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

摘要 三维微生理系统(MPS)是一种功能强大的中间模型系统 使用能够在体外研究和临床试验中架起桥梁的人类细胞和组织。我们建议创建 一个集成的MPS平台，可以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。我们之前生成了一种含有新的细胞外基质的MPS 支持持续胰岛功能和T细胞沿胰岛细胞表面三维迁移的水凝胶(CHIB)， 在第一次此类研究中，我们展示了抗原特异的IGRP反应的人类CD8 T细胞 靶向β细胞杀伤(CMAI)。在这里，我们建议进行跨学科的努力，以整合和扩大MPS 平台(简称胰岛免疫芯片(IChip))以及基于细胞的技术 检测抗原特异性T细胞，能够产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，我们将利用小岛或小岛- 如球体、内皮细胞单层、固有免疫细胞和获得性免疫细胞，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)，以模拟人类T1D所涉及的空间构型和复杂的细胞相互作用 发病机制。我们推测，这种优化的3D iiChip将有助于原位询问Ag- 在T1D发病机制中至关重要的特异性和基因-表型相互作用以及 具有空间和时间控制的免疫调节治疗的机制效应。实验 交付成果将包括使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 IChip的特点将涉及在线传感器和生物记录器的集成，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 T1D风险相关的男性和女性捐赠者的诱导多能干细胞(IPSC)将被 可用于产生免疫细胞、内皮细胞和内分泌细胞，这些细胞对于构建 同基因的“芯片疾病”模式。当负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)，这种iiCHIP将实现对基因表型的动态询问 流体3D中的相互作用、抗原特异性β细胞杀伤和免疫调节治疗的效果 微环境。芯片将使机械研究能够加快临床干预的速度 在抑制免疫介导的β细胞破坏，增强免疫调节和β细胞检测方面 恢复性疗法。

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.