3D Culture of mES Cells in Four-Compartment Bioreactors

描述（由申请人提供）：胚胎干（ES）细胞的研究和扩大规模以开发可能的临床治疗方法受到现有二维培养皿培养方法的限制。我们提出的研究提出了一种新的方法，即在四室中空纤维生物反应器中，在三维介质灌注条件下扩增胚胎干细胞。生物反应器的设计允许完整的氧合和营养物质和废物的有效转移进出细胞，在高密度培养中涉及细胞间隔内最小的溶质梯度。此外，交织纤维提供了一个支架，允许细胞形成3D结构，其中细胞聚集体的大小受到中空纤维之间的间距的限制。我们提出，我们的生物反应器提供的良好控制和通用的培养环境是理想的，既可以大规模扩增未分化的胚胎干细胞，也可以使用多种策略进行胚胎干细胞的定向分化，包括控制细胞暴露于分子试剂和与成熟细胞区分开共培养。这个为期两年的项目的目标是通过在实验室规模的生物反应器中扩展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胚胎干细胞研究的第一步。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环境中增殖和维持，并且在这个3D培养模型中，通过在一个回路内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（区隔共培养），可以维持mES细胞的多能性。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区隔共培养，并允许酶促mES细胞收获。研究计划包括以下任务：1.1建立mES细胞与饲养细胞直接共培养的三维培养模型；1.2建立mES细胞与饲养细胞分区共培养的培养模式；1.3制定从完整生物反应器中获取酶促mES细胞的方案。该项目的完成将为未来的研究提供坚实的基础：1)大规模，潜在的自动化胚胎干细胞生物反应器扩增；2)灌注三维组织密度条件下基于生物反应器的胚胎干细胞定向分化。

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.