Development and functional characterization of a tissue-engineered blood-air barrier model for <i>in vitro</i> applications

The blood-air barrier (BAB) of the lung is a critical interface responsible for gas exchange and protection against external attempts, and acts as a selective barrier. Developing in vitro models that replicate its structural and functional properties is essential in studying pulmonary diseases and their therapy. In this study, a model consisting of alveolar epithelial (A549) and primary endothelial (pHUVEC) cells seeded on opposite sides of a thin (11 ± 4 μm), electrospun poly(ε-caprolactone) mesh of nanofibers (140–800 nm) to represent the basal membrane, and the interstitial matrix of the native BAB when coated with collagen type I, fibronectin, and laminin 511 proteins. The dense, nanofibrous architecture of the mesh enabled the formation of cellular monolayers on opposite sides, allowing gas and nutrient exchange for 14 days at air–liquid interface. The mesh had a Young’s modulus of 8.0 ± 0.8 MPa, and upon coating with proteins, the water contact angles were decreased from 127.5°±2.6 to 94.4°±3.6. Epithelial and endothelial monolayers demonstrated tight junction formation as shown by ZO-1 and CD31 expression. TEER was measured as 44 ± 5.0 Ω·cm2 with a permeability coefficient (Papp) of 2–5 × 10−6 cm/s against fluorescein. This study presents a physiologically relevant in vitro BAB model for respiratory research and therapies.肺的气血屏障（blood-air barrier, BAB）是介导气体交换、抵御外界侵袭并发挥选择性通透屏障作用的关键界面。构建能够复刻其结构与功能特性的体外模型，对肺部疾病的研究及治疗研发均具有重要意义。 本研究构建了一款体外气血屏障模型：将肺泡上皮细胞（alveolar epithelial, A549）与原代内皮细胞（primary endothelial, pHUVEC）接种于超薄（11±4 μm）电纺聚己内酯（poly(ε-caprolactone)）纳米纤维膜的两侧，该膜的纳米纤维直径为140~800 nm；经Ⅰ型胶原蛋白、纤连蛋白与层粘连蛋白511包被后，该膜可模拟天然气血屏障的基底膜与间质基质组分。该膜致密的纳米纤维结构可促使两侧形成细胞单层，使其能够在气液界面培养条件下维持气体与营养物质交换达14天。 该膜的杨氏模量为8.0±0.8 MPa；经蛋白包被后，其水接触角从127.5°±2.6°降至94.4°±3.6°。通过ZO-1与CD31的蛋白表达检测结果可知，上皮与内皮细胞单层成功形成了紧密连接。经检测，该模型的经上皮电阻（transepithelial electrical resistance, TEER）为44±5.0 Ω·cm²，对荧光素的通透系数（permeability coefficient, Papp）为2~5×10⁻⁶ cm/s。 本研究构建的这款体外气血屏障模型具有良好的生理学相关性，可用于呼吸道疾病研究与治疗研发。