Name: primary human nasal epithelial cells: biobanking in the context of precision medicine
Uploaded: 2022-04-22T14:45:00
Description: Watch this Scientific Journal Video about Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine at JoVE.com

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。

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
<ol><li>制备扩增、空气液体和冷冻介质，如 表1所述。</li>
	<li>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...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+patients+with+cystic+fibrosis+homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR.</a> The New England Journal of Medicine. 373 (3), 220-231 (2015).</li><li>Griese, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+elexacaftor/tezacaftor/ivacaftor+for+24+weeks+or+longer+in+people+with+cystic+fibrosis+and+one+or+more+F508del+alleles:+Interim+results+of+an+open-label+phase+3+clinical+trial.">Safety and efficacy of elexacaftor/tezacaftor/ivacaftor for 24 weeks or longer in people with cystic fibrosis and one or more F508del alleles: Interim results of an open-label phase 3 clinical trial.</a> American Journal of Respiratory and Critical Care Medicine. 203 (3), 381-385 (2021).</li><li>Horani, A., Nath, A., Wasserman, M. G., Huang, T., Brody, S. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+CFTR+function+in+nasal+epithelial+cells+from+cystic+fibrosis+patients+predicts+improvement+of+respiratory+function+by+CFTR+modulators.">Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators.</a> Scientific Reports. 7 (1), 7375 (2017).</li><li>Noel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+genotype+with+phenotype+using+CFTR-mediated+whole-cell+Cl&#8722;+currents+in+human+nasal+epithelial+cells.">Correlating genotype with phenotype using CFTR-mediated whole-cell Cl&#8722; currents in human nasal epithelial cells.</a> The Journal of Physiology. , (2021).</li><li>Brewington, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brushed+nasal+epithelial+cells+are+a+surrogate+for+bronchial+epithelial+CFTR+studies.">Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.</a> JCI Insight. 3 (13), 99385 (2018).</li><li>Pranke, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Might+brushed+nasal+cells+be+a+surrogate+for+cftr+modulator+clinical+response.">Might brushed nasal cells be a surrogate for cftr modulator clinical response.</a> American Journal of Respiratory and Critical Care Medicine. 199 (1), 123-126 (2019).</li><li>de Courcey, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+primary+human+nasal+epithelial+cell+cultures+for+the+study+of+cystic+fibrosis+pathophysiology.">Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology.</a> American Journal of Physiology-Cell Physiology. 303 (11), 1173-1179 (2012).</li><li>Devalia, J. L., Sapsford, R. J., Wells, C. W., Richman, P., Davies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+comparison+of+human+bronchial+and+nasal+epithelial+cells+in+vitro.">Culture and comparison of human bronchial and nasal epithelial cells in vitro.</a> Respiratory Medicine. 84 (4), 303-312 (1990).</li><li>McDougall, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+epithelial+cells+as+surrogates+for+bronchial+epithelial+cells+in+airway+inflammation+studies.">Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.</a> American Journal of Respiratory Cell and Molecular Biology. 39 (5), 560-568 (2008).</li><li>Mosler, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+nasal+epithelial+brushing+for+the+study+of+airway+epithelial+functions+in+CF+infants.">Feasibility of nasal epithelial brushing for the study of airway epithelial functions in CF infants.</a> Journal of Cystic Fibrosis: Official Journal of the European Cystic Fibrosis Society. 7 (1), 44-53 (2008).</li><li>Tosoni, K., Cassidy, D., Kerr, B., Land, S. C., Mehta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+drugs+to+probe+the+variability+of+trans-epithelial+airway+resistance.">Using drugs to probe the variability of trans-epithelial airway resistance.</a> PLOS One. 11 (2), 0149550 (2016).</li><li>Sch&#246;gler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pediatric+cystic+fibrosis+airway+epithelial+cell+cultures+at+the+air-liquid+interface+obtained+by+non-invasive+nasal+cytology+brush+sampling.">Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling.</a> Respiratory Research. 18 (1), 215 (2017).</li><li>M&#252;ller, L., Brighton, L. E., Carson, J. L., Fischer, W. A., Jaspers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+human+nasal+epithelial+cells+at+the+air+liquid+interface.">Culturing of human nasal epithelial cells at the air liquid interface.</a> Journal of Visualized Experiments: JoVE. (80), e50646 (2013).</li><li>Awatade, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+functional+responses+in+human+primary+lung+cells+as+a+basis+for+personalized+therapy+for+cystic+fibrosis.">Measurements of functional responses in human primary lung cells as a basis for personalized therapy for cystic fibrosis.</a> EBioMedicine. 2 (2), 147-153 (2014).</li><li>Brewington, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+nasal+epithelial+cell+spheroids+for+individualized+cystic+fibrosis+transmembrane+conductance+regulator+study.">Generation of human nasal epithelial cell spheroids for individualized cystic fibrosis transmembrane conductance regulator study.</a> Journal of Visualized Experiments: JoVE. (134), e57492 (2018).</li><li>Wiszniewski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cultures+of+polarized+airway+epithelial+cells+from+patients+with+cystic+fibrosis.">Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis.</a> American Journal of Respiratory Cell and Molecular Biology. 34 (1), 39-48 (2006).</li><li>Reynolds, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+progenitor+clone+formation+is+enhanced+by+Y-27632-dependent+changes+in+the+transcriptome.">Airway progenitor clone formation is enhanced by Y-27632-dependent changes in the transcriptome.</a> American Journal of Respiratory Cell and Molecular Biology. 55 (3), 323-336 (2016).</li><li>Suprynowicz, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditionally+reprogrammed+cells+represent+a+stem-like+state+of+adult+epithelial+cells.">Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (49), 20035-20040 (2012).</li><li>Awatade, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+functional+differences+in+differentiated+Conditionally+Reprogrammed+(CRC)-+and+Feeder-free+Dual+SMAD+inhibited-expanded+human+nasal+epithelial+cells.">Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.</a> Journal of Cystic Fibrosis. 20 (2), 364-371 (2021).</li><li>Veit, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+folding+correction+of+F508del+and+rare+CFTR+mutants+by+elexacaftor-tezacaftor-ivacaftor+(Trikafta)+combination.">Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.</a> JCI Insight. 5 (18), 139983 (2020).</li><li>Dale, T. P., Borg D'anastasi, E., Haris, M., Forsyth, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rock+Inhibitor+Y-27632+enables+feeder-free,+unlimited+expansion+of+Sus+scrofa+domesticus+swine+airway+stem+cells+to+facilitate+respiratory+research.">Rock Inhibitor Y-27632 enables feeder-free, unlimited expansion of Sus scrofa domesticus swine airway stem cells to facilitate respiratory research.</a> Stem Cells International. 2019, 1-15 (2019).</li><li>Laselva, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+multiple+class+II+CFTR+mutations+by+elexacaftor+tezacaftor+ivacaftor+mediated+in+part+by+the+dual+activities+of+elexacaftor+as+both+corrector+and+potentiator.">Rescue of multiple class II CFTR mutations by elexacaftor+tezacaftor+ivacaftor mediated in part by the dual activities of elexacaftor as both corrector and potentiator.</a> The European Respiratory Journal. 57 (6), 2002774 (2021).</li><li>Li, X., Krawetz, R., Liu, S., Meng, G., Rancourt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+improves+survival+of+cryopreserved+serum/feeder-free+single+human+embryonic+stem+cells.">ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.</a> Human Reproduction. 24 (3), 580-589 (2008).</li><li>Neuberger, T., Burton, B., Clark, H., Van Goor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+primary+cultures+of+human+bronchial+epithelial+cells+isolated+from+cystic+fibrosis+patients+for+the+pre-clinical+testing+of+CFTR+modulators.">Use of primary cultures of human bronchial epithelial cells isolated from cystic fibrosis patients for the pre-clinical testing of CFTR modulators.</a> Methods in Molecular Biology. 741, 39-54 (2011).</li><li>Laselva, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+pre-clinical+modulators+developed+for+F508del-CFTR+have+the+potential+to+be+effective+for+ORKAMBI+resistant+processing+mutants.">Emerging pre-clinical modulators developed for F508del-CFTR have the potential to be effective for ORKAMBI resistant processing mutants.</a> Journal of Cystic Fibrosis. 20 (1), 106-119 (2021).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABBV-2222</td>
			<td>Selleckchem</td>
			<td>S8535</td>
			<td></td>
		</tr>
		<tr>
			<td>ABBV-974</td>
			<td>Selleckchem</td>
			<td>S8698</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488 goat secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 594 goat secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab80779</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFTR monoclonal antibody (24-1)</td>
			<td>R&#38;D Systems</td>
			<td>MAB25031</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cytokeratin 8 antibody</td>
			<td>Progen</td>
			<td>61038</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Muc5AC antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-20118</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ZO-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-10804</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td></td>
			<td>provided by Necker Hospital Pharmacy</td>
			<td></td>
		</tr>
		<tr>
			<td>Colimycin</td>
			<td>Sanofi</td>
			<td>provided by Necker Hospital Pharmacy</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type IV</td>
			<td>Sigma-Aldrich Merck</td>
			<td>C-7521</td>
			<td></td>
		</tr>
		<tr>
			<td>cytology brush</td>
			<td>Laboratory GYNEAS</td>
			<td>02.104</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich Merck</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinephrin</td>
			<td>Sigma-Aldrich Merck</td>
			<td>E4375</td>
			<td></td>
		</tr>
		<tr>
			<td>F12-Nutrient Mixture</td>
			<td>Life Technologies</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferticult</td>
			<td>Fertipro NV</td>
			<td>FLUSH020</td>
			<td></td>
		</tr>
		<tr>
			<td>Flasks 25</td>
			<td>Thermo Scientific</td>
			<td>156.367</td>
			<td></td>
		</tr>
		<tr>
			<td>Flasks 75</td>
			<td>Thermo Scientific</td>
			<td>156.499</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>VWR</td>
			<td>20104.298</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich Merck</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich Merck</td>
			<td>SLCJ0893</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich Merck</td>
			<td>I0516</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2+ and Ca2+-free DPBS</td>
			<td>Life Technologies</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Tazocillin</td>
			<td>Mylan</td>
			<td>provided by Necker Hospital Pharmacy</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell Filters</td>
			<td>Sigma-Aldrich Merck</td>
			<td>CLS3470-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0,25%</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>VX-445</td>
			<td>Selleckchem</td>
			<td>S8851</td>
			<td></td>
		</tr>
		<tr>
			<td>VX-661</td>
			<td>Selleckchem</td>
			<td>S7059</td>
			<td></td>
		</tr>
		<tr>
			<td>VX-770</td>
			<td>Selleckchem</td>
			<td>S1144</td>
			<td></td>
		</tr>
		<tr>
			<td>VX-809</td>
			<td>Selleckchem</td>
			<td>S1565</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylocaine naphazoline 5%</td>
			<td>Aspen France</td>
			<td>provided by Necker Hospital Pharmacy</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary human nasal epithelial cells: biobanking in the context of precision medicine

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。

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?...

Watch this Scientific Journal Video about Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine at JoVE.com

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

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。

原代人鼻上皮细胞：精准医学背景下的生物样本库

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

该方案突出了扩增、分化和冷冻保存原代鼻上皮细胞的关键步骤。在我们的培养条件下，原代鼻上皮细胞模仿肺上皮细胞。这允许患者来源的培养物和来自新鲜或生物银行细胞的个性化医学研究。患者来源的鼻上皮培养物可用于评估CFTR活性，并通过评估患者对新疗法的个体反应来帮助囊性纤维化治疗。演示该程序的将是来自我实验室的工程师Aurelie Hatton。首先，在10毫升扩增培养基中生长鼻上皮或HNE细胞。在37摄氏度和5%二氧化碳下在75平方厘米的胶原蛋白包被烧瓶中孵育，直到达到80%至90%汇合度。每 48 到 72 小时更换一次培养基。之后，用10毫升镁和无钙DPBS洗涤细胞。吸出并丢弃盐水。在加入两毫升0.25%胰蛋白酶并将烧瓶放回培养箱8至12分钟之前。之后，用手掌牢牢地敲击烧瓶，以帮助细胞脱离。加入10毫升扩增培养基以停止酶促反应。使用10毫升移液管大力冲洗烧瓶，以在烧瓶表面拉出并排出扩增培养基，以冲洗和分离细胞并将细胞收集在15毫升管中。将细胞悬浮液以500倍G在4摄氏度下离心5分钟，然后弃去上清液。将沉淀重新悬浮在5至10毫升的扩增培养基中。然后在血细胞计数器上计数细胞。细胞计数后，将细胞以500倍G在4摄氏度下离心5分钟，弃去上清液。然后将沉淀重新悬浮在富集的冷冻培养基中，以获得每毫升10至6个细胞的三至五倍，并置于冷冻小瓶中。通过在零下80摄氏度的适当冷冻容器中将温度降低约一摄氏度/分钟来缓慢冷冻细胞。第二天，将保存的样品移至氮气储存容器中进行长期储存。将新鲜制备的扩增培养基在设定为37摄氏度的水浴中加热。从氮气储存罐中取出冷冻小瓶，并迅速将其放入水浴中，注意不要将整个小瓶浸入水中。当只剩下一小滴冷冻液滴时，从水浴中取出冷冻小瓶。用70%酒精擦拭小瓶，并将其放在引擎盖下。使用一毫升移液器，将解冻的细胞转移到15毫升管中。然后以逐滴方式加入一毫升温扩增培养基。一分钟后，再加入一毫升扩增培养基，等待一分钟。加入10毫升扩增培养基，以500倍G离心2分钟，4摄氏度。吸出并丢弃上清液。将沉淀重新悬浮在所需的扩增培养基中，以达到每毫升6个细胞的一倍10倍的细胞密度。将细胞接种到含有扩增培养基的25平方厘米胶原包被烧瓶上后，将细胞在37摄氏度和5%二氧化碳下孵育。如前所述，在进行细胞扩增之前，在两到三天内目视观察细胞扩张。将细胞以3.3倍的密度接种在0.33平方厘米胶原包被的多孔过滤器上，每个过滤器10至第五个细胞的密度，在顶端侧补充300微升扩增培养基，在基底外侧补充900微升空气液体培养基。三天后，吸出顶端培养基，并在气液界面处培养细胞在气液培养基中三至四周，以建立分化的上皮。每48至72小时更换一次基础培养基。在气液界面处培养的新鲜HNE细胞显示出极化和分化的呼吸道上皮的典型特征。在78个HNE细胞样本中，比较了立即接种的鼻刷和在冲洗培养基中运输一至七天后的成功率。成功培养的初始平均百分比为82%，在0至5天之间播种的样品中达到87%。在新鲜条件下成功区分的样品中，83%在冷冻保存样品中也成功。同样，在新鲜条件下未能区分的样品中，71%在冻融条件下也未成功。此外，50%的样品在2到3倍之间冷冻，每个冷冻瓶的6个细胞在解冻时无法生长，对于低于2倍的10到6个细胞，达到80%。对于在DMSO处理的HNE细胞中响应于毛喉素的短路电流变化和响应于CFTR抑制剂172的短路电流变化，在新鲜或冷冻解冻的HNE之间未观察到显着差异。在治疗后，两种细胞类型都显示出显着的校正，响应于毛喉素的短路电流变化增加以及响应于CFTR抑制剂172的短路电流变化的减少。与DMSO处理的HNE细胞相比，评估CFTR功能双重疗法的纠正反应，以响应毛喉素的短路电流变化，并且VX-809加VX-770和VX-661加VX-770显着改善了对CFTR抑制剂172的反应。比较了来自6名F508del纯合子患者的HNE中的三种不同的CFTR调节剂疗法。当与CFTR校正器和增强器结合使用时，顶点和CFTR调制器，校正短路电流变化响应于毛喉素和短路电流变化，响应于CFTR抑制剂172达到类似程度。冷冻HNE细胞时，重要的是每个冷冻小瓶至少有300万个细胞，以增加解冻后获得良好结果的机会。HNE细胞中的CFTR活性已被证明是评估和调整囊性纤维化个性化治疗的良好预测模型。

Research

JoVE Journal

Biology

Primary Human Bronchial Epithelial Cells Grown from Explants

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

科研

生物学

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...