Abstract

Culture at the air–liquid interface is broadly accepted as necessary for differentiation of cultured epithelial cells towards an in vivo-like phenotype. However, air–liquid interface cultures are expensive, laborious and challenging to scale for increased throughput applications. Deconstructing the microenvironmental parameters that drive these differentiation processes could circumvent these limitations, and here we hypothesize that reduced oxygenation due to diffusion limitations in liquid media limits differentiation in submerged cultures; and that this phenotype can be rescued by recreating normoxic conditions at the epithelial monolayer, even under submerged conditions. Guided by computational models, hyperoxygenation of atmospheric conditions was applied to manipulate oxygenation at the monolayer surface. The impact of this rescue condition was confirmed by assessing protein expression of hypoxia-sensitive markers. Differentiation of primary human bronchial epithelial cells isolated from healthy patients was then assessed in air–liquid interface, submerged and hyperoxygenated submerged culture conditions. Markers of differentiation, including epithelial layer thickness, tight junction formation, ciliated surface area and functional capacity for mucociliary clearance, were assessed and found to improve significantly in hyperoxygenated submerged cultures, beyond standard air–liquid interface or submerged culture conditions. These results demonstrate that an air–liquid interface is not necessary to produce highly differentiated epithelial structures, and that increased availability of oxygen and nutrient media can be leveraged as important strategies to improve epithelial differentiation for applications in respiratory toxicology and therapeutic development.

中文翻译：

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氧合作为气液界面上皮分化的驱动因素

 抽象的

空气-液体界面的培养被广泛认为是培养的上皮细胞向体内样表型分化所必需的。然而，气液界面培养物昂贵、费力且难以扩展以提高通量应用。解构驱动这些分化过程的微环境参数可以规避这些限制，在这里我们假设由于液体培养基中的扩散限制而减少的氧合作用限制了水下培养物的分化；并且这种表型可以通过在上皮单层上重建常氧条件来挽救，即使是在水下条件下。在计算模型的指导下，利用大气条件的过度氧化来控制单层表面的氧化。通过评估缺氧敏感标记物的蛋白质表达证实了这种救援条件的影响。然后在气液界面、浸没和高氧浸没培养条件下评估从健康患者分离的原代人支气管上皮细胞的分化。评估了分化标志物，包括上皮层厚度、紧密连接形成、纤毛表面积和粘液纤毛清除功能能力，发现在高氧浸没培养物中显着改善，超出标准气液界面或浸没培养条件。这些结果表明，空气-液体界面并不是产生高度分化的上皮结构所必需的，并且可以利用增加氧气和营养介质的可用性作为改善上皮分化的重要策略，用于呼吸毒理学和治疗开发的应用。