Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies
Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies
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DOI：
10.1016/j.ejps.2016.02.013
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发表时间：
2016-04-30
影响因子：
4.6
通讯作者：
Rastaldi, Maria Pia
中科院分区：
文献类型：
--
作者：
Li, Min;Corbelli, Alessandro;Rastaldi, Maria Pia
蛋白尿是肾小球疾病的常见症状，并且是由于蛋白质从肾小球滤过屏障渗漏所致，肾小球滤过屏障是由两个有丝分裂后高度特化和相互依赖的细胞群（即肾小球内皮细胞和足细胞）以及其间的基底膜组成的三层结构。尽管近年来取得了巨大的进展，但蛋白尿的发病机制仍有待完全揭示。该领域的研究可以在很大程度上受益于肾小球过滤器的体外模型，但这样的系统已被证明难以实现。在这里，我们描述了一种方法来获得和利用三维足细胞内皮细胞共培养，它可以在很大程度上通过科学界，因为它不依赖于特殊的仪器，也不对专用生物材料的合成。该器械由两面均涂有IV型胶原蛋白的多孔膜组成。足细胞在膜上侧的粘附必须在下侧的VEGF诱导的内皮细胞成熟之前。共培养物可以与足细胞系以及与原代足细胞组装，从而将用途扩展到源自转基因小鼠的细胞。白蛋白渗透性测定已被广泛验证并用作功能读数，从而实现快速药物检测。此外，孔的底部可以填充第三种细胞类型，这增加了分析更复杂的肾小球细胞间信号传导事件的可能性。总之，易于组装和使用的多功能性是该肾小球滤过屏障的三维模型优于现有方法的主要优点。运行可靠测量白蛋白渗透性的功能测试的可能性使该设备成为从药物筛选到细胞间信号传导研究的几种研究应用中的有效伴侣。(C)2016爱思唯尔B.V.保留所有权利。
Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies. (C) 2016 Elsevier B.V. All rights reserved.