Developing an optimized cell based platform for assays of the gastrointestinal enteroendocrine system

项目摘要 人类的肠道是一个非凡的器官，它储存和分泌各种激素 来自肠内分泌(EEC)细胞。这些激素在调节人体健康方面起着关键作用。 进食行为和饱腹感，以及它们的失调会导致暴饮暴食和许多其他 代谢紊乱。出于这些原因，在治疗学市场上存在着对In的需求 体外肠道EEC细胞平台，精确地概括了活体肠道的生理。 为了满足这一需求，阿尔蒂斯生物系统公司，一家早期生物技术公司， 与学术实验室的科学家合作开发了一种新型的原代干细胞-- 基于体外肠道模型(称为RepliGut)。我们已经完成了SBIR第一阶段计划 通过优化RepliGut平台来丰富EEC的一个亚型--肠嗜铬细胞 细胞，并增加屏障的完整性。我们已经开发出一种简单但有效的方法 与起始培养条件相比，显著增加了EEC细胞的形成。 我们已经研究了强制分化为EEC谱系的信号分子 分配，5-羟色胺的量化分析，并调查传代和供体变异。我们 用小型复合屏验证了该平台对EC分泌5-羟色胺的能力 细胞。第一阶段SBIR的所有拟议里程碑都已完成，从而提供了坚实的 为第二阶段SBIR应用奠定基础。这一第二阶段提案的重点是继续 优化EEC构型以满足市场对高通量筛选的需求 化验。除了EC细胞，我们将把我们的研究扩展到另一个重要的亚型， 肠内分泌L细胞，对食物的摄入作出反应，分泌胰升糖蛋白-1和胰淀粉酶。 更多的特征将集中在96孔内电池行为的一致性上 格式化。在作为蜂窝电池使用之前，将确认井与井和板与板之间的低差异 测试平台。将进一步调查潜在的基于区域、性别和年龄的差异 检测来自5个捐赠者和所有6段肠道的干细胞。平台将是 通过筛选大量代谢化合物进行了验证。的性能特征 然后，我们将对该平台进行评估，将我们的内部分析与发货至 合作实验室。

Project Summary The human intestine is a remarkable organ which stores and secretes a variety of hormones from enteroendocrine (EEC) cells. These hormones play critical roles in regulating human feeding behavior and satiety, and their dysregulation leads to overeating and a host of other metabolic disorders. For these reasons, there is a need in the therapeutics marketplace for in vitro intestinal EEC cell platform that precisely recapitulates the physiology of in vivo intestines. To meet this need, Altis Biosystems Inc., an early stage biotechnology company, has collaborated with scientists at academic laboratories to develop a novel, primary-stem-cells- based, in vitro intestinal model (termed RepliGut). We have finished the SBIR Phase I program by optimizing the RepliGut platform to enrich enterochromaffin (EC) cells, a subtype of EEC cells, and increase barrier integrity. We have developed a simple but efficient method that significantly increases the formation of EEC cells compared with the starting culture conditions. We have investigated signaling molecules for forced differentiation towards EEC lineage allocation, quantified assays for serotonin, and investigated passage and donor variation. We validated the platform with a small-scale compound screen for serotonin secretion from EC cells. All proposed milestones in the Phase I SBIR were accomplished, thus providing a solid foundation for this Phase II SBIR application. The focus of this Phase II proposal is to continue the optimization of EEC formation to meet the market needs for high-throughput screening assays. Besides EC cells, we will extend our research to the other important subtype, enteroendocrine L-cells, which secrete GLP-1 and PYY in response to the ingestion of food. Additional characterizations will be focused on the uniformity of cell behaviors within a 96-well format. Low well-to-well and plate-to-plate variation will be confirmed before use as a cellular assay platform. Potential regional-, sex- and age-based variations will be further investigated by testing stem cells derived from 5 donors and all 6 sections of intestine. The platform will be validated by screening a large library of metabolic compounds. Performance characteristics of the platform will then be evaluated in comparisons of our in-house assays vs. kits shipped to collaborating laboratories.