Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA, United States; The Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, United States.
Department of Physiology, Morehouse School of Medicine, Atlanta, GA, United States.
Biomaterials. 2022 Nov;290:121828. doi: 10.1016/j.biomaterials.2022.121828. Epub 2022 Oct 3.
This study reports the cellular self-organization of primary human renal proximal tubule epithelial cells (RPTECs) around a minimal Matrigel scaffold to produce basal-in and apical-out proximal tubule organoids (tubuloids). These tubuloids are produced and maintained in hanging drop cultures for 90+ days, the longest such culture of any kind reported to date. The tubuloids upregulate maturity markers, such as aquaporin-1 (AQP1) and megalin (LRP2), and exhibit less mesenchymal and proliferation markers, such as vimentin and Ki67, compared to 2D cultures. They also experience changes over time as revealed by a comparison of gene expression patterns of cells in 2D culture and in day 31 and day 67 tubuloids. Gene expression analysis and immunohistochemistry reveal an increase in the expression of megalin, an endocytic receptor that can directly bind and uptake protein or potentially assist protein uptake. The tubuloids, including day 90 tubuloids, uptake fluorescent albumin and reveal punctate fluorescent patterns, suggesting functional endocytic uptake through these receptors. Furthermore, the tubuloids release kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, when exposed to albumin in both dose- and time-dependent manners. While this study focuses on potential applications for modeling proteinuric kidney disease, the tubuloids may have broad utility for studies where apical proximal tubule cell access is required.
本研究报告了原代人肾近端小管上皮细胞（RPTEC）在最小 Matrigel 支架周围的细胞自我组织，以产生基底内和顶端外的近端小管类器官（小管）。这些小管在悬滴培养中产生和维持 90 多天，这是迄今为止报道的任何类型的最长培养。与 2D 培养相比，小管上调成熟标志物，如水通道蛋白 1（AQP1）和巨球蛋白（LRP2），并表现出较少的间充质和增殖标志物，如波形蛋白和 Ki67。随着时间的推移，它们还会发生变化，这可以通过比较 2D 培养和第 31 天和第 67 天小管中细胞的基因表达模式来揭示。基因表达分析和免疫组织化学显示，内吞受体巨球蛋白的表达增加，该受体可以直接结合和摄取蛋白质或潜在地协助蛋白质摄取。小管包括第 90 天的小管摄取荧光白蛋白并显示点状荧光模式，表明通过这些受体进行功能性内吞摄取。此外，当小管暴露于白蛋白时，以剂量和时间依赖的方式释放肾脏损伤分子-1（KIM-1），这是肾脏损伤的常见生物标志物。虽然本研究侧重于在蛋白尿性肾病模型中的潜在应用，但小管可能具有广泛的用途，需要获得顶端近端小管细胞。
