Department of Biomedicine and Clinics of Respiratory Medicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031, Basel, Switzerland.
Fraunhofer Institute for Toxicology and Experimental Medicine, 30625, Hannover, Germany.
Respir Res. 2024 Jan 10;25(1):26. doi: 10.1186/s12931-024-02666-9.
Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice.
Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann-Whitney tests were performed using GraphPad Prism software.
Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma.
IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.
蜂窝状囊肿 (HC) 是特发性肺纤维化 (IPF) 患者肺泡区域的独特组织病理学特征。HC 由单层或分层的基底细胞 (BC) 组成，或由具有基底细胞、纤毛细胞和分泌细胞的支气管样上皮组成。本研究通过培养来自 IPF 患者的肺泡 BC，旨在建立一种体外和体内模型，以模拟 IPF 中的 HC 形成。我们：(1) 优化了培养和扩增 IPF 患者来源的肺泡 BC 的条件，(2) 在气液界面 (ALI) 或三维 (3D) 类器官模型上培养细胞，(3) 研究了将细胞注入博来霉素挑战的 NRG 小鼠后的行为。
从外周 IPF 肺组织中分离肺泡 BC，并在组织培养处理的塑料、ALI 或 3D 类器官模型上培养。此外，将细胞注入博来霉素挑战的 NRG 小鼠体内。通过 TaqMan RT-PCR、免疫印迹、免疫细胞化学/免疫荧光 (ICC/IF) 或免疫组织化学 (IHC)/IF 分析样本。使用 GraphPad Prism 软件进行 Mann-Whitney 检验。
培养的肺泡 BC 高表达经典的基底细胞标志物 (TP63、角蛋白 (KRT)5、KRT14、KRT17)，具有强大的增殖能力和伤口闭合能力。细胞可以冷冻保存并传代培养多达 4 代，而基底细胞标志物的损失不明显。当在 ALI 或 3D 类器官模型上培养时，肺泡 BC 分化为纤毛细胞和分泌细胞。当注入博来霉素挑战的小鼠时，人肺泡 BC 细胞在小鼠实质内形成由人基底细胞和分泌细胞组成的 HC 样结构。
在 ALI 上、在 3D 类器官中或在注入博来霉素挑战的小鼠后，来自 IPF 患者的肺泡 BC 形成与 IPF 肺中 HC 非常相似的 HC 样结构。这些模型因此代表了研究蜂窝形成及其在 IPF 中潜在治疗抑制的有力工具。
