A simple method to generate human airway epithelial organoids with externally orientated apical membranes
A simple method to generate human airway epithelial organoids with externally orientated apical membranes
复制标题
DOI：
10.1152/ajplung.00536.2020
复制
发表时间：
2022-03-01
影响因子：
4.9
通讯作者：
Finkbeiner, Walter E.
中科院分区：
文献类型：
--
作者：
Boecking, Carolin A.;Walentek, Peter;Finkbeiner, Walter E.
类器官是一种自组织的三维培养物，提供了复制天然组织的特定细胞成分或器官复杂性方面的模型。我们描述了一种简单的方法，使用分离的人气管支气管上皮细胞在混合基质成分中生长，并在第14天补充Wnt通路激动剂R-spondin 2 （RSPO2）和骨形态发生蛋白拮抗剂Noggin来产生类器官培养。与之前的报道相比，我们的方法在未经预处理的情况下产生了分化的气管支气管球，其顶端膜向外定向，为研究纤毛的形成和功能、疾病发病机制以及病原体与呼吸道粘膜的相互作用提供了一个上皮模型。从3 × 10(5)个细胞开始，28天的类器官产量为1,720 +/- 302。免疫细胞化学证实了气道上皮标志物的细胞定位，包括CFTR、Na+/K+ ATPase、乙酰化的x-微管蛋白、E-cadherin和ZO-1。与天然组织相比，类器官和气液界面（ALI）培养中支气管分化和离子转运相关基因的表达相似。在匹配的原代培养中，类器官纤毛的平均长度为6.1 +/- 0.2 μ m，与ALI培养的5.7 +/- 0.1 μ m相似，类器官培养的纤毛跳动剧烈且协调，频率为7.7 +/- 0.3 Hz， ALI培养的为5.3 +/- 0.8 Hz。渗透诱导的类器官体积变化的功能测量显示水渗透性低。从最小的起始材料生成大量的单个可测试单元是对先前技术的补充。该培养系统可用于研究气道生物学和病理生理学，辅助纤毛病的诊断，并可能用于高通量药物筛选。
Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 x 10(5) cells, organoid yield at day 28 was 1,720 +/- 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-x-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 +/- 0.2 mu m, similar to that of 5.7 +/- 0.1 mu m in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 +/- 0.3 Hz in organoid cultures and 5.3 +/- 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.