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: 10652476
• 负责人: Rebecca R Pompano
• 金额: $ 69.21万
• 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/10652476
• 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; Disease; Endothelial Cells; Engineering; Event; Exclusion; 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; Organizational Models; Outcome; Patients; Pattern; Pharmaceutical Preparations; Physiological; Procedures; Production; Productivity; Qualifying; 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; 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; self organization; 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.

项目概要/摘要 在微芯片上模拟人体的潜力为预测药物测试带来了诱人的希望 以及对机械实验前所未有的控制。然而，现有的器官芯片系统不包括 淋巴结 (LN) 是一种小型且高度组织化的器官，可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。其他可用在 体外 LN 模拟系统尚未解决该系统的关键空间组织和局部微环境问题 组织。由于大多数人都希望保留他们的 LN，因此这是一种经过实验处理的动力学仿生模型 机制研究和测试新疗法都需要该器官的组织。 在这个项目中，我们独特的合格工程师和免疫学家团队将开发和 验证第一个空间组织、3D 培养的淋巴结微生理模型（LN 芯片）， 具有仿生细胞组织和流体流动的特点。在目标 1 中，我们将建立微图案化方法 使用片上光刻技术在微流控芯片内进行 3D 培养的原代人类免疫细胞 可交联凝胶。这种创新方法提供了对细胞分布、局部矩阵的同时控制 成分和流体流动，以复制 3D 细胞间相互作用的扩散和迁移距离。我们将 优化图案化和培养条件以维持 7 – 28 天的活力，保留 T 和 B 细胞反应 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 通过比较获得的微观结构的稳健性来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。我们还将确定最佳 仿生功能的流体流动条件。在目标 3 中，我们将为 T-B 细胞的生产创造条件 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。 LN 芯片上的响应将直接与离体培养的人类扁桃体的响应进行比较，以提供 该模型与人类免疫相关性的明确数据。最后，我们将利用CRISPR/Cas9基因 编辑以测试 LN 芯片在多大程度上重现由结核病相互作用缺陷引起的人类疾病。 总之，这个 U01 项目将产生经过验证的程序，用于稳健且可重复的组装 第一个空间组织的 LN 芯片，包括包含基质细胞和淋巴细胞的具体指南， 并针对明确的人类结核病相互作用进行基准测试。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，并回答有关的机制问题 闪电网络功能。它将与组织芯片联盟的其他片上器官的在线耦合兼容， 并将最终允许在模型组织微环境中直接测试患者淋巴细胞功能 实现基于小分子和 CRISPR/CAS9 遗传的筛选。

项目成果

A Scalable, Modular Degasser for Passive In-Line Removal of Bubbles from Biomicrofluidic Devices.

• DOI: 10.3390/mi14020435
• 发表时间: 2023-02-11
• 期刊: MICROMACHINES
• 影响因子: 3.4
• 作者: Musgrove, Hannah B.;Saleheen, Amirus;Zatorski, Jonathan M.;Arneja, Abhinav;Luckey, Chance John;Pompano, Rebecca R.
• 通讯作者: Pompano, Rebecca R.

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR.

通过优化的茚三酮测定和 1​​ H NMR 对明胶水凝胶的部分和绝对功能化进行定量。

• DOI: 10.1007/s00216-020-02792-5
• 发表时间: 2020
• 期刊: Analytical and bioanalytical chemistry
• 影响因子: 4.3
• 作者: Zatorski,JonathanM;Montalbine,AlyssaN;Ortiz-Cárdenas,JenniferE;Pompano,RebeccaR
• 通讯作者: Pompano,RebeccaR