Engineering a Human Microphysiological System for the Characterization of Islet-Immune Interactions

设计人体微生理系统来表征胰岛免疫相互作用

• 批准号: 10453211
• 负责人: Ashutosh Agarwal
• 金额: $ 102.09万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453211
• 关键词: Engineering; Human; Microphysiological; System; Characterization

Summary Three dimensional (3D) microphysiological systems (MPS) represent a powerful intermediate model system employing human cells and tissues capable of bridging in vitro studies and clinical trials. We propose to create an integrated MPS platform to more accurately model the complex cellular interactions involved in human type 1 diabetes (T1D) pathogenesis. We previously generated an MPS containing novel extracellular matrix hydrogels that support sustained islet function and T cell migration along the islet cell surface in 3D (CHIB), and in first-of-their-kind studies, we demonstrated antigen-specific IGRP-reactive human CD8 T cells resulted in targeted β-cell killing (CMAI). Here, we propose an interdisciplinary effort to integrate and expand the MPS platform (referred to as the islet-immune Chip (iiChip)), as well as the cell-based technologies facilitating testing of antigen-specific T cells, isogenic cellular systems capable of deriving multiple cellular lineages, and genome editing technologies for use by the broader HIRN community. Specifically, we will utilize islets or islet- like spheroids, endothelial cell monolayers, and innate and adaptive immune cells, including dendritic cells (DCs), macrophages, CD4+ conventional T cells (Tconv), CD8+ cytotoxic T cells (CTLs), and regulatory T cells (Tregs), to model the spatial configuration and complex cellular interactions involved in human T1D pathogenesis. We hypothesize that this optimized 3D iiChip will facilitate in situ interrogation of Ag- specific and genotype-phenotype interactions that are essential in T1D pathogenesis as well as the mechanistic effects of immunomodulatory therapies with spatial and temporal control. Experimental deliverables will include the ability to assess islet:immune interactions utilizing real-time high-resolution imaging and quantitation of cellular interactions, trafficking, extravasation, and β-cell function/survival. Key features of the iiChip will involve the integration of in-line sensors and bioreporters, spatial and temporal control of inputs for defined stimulation, and integration of matrices with the capacity for fluidic and cellular recirculation, measurement of soluble and cellular readouts in long-term cell culture. In addition, gene edited induced pluripotent stem cells (iPSC) from male and female donors with T1D-risk associated HLA will be available for the generation of immune, endothelial, and endocrine cells that are essential for building an isogenic “disease-on-a-chip” model. When loaded with primary human cells or isogenic iPSC-derived materials (i.e., endothelial, immune, and β-cells), this iiCHIP will enable dynamic interrogation of genotype-phenotype interactions, antigen-specific β-cell killing, and effects of immunomodulatory therapies within a fluidic 3D microenvironment. The iiChip will enable mechanistic studies capable of expediting clinical interventions aimed at inhibition of immune-mediated β-cell destruction, enhancing immune regulation, and testing of β-cell restorative therapies.

概括 三维 (3D) 微生理系统 (MPS) 代表了强大的中间模型系统 采用能够桥接体外研究和临床试验的人体细胞和组织。我们建议创建 集成 MPS 平台，可更准确地模拟人类类型中涉及的复杂细胞相互作用 1 糖尿病（T1D）发病机制。我们之前生成了包含新型细胞外基质的 MPS 支持持续胰岛功能和 T 细胞沿 3D 胰岛细胞表面迁移的水凝胶 (CHIB)， 在同类首个研究中，我们证明了抗原特异性 IGRP 反应性人类 CD8 T 细胞 靶向β细胞杀伤（CMAI）。在这里，我们提出跨学科的努力来整合和扩展 MPS 平台（称为胰岛免疫芯片（iiChip）），以及基于细胞的技术促进 测试抗原特异性 T 细胞、能够衍生多种细胞谱系的同基因细胞系统，以及 供更广泛的 HIRN 社区使用的基因组编辑技术。具体来说，我们将利用胰岛或胰岛- 如球体、单层内皮细胞​​以及先天性和适应性免疫细胞，包括树突状细胞 (DC)、巨噬细胞、CD4+ 常规 T 细胞 (Tconv)、CD8+ 细胞毒性 T 细胞 (CTL) 和调节性 T 细胞 (Tregs)，模拟人类 T1D 中涉及的空间配置和复杂的细胞相互作用 发病。我们假设这种优化的 3D iiChip 将有助于 Ag-的原位询问 特异性和基因型-表型相互作用对于 T1D 发病机制以及 具有空间和时间控制的免疫调节疗法的机械效应。实验性的 可交付成果将包括利用实时高分辨率评估胰岛：免疫相互作用的能力 细胞相互作用、运输、外渗和 β 细胞功能/存活的成像和定量。钥匙 iiChip 的功能将涉及在线传感器和生物报告器的集成、空间和时间控制 用于定义刺激的输入，以及具有流体和细胞能力的矩阵的集成 再循环，长期细胞培养中可溶性和细胞读数的测量。此外，基因编辑 来自具有 T1D 风险相关 HLA 的男性和女性捐赠者的诱导多能干细胞 (iPSC) 将被 可用于生成免疫细胞、内皮细胞和内分泌细胞，这些细胞对于构建免疫系统至关重要 同基因“芯片疾病”模型。当加载原代人类细胞或同基因 iPSC 衍生材料时 （即内皮细胞、免疫细胞和 β 细胞），该 iiCHIP 将能够动态询问基因型-表型 流体 3D 内的相互作用、抗原特异性 β 细胞杀伤以及免疫调节疗法的效果 微环境。 iiChip 将实现能够加速临床干预的机制研究 抑制免疫介导的 β 细胞破坏、增强免疫调节和 β 细胞测试 恢复性疗法。