Schlemm’s canal on a chip: A platform for screening a novel class of glaucoma medications

施莱姆氏管芯片：用于筛选新型青光眼药物的平台

• 批准号: 10293948
• 负责人: C ROSS ETHIER
• 金额: $ 19.35万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293948
• 关键词: 3-Dimensional; Abbreviations; Animal Model; Anti-glaucoma Agent; Apical; Aqueous Humor; Biological; Biological Assay; Blindness; Blood-Aqueous Barrier; Cell Separation; Cell physiology; Cells; Cellular Assay; Clinical; Cultured Cells; Data; Detection; Development; Drainage procedure; Elements; Endothelial Cells; Endothelium; Ensure; Eye; FDA approved; Functional disorder; Future; Generations; Glaucoma; Glossary; Goals; Harvest; Height; Human; Hyperemia; Impairment; Knowledge; Liquid substance; Magnetism; Measures; Mechanics; Membrane; Microspheres; Molecular; Morphology; Ocular Hypertension; Outcome; Outcome Measure; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Physiologic Intraocular Pressure; Physiological; Physiology; Play; Process; Published Comment; Research; Research Peer Review; Research Personnel; Resistance; Rho-associated kinase; Risk; Risk Factors; Screening procedure; Stimulus; Stretching; Structure of sinus venosus of sclera; Surface; System; Technology; Therapeutic; Tissues; Trabecular meshwork structure; Tracer; Vacuole; Vision; aqueous; automated image analysis; base; chromatin immunoprecipitation; design; drug development; drug discovery; experience; fluid flow; functional outcomes; image processing; improved; in vivo; insight; kinase inhibitor; light microscopy; monolayer; novel; optic nerve disorder; pre-clinical; pressure; rapid detection; rapid testing; response; scale up; screening; side effect; superparamagnetism; therapeutic target; tool

Glaucoma is a major cause of blindness. A major risk factor for glaucoma, and the only treatable risk factor, is elevated intraocular pressure (IOP). IOP is largely determined by the function of the tissues of the conventional outflow pathway, specifically the trabecular meshwork and the inner wall of Schlemm's canal (SC), and increased fluid flow resistance in these tissues causes elevated IOP. The inner wall of SC has been largely ignored as a target for IOP-lowering therapies, in part because our understanding of this tissue's function has been poor. However, recent research findings have greatly improved our understanding and motivate this proposal. The goal of this R21 is to optimize, characterize and validate a rapid and accurate assay system that interrogates the fluid conductivity of cultured SC cells, motivated by the knowledge that increasing SC cellular conductivity will lower IOP. The long-term vision is to use this assay system to serve as a pre-clinical screening tool for drug discovery and thereby accelerate discovery of a novel class of “SC-active” compounds for treating ocular hypertension in glaucoma patients. We expect such SC-active compounds to have fewer side effects compared to other IOP-lowering compounds, including avoidance of conjunctival hyperemia. Because the hydraulic conductivity of SC cells is normally controlled by the formation of membrane-delimited fluid pathways (“pores”), the assay system specifically focuses on detection of such pores in a high-content manner. More particularly, the focus is on intracellular pores (I pores), thought to form an under-exploited secondary aqueous outflow pathway. The key elements of this assay system include: (1) primary SC cell isolation from non-glaucomatous and glaucomatous human eyes; (2) substrate micropatterning to control cultured cell spread/height and enhance assay rigor; (3) delivery of focal mechanical stretch to the apical surface of cultured SC cells using superparamagnetic microspheres and external magnets; (4) a novel fluorescent assay that, together with automated light microscopy and image processing tools, allows the rapid detection of pores that form in SC cells in a manner that minimizes introduction of inadvertent bias; and (5) a high-content format in which cells are cultured in 96 well plates, allowing relatively rapid testing of the effects of agents on SC cells. There is a high degree of rigor in the proposal, which is underpinned by a significant body of peer-reviewed research and strong preliminary data. The applicants have extensive experience in culturing and characterizing human primary SC cells, and the proposal includes a variety of positive and negative controls to validate the assay system. Further, the multi-well assay format has the advantage of naturally allowing multiple technical and biological replicates on each 96 well plate, enabling characterization of repeatability and good statistical rigor. It is expected, as suggested by the proposal's strong preliminary data, that the proposed assay system will be a powerful pre-clinical tool which can in future be used to screen for SC-active compounds and which will also enhance our understanding of SC endothelial cell physiology and pathophysiology.

青光眼是失明的主要原因。青光眼的主要危险因素，也是唯一可治疗的危险因素，是 眼内压（IOP）升高。 IOP在很大程度上取决于常规组织的功能 出口通路，特别是小梁网和Schlemm的内壁（SC）的内壁，并增加了 在这些时期，流体流动性会导致IOP升高。 SC的内壁很大程度上被忽略了 降低IOP疗法的靶标，部分原因是我们对该组织功能的理解很差。 但是，最近的研究结果极大地改善了我们的理解和动机。 该R21的目的是优化，表征和验证快速准确的测定系统，以询问 培养的SC细胞的流体电导率，这是由于增加了SC细胞电导率的知识 会降低IOP。长期的视觉是使用此测定系统作为药物的临床前筛查工具 发现，从而加速了一种新颖的“ SC活性”化合物，以治疗眼部 青光眼患者的高血压。我们期望这种SC活性化合物的副作用较少 到其他降低IOP的化合物，包括避免结膜充血。 因为SC细胞的氢聚电导率通常受到膜限制的形成 液体途径（“孔”），分析系统专门针对检测高素质的毛孔 方式。更特别的是，重点是细胞内毛孔（I孔），被认为形成了一个探索的毛孔 次生水出口通路。该测定系统的关键元素包括：（1）主要SC细胞隔离 从非亮糖果和青光眼的人眼中; （2）底物微图案以控制培养的细胞 分布/高度和增强评估严格； （3）将焦点机械拉伸传递到培养的顶部表面 SC细胞使用超磁微球和外部磁铁； （4）一种新型的荧光测定法， 与自动光学显微镜和图像处理工具一起，可以快速检测到孔 在SC细胞中形成的方式最小化了无意偏见的引入； （5）在 哪些细胞在96个井板中培养，从而可以相对较快地测试药物对SC细胞的影响。 该提案中有高度严格的严格性，这是由大量同行评审的基础的支撑 研究和强大的初步数据。申请人在培养和表征方面有丰富的经验 人类原代SC细胞，该提案包括各种阳性和负面对照，以验证 测定系统。此外，多孔测定格式具有自然允许多种技术和 生物学在每个96井板上重复，从而表征可重复性和良好的统计严格性。 正如提案的强有力数据所建议的那样，预期的是，拟议的测定系统将是一个 功能强大的临床前工具，将来可以用于筛选SC活性化合物，并且还将 增强我们对SC内皮细胞生理和病理生理学的理解。