Barbet Kimberly, Schmitz Mona S, Westhölter Dirk, Kamler Markus, Rütten Stephan, Thiebes Anja L, Sitek Barbara, Bayer Malte, Schedel Michaela, Reuter Sebastian, Darwiche Kaid, Luengen Anja E, Taube Christian
Department of Pulmonary Medicine, University Medical Center Essen, Ruhrlandklinik, Essen, Germany.
Department of Translational Pulmonology, Department of Pulmonary Medicine, University Medical Center Essen, Ruhrlandklinik, Essen, Germany.
Respir Res. 2024 Dec 24;25(1):439. doi: 10.1186/s12931-024-03060-1.
Using primary airway epithelial cells (AEC) is essential to mimic more closely different types and stages of lung disease in humans while reducing or even replacing animal experiments. Access to lung tissue remains limited because these samples are generally obtained from patients who undergo lung transplantation for end-stage lung disease or thoracic surgery for (mostly) lung cancer. We investigated whether forceps or cryo biopsies are a viable alternative source of AEC compared to the conventional technique.
AECs were obtained ex vivo from healthy donor lung tissue using the conventional method and two biopsy procedures (forceps, cryo). The influence of the isolation method on the quality and function of AEC was investigated at different time-points during expansion and differentiation in air-liquid interface cultures. In addition, fully-differentiated AECs were stimulated with house dust mite extract (HDM) to allow functional analyses in an allergic in vitro model. Vitality or differentiation capacity were determined using flow cytometry, scanning electron microscope, periodic acid-Schiff reaction, immunofluorescence staining, and proteomics.
As anticipated, no significant differences between each of the sampling methods were detected for any of the measured outcomes. The proteome composition was comparable for each isolation method, while donor-dependent effects were observed. Treatment with HDM led to minor differences in mucociliary differentiation.
Our findings confirmed the adequacy of these alternative approaches for attaining primary AECs, which can now expand the research for a broader range of lung diseases and for studies at an earlier stage not requiring lung surgery.
使用原代气道上皮细胞（AEC）对于更紧密地模拟人类肺部疾病的不同类型和阶段至关重要，同时可减少甚至取代动物实验。获取肺组织仍然受限，因为这些样本通常来自因终末期肺病接受肺移植或（主要因）肺癌接受胸外科手术的患者。我们研究了与传统技术相比，钳取活检或冷冻活检是否是获取AEC的可行替代来源。
使用传统方法以及两种活检程序（钳取活检、冷冻活检）从健康供体肺组织中离体获取AEC。在气液界面培养的扩增和分化过程中的不同时间点，研究分离方法对AEC质量和功能的影响。此外，用屋尘螨提取物（HDM）刺激完全分化的AEC，以便在过敏性体外模型中进行功能分析。使用流式细胞术、扫描电子显微镜、过碘酸-希夫反应、免疫荧光染色和蛋白质组学来确定活力或分化能力。
正如预期的那样，对于任何测量结果，各采样方法之间均未检测到显著差异。每种分离方法的蛋白质组组成相当，同时观察到了供体依赖性效应。用HDM处理导致黏液纤毛分化存在微小差异。
我们的研究结果证实了这些替代方法获取原代AEC的充分性，这现在可以扩展对更广泛肺部疾病的研究以及对不需要肺手术的早期阶段的研究。
