ES cells are expanded under 3D medium perfusion conditions within four-compartment hollow fiber-based bioreactors. The design of the bioreactors allows integral oxygenation and efficient transfer of nutrients and waste products to and from the cells, cultured at high density involving minimal solute gradients within the cell compartment. Additionally, the interwoven fibers provide a scaffold allowing the cells to form 3D structures where the size of cellular aggregates is limited by the spacing between the hollow fibers. We propose that the well-controlled and versatile culture environment provided by our bioreactor is ideal for both large-scale expansion of undifferentiated ES cells and directed differentiation of ES cells using numerous strategies, including controlled exposure of the cells to molecular reagents and compartmentalized co-culture with mature cells. The objective of this 2-year project is to take the first step toward applying our bioreactor technology to ES cell research, by expanding and maintaining undifferentiated mouse embryonic stem (mES) cells in laboratory-scale versions of our bioreactor. We hypothesize that undifferentiated mES cells can be expanded and maintained in the perfused 3D environment provided by our bioreactor, and that within this 3D culture model mES cell pluripotency can be maintained by culturing mES cells and fibroblast feeder cells in two separate bioreactors perfused within one circuit (compartmentalized co-culture). The specific aim of the project is to: 1. Develop a 3D culture model for bioreactor expansion and maintenance of undifferentiated mES cells, incorporating compartmentalized co-culture of mES cells with fibroblast feeder cells and allowing for enzymatic mES cell harvesting. The research plan consists of the following tasks: 1.1 Develop the 3D culture model incorporating direct co-culture of mES cells and feeder cells; 1.2 Develop the culture model incorporating compartmentalized co-culture of mES cells and feeder cells; and 1.3 Develop a protocol for enzymatic mES cell harvesting from intact bioreactors. Completion of the project will provide a solid foundation for future studies on: 1) large-scale, potentially automated bioreactor expansion of ES cells; and 2) bioreactor-based directed differentiation of ES cells under perfused 3D tissue-density conditions.

描述（由申请人提供）：胚胎干 (ES) 细胞研究和开发可能的临床疗法的规模化受到现有 2D 培养皿培养方法的限制。我们提出的研究提出了一种新方法，其中 ES 细胞在基于四室中空纤维的生物反应器内的 3D 介质灌注条件下进行扩增。生物反应器的设计允许整体充氧，并有效地将营养物质和废物转移到细胞中或从细胞中转移出来，并以高密度培养，涉及细胞室内最小的溶质梯度。此外，交织的纤维提供了一个支架，允许细胞形成 3D 结构，其中细胞聚集体的尺寸受到中空纤维之间的间距的限制。我们认为，我们的生物反应器提供的良好控制和多功能培养环境非常适合未分化 ES 细胞的大规模扩增和使用多种策略定向分化 ES 细胞，包括控制细胞暴露于分子试剂和区室化共培养物。与成熟细胞一起培养。这个为期 2 年的项目的目标是通过在实验室规模的生物反应器中扩增和维持未分化的小鼠胚胎干 (mES) 细胞，迈出将我们的生物反应器技术应用于 ES 细胞研究的第一步。我们假设未分化的 mES 细胞可以在我们的生物反应器提供的灌注 3D 环境中扩增和维持，并且在该 3D 培养模型中，可以通过在一个回路中灌注的两个单独的生物反应器中培养 mES 细胞和成纤维细胞饲养细胞来维持 mES 细胞的多能性（区室化共培养）。该项目的具体目标是： 1. 开发用于生物反应器扩增和维持未分化 mES 细胞的 3D 培养模型，将 mES 细胞与成纤维细胞饲养细胞的区室共培养相结合，并允许酶促 mES 细胞收获。研究计划包括以下任务： 1.1 开发mES细胞和饲养细胞直接共培养的3D培养模型； 1.2 开发mES细胞和饲养细胞分室共培养的培养模型； 1.3 制定从完整生物反应器中收获酶促 mES 细胞的方案。该项目的完成将为未来的研究奠定坚实的基础：1）ES细胞的大规模、潜在自动化生物反应器扩增； 2) 在灌注 3D 组织密度条件下基于生物反应器的 ES 细胞定向分化。