An Integrated Fluorescent Biosensor, Imaging, and Analysis Toolkit for Islet Biologics

摘要 这个为期4年的R 01项目的目标是开发一个创新和全面的工具包，包括 一系列基于荧光蛋白（FP）的生物传感器，微流体芯片上的胰岛，智能手机 荧光显微镜和成像分析应用程序，优化用于人 胰岛作为1型糖尿病（T1 D）的细胞治疗，以及表征表型和 用于未来T1 D治疗的新兴生物制剂的生理学。 我们建议使用基因编码的荧光生物传感器来评估β- 细胞和胰岛，并进一步定义各种有前途的生物制剂的效力、表型和生理学 T1 D细胞替代疗法凭借我们强大的团队和广泛的专业知识， 根据初步结果，我们将追求以下两个具体目标： 1.开发和优化荧光生物传感器，以监测β细胞和胰岛中的关键生理过程。 2.将生物传感器与微流控阵列、智能手机荧光显微镜、定制的 用于胰岛生物制品的标准化、高含量表征的成像分析应用。 本项目的成果将是一个经济和集成的系统，方便和可靠 本发明提供了人胰岛、异种胰岛、包封的胰岛和干细胞衍生的β细胞的表征。这些在 体外试验创新性地评估了β细胞和胰岛的几个关键生理参数， 可用于标准化和质量控制，也可作为重要的筛选工具，以提高生成， 储存和运输下一代生物制剂。我们建议的工作将产生重大影响， 目前和未来的糖尿病细胞替代疗法的发展。

Abstract The objective of this 4-year R01 project is to develop an innovative and comprehensive toolkit, including a collection of fluorescent protein (FP) based biosensors, a microfluidic Islet-On-a-Chip, a smartphone fluorescence microscope, and an imaging analysis application, optimized for the potency evaluation of human islets as cell-based therapy in Type 1 diabetes (T1D), as well as for characterization of the phenotype and physiology of emerging new biologics for future T1D treatment. We propose to use genetically encoded fluorescent biosensors to evaluate key physiological parameters of β- cells and islets, and furthermore, to define the potency, phenotype, and physiology of various promising biologics for T1D cell replacement therapy. Leveraging the complementary expertise of our strong team and our extensive preliminary results, we will pursue the following two Specific Aims: 1. Develop and optimize fluorescent biosensors to monitor key physiological processes in β-cells and islets. 2. Integrate biosensors with microfluidic arrays, a smartphone fluorescence microscope, a customized imaging analysis application for standardized, high-content characterization of islet biologics. The outcome of this project will be an economic and integrated system for convenient and reliable characterization of human islets, xenogeneic islets, encapsulated islets, and stem-cell-derived β-cells. These in vitro assays, which innovatively evaluate several key physiological parameters of β-cells and islets, can not only be used for standardization and quality control, but also be important screening tools to improve the generation, storage, and transport of next-generation biologics. Our proposed work will generate significant impacts on development of current and future cell replacement therapy for diabetes.