3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, Portugal.
ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
Acta Physiol (Oxf). 2020 Sep;230(1):e13491. doi: 10.1111/apha.13491. Epub 2020 May 14.
Herein we propose creating a bilayer tubular kidney in-vitro model. It is hypothesized that membranes composed of decellularized porcine kidney extracellular matrix are valid substitutes of the tubular basement membrane by mimicking the physiological relevance of the in vivo environment and disease phenotypes.
Extracellular matrix was obtained from decellularized porcine kidneys. After processing by lyophilization and milling, it was dissolved in an organic solvent and blended with poly(caprolactone). Porous membranes were obtained by electrospinning and seeded with human primary renal progenitor cells to evaluate phenotypic alterations. To create a bilayer model of the in vivo tubule, the same cells were differentiated into epithelial tubular cells and co-cultured with endothelial cells in opposite sites.
Our results demonstrate increasing metabolic activity, proliferation and total protein content of renal progenitors over time. We confirmed the expression of several genes encoding epithelial transport proteins and we could also detect tubular-specific proteins by immunofluorescence stainings. Functional and transport assays were performed trough the bilayer by quantifying both human serum albumin uptake and inulin leakage. Furthermore, we validated the chemical modulation of nephrotoxicity on this epithelium-endothelium model by cisplatin exposure.
The use of decellularized matrices in combination with primary renal cells was shown to be a valuable tool for modelling renal function and disease in vitro. We successfully validated our hypothesis by replicating the physiological conditions of an in vitro tubular bilayer model. The developed system may contribute significantly for the future investigation of advanced therapies for kidney diseases.
本研究旨在构建一种双层管状肾体外模型。我们假设，由脱细胞化猪肾细胞外基质组成的双层膜通过模拟体内环境的生理相关性和疾病表型，可以作为管状基底膜的有效替代品。
从脱细胞化的猪肾中提取细胞外基质。经过冻干和研磨处理后，将其溶解在有机溶剂中并与聚己内酯混合。通过静电纺丝获得多孔膜，并接种人原代肾祖细胞以评估表型改变。为了构建体内管状的双层模型，将相同的细胞分化为上皮管状细胞，并与内皮细胞在相反的位置共培养。
我们的结果表明，肾祖细胞的代谢活性、增殖和总蛋白含量随时间增加。我们证实了编码上皮转运蛋白的几个基因的表达，并且通过免疫荧光染色也可以检测到管状特异性蛋白。通过定量人血清白蛋白摄取和菊粉渗漏，在双层模型中进行了功能和转运测定。此外，我们通过顺铂暴露验证了在该上皮-内皮模型上对肾毒性的化学调节。
使用脱细胞基质结合原代肾细胞被证明是体外模拟肾功能和疾病的有价值的工具。我们通过复制体外管状双层模型的生理条件成功验证了我们的假设。所开发的系统可能为未来对肾脏疾病的先进治疗方法的研究做出重大贡献。
