From single molecule to microfluidic 3D tissue platforms: novel multiscale tools to investigate hyper-stimulated immune cells in the circulation

从单分子到微流体 3D 组织平台：研究循环中过度刺激免疫细胞的新型多尺度工具

• 批准号: 10358578
• 负责人: Lane A. Baker
• 金额: $ 43.8万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10358578
• 关键词: 3-Dimensional; 3D Print; Abnormal Red Blood Cell; Adhesions; Affect; Autoimmune Diseases; Biological; Biosensor; Blood Circulation; Blood Vessels; Carrier Proteins; Cell membrane; Cell model; Cell physiology; Cells; Cellular Assay; Cellular Metabolic Process; Centers for Disease Control and Prevention (U.S.); Cicatrix; Cross-Sectional Studies; Data; Demyelinations; Development; Diagnosis; Disease; Environment; Erythrocytes; Extravasation; Fiber; Glucose; Glucose Transporter; Immobilization; Immune; Immune system; In Vitro; Inflammatory Response; Ion Channel; Lead; Lesion; Link; Liquid substance; Measurement; Mediating; Membrane; Metabolic; Methods; Microfluidics; Modeling; Molecular; Monitor; Multiple Sclerosis; Myelin; Myelin Proteins; Nerve; Nerve Fibers; Neuraxis; Nucleotides; Pathway interactions; Patients; Persons; Pharmacologic Substance; Process; Production; Records; Registries; Research Personnel; SLC2A1 gene; Sampling; Solid; Symptoms; T-Cell Activation; T-Lymphocyte; Technology; Testing; Tissues; United States; Western Blotting; analytical tool; base; cell mediated immune response; extracellular; gel electrophoresis; glucose monitor; glucose transport; glucose uptake; improved; in vivo; innovation; insight; molecular scale; multiple sclerosis patient; nanoscale; nerve damage; novel; overexpression; response; sensor; single molecule; stem; tool; uptake

PROJECT SUMMARY The exact mechanism underlying the onset of Multiple Sclerosis (MS), a disease that affects over 2 million people worldwide and ~ 400,000 in the United States, is unknown although most experts in the field agree that MS involves an abnormal immune-mediated response against the body’s central nervous system (CNS). Specifically, in the CNS, components of the immune system attack myelin, the protein-based substance that surrounds nerve fiber. This attack on myelin results in multiple scar lesions (hence, Multiple Sclerosis) that lead to disease symptoms. Within the immune system, evidence continues to mount that T-cells are the bloodstream components responsible for the demyelination of the nerve fiber. While it is not clear what triggers the T-cells to attack myelin, it is becoming increasingly clear that in vivo, extracellular ATP may be a major determinant in controlling T-cell function and their passage from the bloodstream to the CNS where they participate in the damage to myelin. Innovative analytical tools are needed to investigate the mechanism of T- cell activation, adhesion, and transfer (extravasation) from the bloodstream to the CNS, at the tissue, cellular, and molecular scales. To meet this need, an investigative team consisting of multiple investigators with expertise in fluidic platforms, nanoscale biosensors, and biological samples, proposes a set of specific aims that will prove that the T-cell activation in MS is due to abnormal glucose processing and ATP production and release by the MS red blood cell. We propose that the development of an innovative microfluidic platform with electrospun fibers will create a unique 3D-environment on a controlled in vitro platform for improved monitoring of T-cell activation/adhesion and extravasation across a tissue cultured to a membrane. Next, we will employ classical cell assay methods to establish that the MS red blood cell has unique glucose processing capabilities stemming from an overexpression of the glucose transporter found in the red blood cell (GLUT1). In aim 3, ion channel modified nanopipettes will be developed to perform quantitative, nanoscale determinations on glucose and ATP transport at the single red blood cell level. These nanoscale sensors will confirm that the somewhat “global” findings in aim 2 (increased glucose transporter and overproduction of ATP) are indeed affecting glucose uptake, and that the uptake is linked to ATP release, thus providing unprecedented metabolic insight on the genesis of inflammatory response. In the final aim of the proposal, we will combine our tools and discoveries from aims 1-3 with pharmaceutical manipulation of red blood cells obtained from MS patients and controls to determine if abnormal glucose transport in the MS red blood cell is the origin of extracellular ATP production and dysregulation of T-cell activation and adhesion. The successful completion of these aims will not only provide insight into factors affecting demyelination in MS, but also provide platform technologies for cellular analyses across multiple fields.

项目概要 多发性硬化症 (MS) 发病的确切机制，这种疾病影响了超过 200 万人 尽管该领域的大多数专家都同意这一点，但全球范围内的人数和美国约 400,000 人的人数仍不得而知。 MS 涉及针对人体中枢神经系统 (CNS) 的异常免疫介导反应。 具体来说，在中枢神经系统中，免疫系统的成分会攻击髓磷脂，髓磷脂是一种基于蛋白质的物质， 这种对髓磷脂的攻击会导致多处病变疤痕（因此，多发性硬化症）。 在免疫系统中，越来越多的证据表明 T 细胞是导致疾病症状的原因。 负责神经纤维脱髓鞘的血液成分虽然尚不清楚。 触发 T 细胞攻击髓磷脂，越来越清楚的是，在体内，细胞外 ATP 可能是一种 控制 T 细胞功能及其从血流到 CNS 的主要决定因素 需要创新的分析工具来研究 T- 的机制。 细胞活化、粘附和从血流转移（外渗）到中枢神经系统、组织、细胞、 为了满足这一需求，由多名研究人员组成的研究小组 流体平台、纳米级生物传感器和生物样本方面的专业知识提出了一系列具体目标 这将证明多发性硬化症中 T 细胞的激活是由于异常的葡萄糖加工和 ATP 产生造成的 我们建议开发一种创新的微流体平台。 电纺纤维将在受控体外平台上创建独特的 3D 环境，以改善监测 接下来，我们将利用培养到膜上的 T 细胞活化/粘附和外渗。 经典的细胞测定方法，以确定 MS 红细胞具有独特的葡萄糖处理能力 源于红细胞中葡萄糖转运蛋白 (GLUT1) 的过度表达。在目标 3 中，离子。 将开发通道改良的纳米移液器以对葡萄糖进行定量、纳米级测定 这些纳米级传感器将在某种程度上证实这一点。 目标 2（增加葡萄糖转运蛋白和 ATP 过量产生）的“全球”发现确实正在影响 葡萄糖摄取，并且摄取与 ATP 释放相关，从而提供前所未有的代谢洞察 在该提案的最终目标中，我们将结合我们的工具和炎症反应的起源。 通过对从多发性硬化症患者获得的红细胞进行药物操作，实现目标 1-3 的发现，以及 对照以确定 MS 红细胞中的异常葡萄糖转运是否是细胞外 ATP 的来源 T 细胞活化和粘附的产生和失调将成功完成这些目标。 不仅可以深入了解影响多发性硬化症脱髓鞘的因素，还可以提供平台技术 跨多个领域的细胞分析。

项目成果

Perspective and Prospectus on Single-Entity Electrochemistry.

单实体电化学的观点和简介。

• DOI: 10.1021/jacs.8b09747
• 发表时间: 2018-11-02
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: L. A. Baker

Evaluation and optimization of PolyJet 3D-printed materials for cell culture studies.

用于细胞培养研究的 PolyJet 3D 打印材料的评估和优化。

• 发表时间: 2022-05
• 影响因子: 4.3
• 作者: Currens, Emily R;Armbruster, Michael R;Castiaux, Andre D;Edwards, James L;Martin, R Scott
• 通讯作者: Martin, R Scott

A 3D-printed, multi-modal microfluidic device for measuring nitric oxide and ATP release from flowing red blood cells.

一种 3D 打印的多模式微流体装置，用于测量流动红细胞释放的一氧化氮和 ATP。

• 发表时间: 2022-08-25
• 作者: Hayter, Elizabeth A;Azibere, Samuel;Skrajewski, Lauren A;Soule, Logan D;Spence, Dana M;Martin, R Scott
• 通讯作者: Martin, R Scott

A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.

一种新型 3D 打印离心超滤方法揭示了人血清白蛋白的体内糖化降低了其与锌的结合亲和力。

• 发表时间: 2020
• 作者: Jacobs, Monica J;Pinger, Cody W;Castiaux, Andre D;Maloney, Konnor J;Spence, Dana M
• 通讯作者: Spence, Dana M

Imaging with Ion Channels.

使用离子通道成像。

• 发表时间: 2021
• 影响因子: 7.4
• 作者: Zhu, Cheng;Huang, Kaixiang;Wang, Yunong;Alanis, Kristen;Shi, Wenqing;Baker, Lane A
• 通讯作者: Baker, Lane A