Department of Chemical Engineering, McGill University, Montreal, Canada.
Department of Chemical Engineering, McMaster University, Hamilton, Canada.
Integr Biol (Camb). 2021 Mar 17;13(3):61-72. doi: 10.1093/intbio/zyab002.
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.
在空气-液体界面培养被广泛认为是培养上皮细胞向类似于体内表型分化所必需的。然而，空气-液体界面培养昂贵、费力且难以扩大规模以满足高通量应用的需求。解构驱动这些分化过程的微环境参数可以规避这些限制，在这里我们假设液体培养基中的扩散限制导致的低氧环境限制了浸没培养中的分化；并且即使在浸没条件下，通过在上皮单层中重新创造正常氧条件可以挽救这种表型。在计算模型的指导下，采用超氧条件来调节单层表面的氧合作用。通过评估缺氧敏感标志物的蛋白表达来确认这种挽救条件的影响。然后在空气-液体界面、浸没和超氧浸没培养条件下评估分离自健康患者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、纤毛表面面积和黏液纤毛清除功能，发现超氧浸没培养条件下显著改善，超过标准空气-液体界面或浸没培养条件。这些结果表明，产生高度分化的上皮结构并不需要空气-液体界面，并且增加氧气和营养培养基的可用性可以作为提高上皮分化的重要策略，用于呼吸毒理学和治疗开发中的应用。
