A perfluorocarbon-based culture device for beta cell biology applications (Phase

摘要 传统的培养容器不是为生理氧气输送而设计的。在普通塑料器皿中培养细胞和组织时，通常会观察到高氧和缺氧，这两者都与细胞功能降低和死亡有关。提供氧合的适当手段对于其中分化结果取决于氧张力水平的干细胞应用也是至关重要的。我们通过设计一种新的培养装置“氧气三明治”来解决这个问题。这个简单的系统被设计成通过基底透气的全氟化碳-硅（PFC/Si）膜以准生理方式输送氧气。我们的长期目标是将该系统建立为组织培养的新标准，既适用于氧扩散速率受限的应用（如3D培养），也适用于需要精确调节氧合以将干细胞分化引导到所需方向的应用。我们将把我们的概念验证工作集中在胰岛/β细胞生物学上。这是一个迅速扩大的市场，包括临床应用（胰岛移植），对几个物种的成人/胎儿胰岛的积极体外研究，以及临床前干细胞研究，有可能在未来5-10年内彻底改变糖尿病的治疗。我们模型选择的相关性通过两个有据可查的观察结果得到了强调：[1]胰腺β细胞（目前用于糖尿病临床治疗的最终产品）对亚生理氧浓度和超生理氧浓度高度敏感;和[2]干细胞分化为β细胞（世界范围内研究的主题是替代尸体胰岛用于未来的临床用途）非常依赖于不断变化的氧张力。我们的I期研究旨在证明，在PFC/Si培养的胰岛中观察到的体外存活和功能增强也导致使用边缘质量异种移植模型（人胰岛移植到糖尿病nu/nu免疫缺陷小鼠中）的临床前移植结局更好。在成功完成这些研究后，我们的II期提案基于以下具体目标：（1）规模扩大和定义PFC/Si培养装置大规模生产的制造工艺（QA和QC，灭菌）;以及（2）人类胰岛和胚胎干细胞（hESc）的生物学测试和再现性研究。这种应用程序得益于具有高度互补专业知识的一流团队的组装。我们的第一阶段结果强烈支持这一建议的可行性。我们研究的成功将填补我们在体外保存胰岛细胞功能和存活能力方面的一个广泛公认的空白，证实该系统是β细胞生物学和分化研究的潜在新标准。由于这种积极的结果可能最终加速新一代β细胞替代疗法的适用性，因此该项目与美国国立糖尿病、消化和肾脏疾病研究所（NIDDK）的使命密切相关。这些研究的成功也将为我们的氧增强技术在许多其他细胞培养应用中的优越性提供原则证明。

ABSTRACT Conventional culture vessels are not designed for physiological oxygen delivery. Both hyperoxia and hypoxia - commonly observed when culturing cells and tissues in regular plasticware- have been linked to reduced cellular function and death. An adequate means to provide oxygenation is also critical for stem cell applications in which the differentiation outcome is dependent on oxygen tension levels. We have addressed this problem by devising a novel culture device, the "oxygen sandwich". This simple system is designed to deliver oxygen in a quasi-physiological fashion by means of a basal air-permeable perfluorocarbon-silicone (PFC/Si) membrane. Our long-term goal is to establish this system as a new standard for tissue culture, both for applications in which oxygen diffusion rates become limiting (such as 3D culture) and for those that require precise adjustments of oxygenation to steer stem cell differentiation in the desired direction. We will focus our proof-of- concept work on islet/beta cell biology. This is a rapidly expanding market that includes clinical uses (islet transplantation), active in vitro research on adult/fetal islets of several species, and pre-clinicl stem cell research with the potential to revolutionize the treatment of diabetes within the next 5-10 years. The pertinence of our model choice is highlighted by two well-documented observations: [1] Pancreatic beta cells (the current end product used in clinical therapies for diabetes) are highly sensitive to sub- and super-physiological oxygen concentrations; and [2] Stem cell differentiation into beta cells (the subject of worldwide research to replace cadaveric islets for future clinical uses) is exquisitely dependent on evolving oxygen tensions. Our Phase I studies aimed at demonstrating that the enhanced in vitro survival and function observed in PFC/Si-cultured islets also resulted in better pre-clinical transplantation outcomes using a marginal mass xenotransplantation model (human islets into diabetic nu/nu immunodeficient mice). After the successful completion of these studies, our Phase II proposal is based on the following specific aims: (1) Scaling-up and definition of manufacturing process (QA & QC, sterilization) for mass production of PFC/Si culture devices; and (2) biological testing and reproducibility studies in human islets and embryonic stem cells (hESc). This application benefits from the assembly of first-rate teams with highly complementary expertise. Our Phase I results are strongly supportive of the feasibility of this proposal. Success in our research would fill a widely acknowledged gap in our ability to preserve islet cell function and survival in vitro confirming this system as a potential new standard for beta cell biology and differentiation studies. As such positive outcome might ultimately speed up the applicability of new-generation beta cell replacement therapies, this project is greatly relevant to the mission of the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK). Success in these studies will also provide proof of principle of the superiority of our oxygen enhancing technology for many other cell culture applications.