A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia.
A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia.
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DOI：
10.3390/jcm9113753
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发表时间：
2020-11-21
影响因子：
3.9
通讯作者：
Jackson CL
中科院分区：
文献类型：
--
作者：
Coles JL;Thompson J;Horton KL;Hirst RA;Griffin P;Williams GM;Goggin P;Doherty R;Lackie PM;Harris A;Walker WT;O'Callaghan C;Hogg C;Lucas JS;Blume C;Jackson CL
鼻上皮细胞气液界面（ALI）培养是原发性纤毛运动障碍（PCD）诊断和研究的重要工具。离体样品通常显示继发性运动障碍，因为在取样期间细胞损伤，感染或炎症混淆PCD诊断结果。ALI培养可使健康纤毛再生，促进原发性纤毛运动障碍与继发性纤毛运动障碍的分化。我们描述了2018年4月在三个合作的PCD诊断站点采用的经修订的ALI培养方法，包括目前南安普敦大学医院的COVID-19风险缓解措施，以及目前的结果。240个鼻上皮细胞样本用于ALI培养，199个（82.9%）被纤毛。83份离体样本中有54份（63.9%）在体外培养后提供了最初模棱两可或不充分的诊断信息。181例鼻刷样本中多余的基底上皮细胞冷冻于液氮中；39例经低温培养，均有纤毛。通过高速视频显微镜再现离体样品的纤毛跳动模式，扫描电镜显示良好的纤毛化，并且在冷冻后ali培养的代表性病例中，纤毛可以进行免疫荧光标记（抗α -微管蛋白和抗rsph4a）。总之，我们的ALI培养方案在三个中心提供了高调解率，最大限度地减少了患者重复刷牙活检的回忆，提高了诊断的确定性。剩余诊断样本的冷冻保存成功，为PCD研究提供了便利。
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.
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DOI：
10.1534/g3.115.019851
发表时间：
2015-07-02
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
Marshall CR;Scherer SW;Zariwala MA;Lau L;Paton TA;Stockley T;Jobling RK;Ray PN;Knowles MR;FORGE Canada Consortium;Hall DA;Dell SD;Kim RH
通讯作者：
Kim RH
影响因子：
4
作者：
Lai, Michele;Pifferi, Massimo;Pistello, Mauro
通讯作者：
Pistello, Mauro
影响因子：
--
作者：
Marthin, June Kehlet;Stevens, Elizabeth Munkebjerg;Nielsen, Kim Gjerum
通讯作者：
Nielsen, Kim Gjerum
影响因子：
24.3
作者：
Jackson, Claire L.;Behan, Laura;Lucas, Jane S.
通讯作者：
Lucas, Jane S.
影响因子：
9.6
作者：
Kempeneers, Celine;Seaton, Claire;Chilvers, Mark A.
通讯作者：
Chilvers, Mark A.