Development of a 3D cell culture model of the glomerular filtration barrier

描述（由申请人提供）：该R21申请的目标是开发肾小球滤过屏障的三维（3D）模型，该模型包含关键细胞成分，足细胞和内皮细胞，以及模拟肾小球基底膜（GBM）的基质。使用一种新颖的方法，我们开发了一种基于胶原蛋白的水凝胶支架来复制基底膜的组成和结构，并将这种支架插入到一个允许足细胞和内皮细胞的相对单层生长的装置中。该应用程序的具体目标将开发和表征该3D模型，并将测试成熟细胞结构形成的要求：足突、狭缝隔膜和窗。具体目标1将研究细胞基质相互作用和旁分泌信号事件在成熟细胞表型发育中的作用。这些研究将优化支架的厚度，并纳入相关的GBM肽用于天然细胞附着。水凝胶支架重塑将通过天然GBM蛋白的Western分析和降解支架材料释放的二酪胺加合物的HPLC定量来评估。细胞表型将通过共聚焦免疫荧光和电子显微镜分析。具体目标2将侧重于脱细胞水凝胶支架的物理特性，并通过无侧限压缩测试和流室断裂容限测试测试其承受压力和剪切应力载荷的能力。此外，支架的结构将被优化，以适应通过支架材料浓度和交联密度在体内条件下复制的力。然后，具体目标3将通过将3D细胞培养模型放入流动室并测试其对过滤物理力的响应，将目标1中的细胞优化工作与目标2中的支架优化工作整合在一起。这些研究将允许对足突、狭缝膈和内皮窗形成的细胞-细胞、细胞-基质和水动力进行综合分析。我们的长期目标是开发一种系统，该系统可以复制存在于过滤屏障中的所有“生化”（细胞-细胞和细胞-基质）和“机械”（流体流动和压力）信号，并将这些信号整合到一个系统中，该系统可以重现过滤屏障结构和功能的体内动态。我们设想该模型可用于测试内皮-足细胞相互作用的生物学和病理机制，并模拟肾小球疾病特征的物理力改变。公共卫生相关性：我们正在设计一种体外模型系统，可用于以受控方式测试特定疾病诱导事件对肾脏过滤屏障结构和功能的作用。通过确定哪些生化或机械压力导致异常细胞行为，将使我们能够专注于疾病中的这些过程，这可能为治疗的新方向奠定基础。这些研究将使我们不仅了解滤过屏障的各个组成部分是如何工作的，而且还了解它们如何与正常肾功能相互作用，以及肾脏如何在疾病中受损。

DESCRIPTION (provided by applicant): The goal of this R21 application is to develop a three-dimensional (3D) model of the glomerular filtration barrier that incorporates the key cellular components, podocytes and endothelial cells, and matrix that models the glomerular basement membrane (GBM). Using a novel method, we have developed a collagen-based hydrogel scaffold to replicate the basement membrane composition and structure, and have inserted this scaffold into an apparatus that permits the growth of apposing monolayers of podocytes and endothelial cells. The specific aims of this application will develop and characterize this 3D model, and will test requirements for the formation of mature cellular structures: foot processes, slit diaphragms, and fenestrae. Specific aim 1 will examine the role of cell matrix interactions and paracrine signaling events on the development of mature cell phenotypes. These studies will optimize thickness of the scaffold and incorporate relevant GBM peptides for native cell attachments. Hydrogel scaffold remodeling will be evaluated by Western analysis of native GBM proteins and HPLC quantitation of released di-tyramine adducts from degraded scaffold material. Cell phenotype will be analyzed by confocal immunofluorescence and electron microscopy. Specific aim 2 will focus on the physical properties of the acellular hydrogel scaffold and testing its ability to withstand pressure and shear stress loads using unconfined compression testing, and break tolerance testing in a flow chamber. In addition, the structure of the scaffold will be optimized to accommodate forces that replicate in vivo conditions through scaffold material concentration and crosslink density. Specific aim 3 then will integrate the cell optimization work in aim 1 with the scaffold optimization work in aim 2, by placing the 3D cell culture model into a flow chamber and testing its responses to the physical forces of filtration. These studies will allow integrated analysis of cell-cell, cell-matrix and hydrodynamic forces on the formation of foot processes, slit diaphragms and endothelial fenestrae. Our long-term goal is to develop a system that replicates all the "biochemical" (cell-cell and cell-matrix) and "mechanical" (fluid flow and pressure) signals that exist in the filtration barrier, as well as the integration of these signals into a system that recreates the in vivo dynamics of filtration barrier structure and function. We envision that this model could be used to test biological and pathological mechanisms of endothelial-podocyte interactions and model altered physical forces characteristic of glomerular disease. PUBLIC HEALTH RELEVANCE: We are designing an in vitro model system that can be used to test in a controlled manner the role of specific disease-inducing events on the structure and function of the kidney's filtration barrier. By determining which biochemical or mechanical stresses lead to abnormal cellular behavior will allow us to focus on these processes in disease, which may lay the ground work for new directions in therapy. These studies will allow us to understand not only how individual components of the filtration barrier work, but also how they interact with regard to both normal kidney function and how it is injured in disease.