BACKGROUND Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation. METHODS Nasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition" (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult™-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADiALI under basal and CFTR corrector treated (VX-809) conditions. RESULTS No significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. CONCLUSION Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.

中文翻译：

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分化的条件重编程 (CRC) 和无饲养层双 SMAD 抑制扩增的人鼻上皮细胞的显着功能差异

背景技术在气液界面 (ALI) 处分化的源自患者的气道细胞是用于囊性纤维化 (CF) 精准治疗的有价值的模型。已经建立了不同的培养扩增方法来扩展气道基底细胞的扩增能力，同时保留功能性气道上皮生理学。即使在相同的 CFTR 基因型内并且尽管使用了类似的 ALI 培养技术，但在培养物中观察到对 CFTR 调节剂的反应有相当大的变化。我们旨在解决文化扩展方法对分化的影响。方法 收集来自 14 个人 (CF=9; 非 CF=5) 的鼻上皮刷，然后在条件重编程培养 (CRC) 和无饲养血清“双 SMAD 抑制” (SMADi) 方法下等分和扩大。使用专有的 PneumaCult™-ALI 培养基对来自每种培养物的扩增细胞进行分化。在基础和 CFTR 校正剂处理 (VX-809) 条件下，比较了 CRCALI 和 SMADiALI 的形态学（免疫荧光）、整体蛋白质组学（LC-MS/MS）和功能（屏障完整性、纤毛运动和离子转运）。结果没有观察到结构形态或基线全球蛋白质组学特征的显着差异。尽管细胞连接形态或纤毛丰度没有差异，但屏障完整性和纤毛运动有显着差异。上皮钠通道和钙激活的氯化物通道活性没有差异，但与 CRCALI 对应物相比，SMADiALI 中 CFTR 介导的氯化物电流显着降低。结论体外细胞生理功能的改变比气道ALI的结构和分化潜能更显着。由于初始扩增培养条件显着影响 CFTR 活性，如果在没有特定参考范围的情况下将来自不同实验室的数据相互比较，这可能会导致错误的结论。