A spatially organized microphysiological model of a human lymph node

人体淋巴结的空间组织微生理模型

• 批准号: 10239046
• 负责人: Rebecca R Pompano
• 金额: $ 64.04万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239046
• 关键词: 3-Dimensional; Address; Antibodies; Antibody Affinity; Antibody Formation; Autoimmune Diseases; B cell differentiation; B-Cell Activation; B-Lymphocytes; Benchmarking; Biology; Biomimetics; CRISPR/Cas technology; Cell Communication; Cell physiology; Cells; Cellular Immunity; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complex; Coupling; Culture Media; Data; Defect; Development; Diffusion; Endothelial Cells; Engineering; Event; Foundations; Future; Gel; Generations; Genes; Genetic; Guidelines; Helper-Inducer T-Lymphocyte; Housing; Human; Human body; Image; Immune; Immunity; Immunologist; In Vitro; Inflammatory; Investigation; Left; Leukocytes; Ligands; Lymph Node Tissue; Lymphatic Endothelial Cells; Lymphocyte; Lymphocyte Function; Maintenance; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Monitor; Organ; Outcome; Patients; Pattern; Pharmaceutical Preparations; Physiological; Procedures; Production; Reproducibility; Research Personnel; Reticular Cell; Sinus; Slice; Stimulus; Stromal Cells; Structure; System; T-Lymphocyte; Testing; Therapeutic; Tissue Microarray; Tissue Model; Tissues; Tonsil; Vaccinated; Work; adaptive immune response; adaptive immunity; base; cell mediated immune response; cell motility; chemokine; cytokine; design; drug testing; experimental study; fluid flow; human disease; human model; immune function; in vivo; innovation; lymph nodes; lymphatic vessel; microchip; microfluidic technology; microphysiology system; migration; mimetics; novel therapeutics; organ on a chip; polydimethylsiloxane; preservation; receptor; recruit; response; screening; small molecule; three dimensional cell culture; vaccination strategy

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.

项目摘要/摘要 在微芯片上建模人体的潜力提供了预测药物测试的诱人希望 和机械实验的前所未有的控制。但是，现有的芯片系统排除了 LN（LN），启动适应性免疫调查的小型且高度组织的器官。没有LN， 抗体或细胞介导的免疫的诱导和发育也很大程度上不存在。其他可用 体外LN模拟系统尚未解决关键的空间组织和本地微环境 组织。由于大多数人都想保留其LN，这是一个可以实验的动力学模型 对于机械研究和测试新疗法，需要组织这种器官。 在这个项目中，我们独特合格的工程师和免疫学家团队将发展和 验证淋巴结（LN-CHIP）的第一个空间组织的3D培养微生物生理模型， 具有仿生细胞组织和流体流动。在AIM 1中，我们将建立Micropattern的方法 在微流体芯片内的3D培养物中的原代人免疫球，使用光片的光刻图 可以交联的凝胶。这种创新的方法可简单地控制细胞分布，局部矩阵 组成和流体流，以复制3D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天的生存能力，保留T和B细胞响应 进行简单的刺激，并测试微流体外壳的多种材料。在AIM 2中，我们将确定最佳策略 通过比较通过 图案化趋化因子梯度，基质/内皮细胞或淋巴细胞。我们还将确定最佳 仿生功能的流体流量条件。在AIM 3中，我们将建立生产性T-B细胞的条件 LN芯片上的相互作用，导致长寿命高亲和力抗体的分化和产生。 在LN-Chip上的反应将直接与体内培养的人扁桃体的响应进行比较，以提供 关于模型与人类免疫力相关性的确定数据。最后，我们将使用CRISPR/CAS9基因 编辑以测试LN-Chip在t -B相互作用中缺陷引起的人类疾病的程度。 总而言之，该U01项目将制定经过验证的程序，以实现强大和可重复的组装 第一个空间组织的LN芯片，包括包含基质细胞和淋巴细胞的特定指南， 并针对明确的人类T-B相互作用进行基准测试。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有关机械性问题 LN功能。它将与组织芯片联盟中的其他芯片上的其他芯片兼容， 并将允许在模型组织微环境中直接测试患者淋巴细胞功能，最终 可以启用小分子和CRISPR/CAS9基于遗传的筛选。