BACKGROUND In vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators. METHODS Primary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor). RESULTS CF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661. CONCLUSIONS CF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.

中文翻译：

---

来自扩大的原发性囊性纤维化人气道细胞的体外 3D 培养肺模型

背景 体外囊性纤维化 (CF) 模型对于了解疾病的机制和后果至关重要。它们也是当前和新型 CF 药物临床前疗效研究的金标准。然而，很少有研究调查原代 CF 人支气管上皮 (CF-HBE) 细胞的扩增和分化。在这里，我们描述了扩大原代 CF 气道细胞的培养条件，同时保留了它们分化为表达功能性囊性纤维化跨膜电导调节器 (CFTR) 离子通道的 3D 上皮培养物的能力，该离子通道对 CFTR 调节剂有反应。方法 使用不含饲养细胞或添加 Rho 激酶 (ROCK) 抑制剂的 PneumaCultTM-Ex Plus (StemCell Technologies) 培养基扩增原代 CF 气道细胞。在气液界面 (ALI) 上差异传代的 CF-HBE 细胞在表型和功能上表征为响应 CFTR 校正药物 VX-661 (Tezacaftor)。结果 CF-HBE 原代细胞扩增至 6 代（约 25 次群体倍增），分化为 3D 上皮培养物，如跨上皮电阻 (TEER) > 400 Ohms∙cm2 和具有高脚杯状假复层柱状纤毛上皮的存在所证明细胞。然而，当用校正药物 VX-661 处理时，来自大多数供体的最多 5 代细胞显示出增加的 CFTR 介导的短路电流。纤毛搏动频率 (CBF) 也随着校正器 VX-661 的增加而增加。结论 CF 供体来源的气道细胞可以在不使用饲养细胞或额外的 ROCK 抑制剂的情况下进行扩增，并且仍然实现对 CFTR 调节剂有反应的最佳 3D 上皮培养物。对罕见 CF 突变的研究可以从细胞扩增中受益，并可能导致个性化药物/治疗的设计。