原代人鼻上皮细胞：精准医学背景下的生物样本库
Summary
在这里，我们描述了原代人鼻上皮（HNE）细胞在气液界面处的分离，扩增和分化，以及允许成功冷冻和解冻扩增的HNE的生物样本库方案。该方案分析了不同调节剂处理下分化HNE细胞的电生理特性和CFTR相关氯离子分泌校正。
Abstract
人鼻上皮 （HNE） 细胞易于通过简单的非侵入性鼻刷收集。在气液界面条件下，患者来源的原代 HNE 细胞可以被扩增并分化为假分层上皮，以量化环 AMP 介导的氯化物 （Cl–） 转运作为囊性纤维化跨膜传导调节因子 （CFTR） 功能的指标。如果有效地执行诸如鼻刷质量和冷冻保存时的细胞密度等关键步骤，则可以成功地对HNE细胞进行生物银行分析。此外，短路电流研究表明，冻融不会显着改变HNE电池的电生理特性和对CFTR调节剂的反应。在这项研究中使用的培养条件下，当每个冷冻管冷冻少于2×106 个细胞时，失败率非常高。我们建议每个冷冻管至少冷冻3×106 个细胞。我们表明，将CFTR校正剂与CFTR增强剂相结合的双重疗法对F508del纯合子HNE细胞中的CFTR活性具有相当的校正效果。与双重治疗VX-809 + VX-770相比，三联疗VX-445 + VX-661 + VX-770显着增加了CFTR活性的校正。HNE细胞中CFTR活性的测量是一种有前途的临床前生物标志物，可用于指导CFTR调节剂治疗。
Introduction
囊性纤维化（CF）是一种常染色体隐性遗传性疾病，由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起，导致CFTR蛋白（位于上皮1，2的阴离子通道）的缺失或功能障碍。CFTR治疗的最新进展改善了疾病的预后，最后批准的结合CFTR校正剂和CFTR增强剂的药物导致携带最常见突变p.Phe508del突变（F508del）3，4的CF患者的肺功能和生活质量的重大改善。尽管有这种有希望的治疗进展，但大约10%的CF患者没有资格，因为他们携带这些CFTR调节剂无法挽救的突变。对于这些患者，需要测试其他药物或药物组合，以找到针对特定突变的最有效组合，这突出了个性化治疗的重要性。
人鼻上皮 （HNE） 细胞易于通过简单的非侵入性鼻刷收集，并允许定量循环 AMP 介导的氯化物 （Cl−） 转运作为 CFTR 功能的指标。HNE细胞产生准确的人类气道模型，但它们的寿命在培养物中受到限制。由于培养技术的优化，患者来源的原代HNE细胞可以用Rho相关激酶抑制剂（ROCKi）有条件地重新编程，扩增，并在微孔过滤器5，6上的空液界面（ALI）条件下分化成假分层上皮。存在许多用于HNE培养的培养方案（市售，无血清，“自制”，与饲养层细胞共培养等），并且已经描述了培养基和培养条件的选择影响生长，细胞群分化和上皮功能7，8。本文的实验方案提出了一种简化的、无饲养层的 ROCKi 扩增方法，该方法允许成功获得大量 HNE 细胞，然后在 ALI 上进行分化以进行 CFTR 功能测定。
我们已经证明，在分化的HNE细胞中，用CFTR调节剂治疗48小时足以诱导CFTR依赖性Cl– 电流的电生理校正，并且在 体外 观察到的校正可能与患者的临床改善相关9。因此，HNE细胞不仅代表了基础CF研究的合适模型，而且代表了具有患者特异性CFTR调节剂测试的临床前研究的合适模型。在这种个性化治疗的背景下，该方案的目标是验证来自CF患者的冷冻保存的HNE细胞，在我们的条件下生长，是CFTR校正研究的合适模型，并且在比较来自新鲜和冷冻解冻细胞的CFTR依赖性Cl– 转运时，可以预期类似的结果。该研究还评估了使用双重和三联疗法时不同CFTR调节剂的疗效。
Protocol
所有实验均按照《赫尔辛基宣言》和《Huriet-Serusclat人类研究伦理法》所述的指导方针和规定进行。 1. 烧瓶和不同培养基的制备 制备扩增、空气液体和冷冻介质，如 表1所述。 通过将50mg胶原蛋白溶解在100mL的0.2%冰醋酸中来制备人胶原蛋白IV的储备溶液。在磁力搅拌器上混合至少1小时，然后过滤灭菌溶液。在4°C下储存在玻璃瓶中长达6个…
Representative Results
在气液界面处培养的新鲜HNE细胞显示出通过免疫染色评估的极化和分化的呼吸道上皮的典型特征（图1）。HNE细胞重新分化成异质层上皮细胞（角蛋白8免疫染色阳性），模仿由纤毛（阳性α-微管蛋白染色）和非纤毛产生粘液的杯状细胞（Muc5Ac免疫染色阳性）组成的假分层呼吸道上皮的 体内 情况。整体上皮呈现紧密连接（通过 ZO-1、带状动脉闭塞-1 染色评估）。在野生?…
Discussion
在个性化医疗的背景下，使用患者来源的鼻上皮细胞作为人支气管上皮（HBE）细胞的替代物来测量CFTR活性已被提出，因为HNE在培养物中复制细胞的特性9，11。与HBE细胞培养物相比，HNE的强大优势在于它们易于且非侵入性地取样。HNE细胞培养物中的短路电流测量能够评估CFTR依赖性Cl– 通过上皮的运输，其显示与通过各种基因型患者的鼻电位差异评…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们衷心感谢所有患者及其家属参与这项研究。这项工作得到了法国Mucoviscidose Vaincre la Mucoviscidose协会的资助;法国协会ABCF 2和Vertex Pharmaceuticals创新奖。
Materials
| ABBV-2222 | Selleckchem | S8535 | |
| ABBV-974 | Selleckchem | S8698 | |
| Advanced DMEM/F-12 | Life Technologies | 12634010 | |
| Alexa 488 goat secondary antibody | Invitrogen | A11001 | |
| Alexa 594 goat secondary antibody | Invitrogen | A11012 | |
| Amphotericin B | Life Technologies | 15290026 | |
| Anti-alpha-tubulin antibody | Abcam | ab80779 | |
| Anti-CFTR monoclonal antibody (24-1) | R&D Systems | MAB25031 | |
| Anti-cytokeratin 8 antibody | Progen | 61038 | |
| Anti-Muc5AC antibody | Santa Cruz Biotech | sc-20118 | |
| Anti-ZO-1 antibody | Santa Cruz Biotech | sc-10804 | |
| Ciprofloxacin | provided by Necker Hospital Pharmacy | ||
| Colimycin | Sanofi | provided by Necker Hospital Pharmacy | |
| Collagen type IV | Sigma-Aldrich Merck | C-7521 | |
| cytology brush | Laboratory GYNEAS | 02.104 | |
| DMSO | Sigma-Aldrich Merck | D2650 | |
| EGF | Life Technologies | PHG0311 | |
| Epinephrin | Sigma-Aldrich Merck | E4375 | |
| F12-Nutrient Mixture | Life Technologies | 11765054 | |
| FBS | Life Technologies | 10270106 | |
| Ferticult | Fertipro NV | FLUSH020 | |
| Flasks 25 | Thermo Scientific | 156.367 | |
| Flasks 75 | Thermo Scientific | 156.499 | |
| Glacial acetic acid | VWR | 20104.298 | |
| HEPES | Sigma-Aldrich Merck | H3375 | |
| Hydrocortisone | Sigma-Aldrich Merck | SLCJ0893 | |
| Insulin | Sigma-Aldrich Merck | I0516 | |
| Mg2+ and Ca2+-free DPBS | Life Technologies | 14190094 | |
| Penicillin/Streptomycin | Life Technologies | 15140130 | |
| Tazocillin | Mylan | provided by Necker Hospital Pharmacy | |
| Transwell Filters | Sigma-Aldrich Merck | CLS3470-48EA | |
| Triton-X100 | Sigma-Aldrich Merck | T8787 | |
| Trypsin 0,25% | Life Technologies | 25200056 | |
| Vectashield mounting medium with DAPI | Vector Laboratories | H-1200 | |
| VX-445 | Selleckchem | S8851 | |
| VX-661 | Selleckchem | S7059 | |
| VX-770 | Selleckchem | S1144 | |
| VX-809 | Selleckchem | S1565 | |
| Xylocaine naphazoline 5% | Aspen France | provided by Necker Hospital Pharmacy | |
| Y-27632 | Selleckchem | S1049 |
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Cite This Article
Kelly, M., Dreano, E., Hatton, A., Lepissier, A., Golec, A., Sermet-Gaudelus, I., Pranke, I. Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine. J. Vis. Exp. (182), e63409, doi:10.3791/63409 (2022).