A spatially organized microphysiological model of a human lymph node

项目总结/摘要 在微芯片上模拟人体的潜力为预测性药物测试提供了诱人的希望 和前所未有的控制机制实验。然而，现有的芯片上器官系统排除了 淋巴结（LN），小而高度组织化的器官，启动适应性免疫反应。如果没有LN， 抗体或细胞介导的免疫的诱导和发展也基本上不存在。其他可用 体外LN模拟系统尚未解决这一关键的空间组织和局部微环境， 组织.由于大多数人都想保留他们的LNs，一个实验上易于处理的动态仿生模型 这个器官的组织对于机制研究和测试新疗法都是必要的。 在这个项目中，我们独特的合格的工程师和免疫学家团队将开发和 验证淋巴结的第一个空间组织的3D培养的微生理模型（LN芯片）， 具有仿生细胞组织和流体流动。在目标1中，我们将建立微图案化方法 在微流控芯片内的3D培养中的原代人类免疫细胞，使用片上光刻技术， 可交联凝胶。这种创新的方法提供了同时控制蜂窝分布，局部矩阵 组合物和流体流动，以复制3D细胞-细胞相互作用的扩散和迁移距离。我们将 优化模式和培养条件，以维持7 - 28天的活力，保持T和B细胞应答 简单的刺激，并测试微流体外壳的多种材料。在目标2中，我们将确定最佳策略 为了实现仿生淋巴结组织， 图案化趋化因子梯度、基质/内皮细胞或淋巴细胞。我们还将确定 仿生功能的流体流动条件。在目标3中，我们将建立生产性T-B细胞的条件， 在LN-芯片上的相互作用导致分化和长寿命、高亲和力抗体的产生。 LN芯片上的反应将直接与离体培养的人扁桃体的反应进行比较，以提供 该模型与人体免疫相关性的确定性数据。最后，我们将CRISPR/Cas9基因 编辑以测试LN芯片再现由T-B相互作用缺陷引起的人类疾病的程度。 总之，该U 01项目将制定经过验证的程序，用于稳健且可重复的组装 第一个空间组织的LN芯片，包括用于包含基质细胞和淋巴细胞的特定指南， 并以明确定义的人类T- B相互作用为基准。该平台将广泛适用于模型 炎症和自身免疫性疾病，测试疫苗接种策略，并回答有关 LN函数。它将与来自组织芯片联盟的其他芯片上器官的在线耦合兼容， 并且最终将允许在模型组织微环境内直接测试患者淋巴细胞功能， 从而实现基于小分子和CRISPR/CAS9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.