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

总结 三维（3D）微生理系统（MPS）代表了一个强大的中间模型系统 使用能够桥接体外研究和临床试验的人细胞和组织。我们建议开设 一个集成的MPS平台，可以更准确地模拟人体类型中涉及的复杂细胞相互作用， 1型糖尿病（T1 D）发病机制。我们先前产生了含有新型细胞外基质的MPS 支持持续的胰岛功能和T细胞沿着胰岛细胞表面在3D（CHIB）中迁移的水凝胶， 在首次的研究中，我们证明了抗原特异性IGRP反应性人CD 8 T细胞的产生， 靶向β细胞杀伤（CMAI）。在这里，我们提出了一个跨学科的努力，整合和扩大MPS 平台（称为胰岛免疫芯片（iiChip）），以及基于细胞的技术， 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，和 基因组编辑技术，供更广泛的HIRN社区使用。具体来说，我们将利用胰岛或胰岛- 如球状体、内皮细胞单层以及先天性和适应性免疫细胞，包括树突状细胞 (DCs)巨噬细胞、CD 4+常规T细胞（Tconv）、CD 8+细胞毒性T细胞（CTL）和调节性T细胞 （TlD），以模拟人类T1 D中涉及的空间构型和复杂的细胞相互作用。 发病机制我们假设这种优化的3D iiChip将有助于原位询问Ag- 特异性和基因型-表型相互作用在T1 D发病机制中至关重要， 具有空间和时间控制的免疫调节疗法的机制作用。实验 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。关键 iiChip的特点将包括集成在线传感器和生物报告器，空间和时间控制 用于确定刺激的输入，以及具有流体和细胞的能力的基质的整合 再循环，测量长期细胞培养中的可溶性和细胞读数。此外，基因编辑 来自具有T1 D风险相关HLA的男性和女性供体的诱导多能干细胞（iPSC）将被 可用于产生免疫，内皮和内分泌细胞，这些细胞对于建立一个 同基因“芯片上的疾病”模型。当装载原代人细胞或等基因iPSC衍生材料时 (i.e.,内皮、免疫和β细胞），这种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细胞杀伤和免疫调节治疗的效果 微环境iiChip将使机制研究能够加速临床干预， 在抑制免疫介导的β细胞破坏，增强免疫调节和检测β细胞 恢复性治疗

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.