Matrigel-Free Laminin-Entactin Matrix to Induce Human Renal Proximal Tubule Structure Formation In Vitro
Matrigel-Free Laminin-Entactin Matrix to Induce Human Renal Proximal Tubule Structure Formation In Vitro
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DOI：
10.1021/acsbiomaterials.0c01385
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发表时间：
2020-12-01
影响因子：
5.8
通讯作者：
Kaplan, D. L.
中科院分区：
文献类型：
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作者：
Adelfio, M.;Szymkowiak, S.;Kaplan, D. L.
成功的体外组织模型必须重现天然组织特征，同时也是可重现的。目前，Matrigel是用于在体外诱导近曲小管（PCT）形成的主要生物材料，因为其与肾小管基底膜相似的组成和结构以及关键生长因子的存在。然而，基质胶并没有很好地定义，并且批次间的可变性是一个重要的问题。在这里，我们定义了一种无基质胶的方法，使用层粘连蛋白-巢蛋白（L-E）基质，支持在体外使用永生化的人肾上皮细胞（RPTEC/TERT 1）与鼠成纤维细胞基质细胞（FOXD 1（lacZ+））共培养的近端小管样结构的形成。除了成纤维细胞生长因子8a（FGF-8a）外，基质还支持肾小管基底膜的特定组分（层粘连蛋白、巢蛋白/巢蛋白和硫酸乙酰肝素蛋白聚糖）的存在。基质还诱导小管形成，导致基于几个关键标志物的PCT的形成，包括E-钙粘蛋白、水通道蛋白-1和Na+/K+ ATP酶。此外，这些PCT结构在培养18天后显示出细胞极性和明确的管腔。这种层粘连蛋白-巢蛋白（L-E）基质构成了一种确定的和一致的生物材料，可用于肾脏组织工程，以了解体外近端小管发育和肾毒性研究。
A successful in vitro tissue model must recapitulate the native tissue features while also being reproducible. Currently, Matrigel is the principal biomaterial used to induce the formation of proximal convoluted tubules (PCTs) in vitro, because of its similar composition and structure with the kidney tubular basement membrane and the presence of critical growth factors. However, Matrigel is not well-defined, and batch-to-batch variability is a significant issue. Here, we define a Matrigel-free method, using a laminin-entactin (L-E) matrix to support the formation of proximal tubular-like structures in vitro using immortalized human renal epithelial cells (RPTEC/TERT1) cocultured with murine fibroblast stromal cells (FOXD1(lacZ+)). The matrix supports the presence of specific components of the tubular basement membrane (laminin, entactin/nidogen, and heparan sulfate proteoglycan) in addition to fibroblast growth factor 8a (FGF-8a). The matrix also induces tubulogenesis, leading to the formation of PCTs based on several key markers, including E-cadherin, aquaporin-1, and Na+/K+ ATPase. Moreover, these PCT structures displayed cell polarity and a well-defined lumen after 18 days in culture. This laminin-entactin (L-E) matrix constitutes a defined and consistent biomaterial that can be used in kidney tissue engineering for understanding in vitro proximal tubule development and for nephrotoxicity studies.