A spatially organized microphysiological model of a human lymph node

项目摘要/摘要 在微芯片上建立人体模型的可能性为预测性药物测试带来了诱人的希望 对机械实验进行前所未有的控制。然而，现有的芯片上器官系统排除了 淋巴结节(LN)，是启动适应性免疫反应的高度组织化的小器官。如果没有LN， 抗体或细胞免疫的诱导和发展在很大程度上也是缺乏的。其他可用地址： 体外LN模拟系统还没有解决这一关键的空间组织和局部微环境 组织。因为大多数人类想要保留他们的LNS，一个实验上容易处理的，仿生的动力学模型 而这个器官的组织是机械研究和测试新疗法所必需的。 在这个项目中，我们唯一合格的工程师和免疫学家团队将开发和 验证第一个空间组织的、3D培养的淋巴结微生理模型(LN-CHIP)， 具有仿生细胞组织和流体流动的特点。在目标1中，我们将建立微图案的方法 原代人体免疫细胞在微流控芯片内进行3D培养，使用芯片上的光刻技术进行光刻- 可交联性凝胶。这一创新方法可同时控制蜂窝分布、本地矩阵 成分和流体流动，以复制3D细胞-细胞相互作用的扩散和迁移距离。我们会 优化构图和培养条件以维持7-28天的活性，保持T和B细胞的应答 以简单的刺激，并测试多种材料的微流控外壳。在目标2中，我们将确定最佳策略 通过比较以下方法获得的微结构的稳健性来实现仿生淋巴组织 图案化的趋化因子梯度、间质/内皮细胞或淋巴细胞。我们还将确定最优的 仿生功能的流体流动条件。在目标3中，我们将为生产T-B细胞创造条件 LN芯片上的相互作用导致分化和产生长寿命、高亲和力的抗体。 LN芯片上的反应将直接与体外培养的人类扁桃体的反应进行比较，以提供 关于该模型与人类免疫力相关性的确凿数据。最后，我们将使用CRISPR/Cas9基因 编辑以测试LN-芯片在多大程度上概括了T-B相互作用中的缺陷导致的人类疾病。 总之，这个U01项目将为健壮的和可重复的组装产生经过验证的程序 第一个空间组织的LN芯片，包括包括基质细胞和淋巴细胞的具体指南， 并对照定义明确的人类T-B互动进行基准测试。该平台将广泛适用于模型 炎症性和自身免疫性疾病，测试疫苗接种策略，并回答关于 Ln函数。它将兼容与来自组织芯片联盟的其他芯片上器官的在线耦合， 并最终将允许在模型组织微环境中直接测试患者的淋巴细胞功能 支持小分子和CRISPR/Cas9基于基因的筛选。

PROJECT SUMMARY/ABSTRACT The potential to model the human body on a microchip offers tantalizing hope of predictive drug testing and unprecedented control for mechanistic experiments. However, existing organ-on-chip systems exclude the lymph node (LN), the small and highly organized organ that initiates adaptive immune responses. Without a LN, the induction and development of antibody- or cell-mediated immunity is also largely absent. Other available in vitro LN-mimetic systems do not yet address the crucial spatial organization and local microenvironment of this tissue. As most humans want to keep their LNs, an experimentally tractable, biomimetic model of the dynamics and organization of this organ is needed both for mechanistic studies and to test new therapies. In this project, our uniquely qualified team of engineers and immunologists will develop and validate the first spatially organized, 3D-cultured microphysiological model of a lymph node (LN-chip), featuring biomimetic cellular organization and fluid flow. In Aim 1, we will establish methods to micropattern primary human immune cells in 3D culture inside a microfluidic chip, using on-chip photolithography of photo- crosslinkable gels. This innovative approach provides simultaneous control over cellular distribution, local matrix composition, and fluid flow, to replicate diffusion and migration distances for 3D cell-cell interactions. We will optimize patterning and culture conditions to maintain viability for 7 – 28 days, preserve T and B cell response to simple stimuli, and test multiple materials for the microfluidic housing. In Aim 2, we will identify the best strategy to achieve biomimetic lymph node organization by comparing the robustness of microstructure obtained by patterning chemokine gradients, stromal/endothelial cells, or lymphocytes. We will also determine the optimal fluid flow conditions for biomimetic function. In Aim 3, we will establish conditions for productive T-B cell interactions on the LN-chip leading to differentiation and production of long-lived, high-affinity antibodies. Responses on the LN-chip will be directly compared to those of ex vivo cultured human tonsils, to provide definitive data on the relevance of the model to human immunity. Finally, we will employ CRISPR/Cas9 gene editing to test the extent to which the LN-chip recapitulates human disease caused by defects in T—B interaction. In summary, this U01 project will produce validated procedures for robust and reproducible assembly of the first spatially organized LN-chip, including specific guidelines for inclusion of stromal cells and lymphocytes, and benchmarking against well-defined human T- B interactions. The platform will be broadly applicable to model inflammatory and autoimmune diseases, test vaccination strategies, and answer mechanistic questions about LN function. It will be compatible with in-line coupling to other organs-on-chip from the Tissue Chip consortium, and will allow for direct testing of patient lymphocyte function within a model tissue microenvironment, ultimately enabling both small molecule and CRISPR/CAS9 genetic based screens.