Coles Janice L, Thompson James, Horton Katie L, Hirst Robert A, Griffin Paul, Williams Gwyneth M, Goggin Patricia, Doherty Regan, Lackie Peter M, Harris Amanda, Walker Woolf T, O'Callaghan Christopher, Hogg Claire, Lucas Jane S, Blume Cornelia, Jackson Claire L
Primary Ciliary Dyskinesia Centre, NIHR Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK.
School of Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK.
J Clin Med. 2020 Nov 21;9(11):3753. doi: 10.3390/jcm9113753.
Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.
鼻上皮细胞的气液界面（ALI）培养是原发性纤毛运动障碍（PCD）诊断和研究中的一种重要工具。体外样本在采样、感染或炎症过程中常因细胞损伤而出现继发性运动障碍，这会混淆PCD的诊断结果。ALI培养能够使健康纤毛再生，有助于区分原发性和继发性纤毛运动障碍。我们描述了一种自2018年4月起在三个合作的PCD诊断中心采用的改良ALI培养方法，包括当前南安普敦大学医院针对新冠病毒的风险缓解措施，并展示了结果。共接种240个鼻上皮细胞样本进行ALI培养，其中199个（82.9%）形成了纤毛。83个最初结果不明确或样本不足的体外样本中，有54个（63.9%）在体外培养后提供了诊断信息。181个鼻刷样本中剩余的基底上皮细胞被液氮冷冻保存；39个样本在冷冻保存后进行了ALI培养，且均形成了纤毛。体外样本的纤毛摆动模式（通过高速视频显微镜观察）得以重现，扫描电子显微镜显示纤毛形成良好，并且在冷冻保存后进行ALI培养的代表性病例中，纤毛可以用免疫荧光标记（抗α微管蛋白和抗RSPH4a）。总之，我们的ALI培养方案在三个中心都实现了高纤毛形成率，减少了患者因重复刷检活检的召回次数，并提高了诊断的确定性。剩余诊断样本的冷冻保存取得成功，促进了PCD研究。
