Human proximal tubule cells form functional microtissues
Human proximal tubule cells form functional microtissues
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DOI：
10.1007/s00424-015-1771-8
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发表时间：
2016-04-01
影响因子：
4.5
通讯作者：
Devuyst, Olivier
中科院分区：
文献类型：
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作者：
Prange, Jenny A.;Bieri, Manuela;Devuyst, Olivier
肾脏近端小管的上皮细胞介导复杂的转运过程，特别容易受到药物毒性的影响。药物毒性研究通常基于永生化近端肾小管细胞的二维培养。这样的永生化细胞是去分化的，并且失去在体内遇到的转运特性（包括饱和的内吞摄取）。产生分化的器官型人类微组织可能会缓解这些限制，并促进药物毒性研究。在这里，我们描述了在明确的条件下永生化（HK-2）和原代（HRPTEpic）人肾近端肾小管上皮细胞的肾脏微组织的生成和表征。在悬滴GravityPLUS（TM）培养板中进行微组织培养，并通过监测白蛋白的内吞摄取来表征形态学、增殖和分化标记。通过将HK-2或HRPTEpic C细胞与成纤维细胞共培养成功地获得了肾脏微组织。HK-2微组织在培养10天内形成高度增殖但去分化的微组织，而与成纤维细胞共培养2天后已经产生球形结构。低传代HRPTEpic C微组织（单培养和共培养）的增殖性较低，并表达组织特异性分化标志物。电子显微镜证明上皮分化标志物，包括微绒毛，紧密连接，内涵体，和溶酶体在共培养的HRPTEpic微组织。共培养的HRPTEpic C微组织表现出特异性的白蛋白摄取，镉和庆大霉素可以抑制。总之，我们建立了一个可靠的悬滴方案，以获得功能性肾微组织与近端肾小管上皮细胞系。这些微组织可用于高通量药物和毒理学筛选，内吞作用作为功能读数。
The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS (TM) culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.