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

青光眼是导致失明的主要原因。青光眼的一个主要危险因素，也是唯一可治疗的危险因素是 眼压升高。眼压在很大程度上是由传统眼组织的功能决定的 流出途径，特别是小梁网和Schlemm管(SC)内壁，并增加 这些组织中的液体流动阻力会导致眼压升高。SC的内壁在很大程度上被忽视为一种 降低眼压治疗的目标，部分原因是我们对这种组织的功能了解很差。 然而，最近的研究结果极大地提高了我们的理解，并推动了这一提议。 此R21的目标是优化、表征和验证一种快速、准确的检测系统，该系统可以 培养的干细胞的液体电导率，其动机是增加干细胞的电导率 会降低眼压。我们的长期目标是使用这个检测系统作为药物的临床前筛选工具。 发现并从而加速发现一类新型的治疗眼科疾病的“SC活性”化合物 青光眼患者的高血压。我们预计这类SC活性化合物的副作用会比 对其他降眼压化合物，包括避免结膜充血。 因为SC电池的水力传导性通常是由膜分隔的形成控制的 流体通道(“毛孔”)，该检测系统专门专注于检测此类毛孔中的高含量 举止。更具体地说，重点是细胞内的毛孔(i毛孔)，被认为形成了一个未被充分开发的 二次房水流出通道。该检测系统的关键技术包括：(1)干细胞的原代分离 来自非青光眼和青光眼的；(2)底物微图案化以控制培养的细胞 铺展/高度和提高分析的严谨性；(3)将局部机械拉伸输送到培养的根尖表面 使用超顺磁性微球和外部磁铁的SC细胞；(4)一种新的荧光检测方法， 与自动光学显微镜和图像处理工具相结合，可以快速检测 在SC细胞中以最大限度地减少无意偏向引入的形式；以及(5)在 这些细胞被培养在96孔板中，允许相对快速地测试药物对SC细胞的影响。 该提案具有高度的严谨性，得到了大量同行评审的支持 研究和强劲的初步数据。申请者在培养和塑造个性方面有丰富的经验 人类原代干细胞，该提案包括各种阳性和阴性对照，以验证 化验系统。此外，多孔化验格式的优点是自然允许多种技术和 在每个96孔板上进行生物复制，能够表征重复性和良好的统计精确度。 正如该提案的强劲初步数据所表明的那样，预计拟议的化验系统将是一个 强大的临床前工具，将来可以用来筛选SC活性化合物，它还将 加深对干细胞内皮细胞生理学和病理生理学的了解。

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.