University Clinic for Cardiac and Thoracic Surgery, Otto-von-Guericke-University Magdeburg, D-39120 Magdeburg, Germany.
Int J Mol Sci. 2023 Feb 18;24(4):4113. doi: 10.3390/ijms24044113.
In vitro airway models are increasingly important for pathomechanistic analyses of respiratory diseases. Existing models are limited in their validity by their incomplete cellular complexity. We therefore aimed to generate a more complex and meaningful three-dimensional (3D) airway model. Primary human bronchial epithelial cells (hbEC) were propagated in airway epithelial cell growth (AECG) or PneumaCult ExPlus medium. Generating 3D models, hbEC were airlifted and cultured on a collagen matrix with donor-matched bronchial fibroblasts for 21 days comparing two media (AECG or PneumaCult ALI (PC ALI)). 3D models were characterized by histology and immunofluorescence staining. The epithelial barrier function was quantified by transepithelial electrical resistance (TEER) measurements. The presence and function of ciliated epithelium were determined by Western blot and microscopy with high-speed camera. In 2D cultures, an increased number of cytokeratin 14-positive hbEC was present with AECG medium. In 3D models, AECG medium accounted for high proliferation, resulting in hypertrophic epithelium and fluctuating TEER values. Models cultured with PC ALI medium developed a functional ciliated epithelium with a stable epithelial barrier. Here, we established a 3D model with high in vivo-in vitro correlation, which has the potential to close the translational gap for investigations of the human respiratory epithelium in pharmacological, infectiological, and inflammatory research.
体外气道模型对于呼吸道疾病的病理机制分析越来越重要。现有的模型由于其细胞复杂性的不完全而存在局限性。因此，我们旨在生成一个更复杂和更有意义的三维（3D）气道模型。原代人支气管上皮细胞（hbEC）在气道上皮细胞生长（AECG）或 PneumaCult ExPlus 培养基中繁殖。生成 3D 模型时，将 hbEC 空运并在含有供体匹配的支气管成纤维细胞的胶原基质上培养 21 天，比较两种培养基（AECG 或 PneumaCult ALI（PC ALI））。通过组织学和免疫荧光染色对 3D 模型进行特征描述。通过跨上皮电阻（TEER）测量来量化上皮屏障功能。通过 Western blot 和带有高速摄像机的显微镜确定纤毛上皮的存在和功能。在 2D 培养中，AECG 培养基中存在更多的角蛋白 14 阳性 hbEC。在 3D 模型中，AECG 培养基促进了高增殖，导致肥大的上皮和波动的 TEER 值。用 PC ALI 培养基培养的模型形成了具有稳定上皮屏障的功能性纤毛上皮。在这里，我们建立了一种具有高体内-体外相关性的 3D 模型，该模型有可能缩小人类呼吸道上皮在药理学、感染学和炎症研究中的转化差距。
