3D Culture of mES Cells in Four-Compartment Bioreactors

描述(申请人提供)：胚胎干细胞(ES)研究和放大以开发可能的临床疗法受到现有2D培养皿方法的限制。我们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3D介质灌流条件下扩增ES细胞。生物反应器的设计允许完整的氧合作用和营养物质和废物进出细胞的有效转移，以高密度培养，涉及细胞内最小的溶质梯度。此外，交织纤维提供了允许细胞形成3D结构的支架，其中细胞聚集体的大小受到中空纤维之间的间隔的限制。我们认为，我们的生物反应器提供的良好控制和多功能的培养环境对于大规模扩增未分化的ES细胞和使用多种策略定向分化ES细胞是理想的，包括控制细胞暴露于分子试剂和与成熟细胞隔室共培养。这个为期两年的项目的目标是通过在我们的生物反应器的实验室规模版本中扩大和维护未分化的小鼠胚胎干细胞(MES)，向将我们的生物反应器技术应用于ES细胞研究迈出第一步。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流3D环境中扩增和维持，并且在这个3D培养模型中，MES细胞和成纤维细胞饲养层细胞可以通过在一个循环内灌流的两个单独的生物反应器中培养来维持MES细胞的多能性(分区共培养)。该项目的具体目标是：1.开发一种用于未分化MES细胞的生物反应器扩增和维护的3D培养模型，将MES细胞与成纤维细胞饲养层细胞隔室共培养，并允许酶促MES细胞的采集。该研究计划包括以下任务：1.1开发MES细胞和饲养层细胞直接共培养的3D培养模型；1.2开发MES细胞和饲养层细胞分区共培养的培养模型；1.3开发从完整的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、潜在的自动化生物反应器扩增胚胎干细胞；以及2)在灌流的3D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.