作者要感谢丽莎戴利的帮助输入。 这项工作是由美国国立卫生研究院（ES013611和HL095163）的资金支持，乘务员医学研究所（FAMRI）和环境保护署（CR83346301），并宣称没有利益冲突。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。虽然本文中描述的研究已经通过合作协议CR83346301与中心环境医学，哮喘和肺癌生物学在北卡罗莱纳州教堂山大学资助部分由美国环境保护署，它并没有被受到机构的要求同行和政策审查，因此并不一定反映该机构的意见，并没有正式认可应被推断。 Ø提号贸易名称或商业产品并不构成认可或推荐使用。洛雷塔穆勒是由瑞士国家科学基金会与个人的资助。

1。准备塑胶制品：大衣板<ol><li>加入500μlPureCol（1:100稀释，用无菌水）的12孔板的每个孔中。 </li><li>在孵化器30分钟，在37℃，取出PureCol和冲洗用HBSS右侧的单元格将被添加到板前（参见下面的步骤3.1）。 </li></ol> 2。获取和人类长者邻舍中心预处理活检<ol><li>鼻刮活检<ol><li>获得肤浅的内皮细胞通过9毫米可重复使用的聚丙烯鼻窥器（型号22009）上的操作与耳镜衬窥器直视下鼻甲（型号21700）。该器件提供了鼻甲和运动灵活性的最佳视觉效果。 </li><li>在头往后仰稍或卧在检查表中的仰卧位太师椅坐在直立的主题进行活组织切片检查。 </li><li>将耳镜窥器插入鼻孔和下turbi内特 - 可视化与照明。 </li><li>通过窥镜插入无菌热塑性刮匙与向远侧延伸至鼻甲背面的小费。 </li><li>使用上的鼻甲的下表面温和的压力，刮匙被吸入穿过粘膜表面5倍和缩回。通过毛细管作用保持黏膜细胞的成功提取将是显而易见的，在杯子的刮匙。 </li></ol></li><li>把样品在15毫升锥形管中装有8毫升的RPMI 1640中，传输在冰上。 </li><li>使用镊子检索探头，打跑组织从犀牛探头与P-1000移液器，丢弃探头</li><li>离心沉淀（4℃，400×g离心5分钟），吸出上清液（注意：注意不要吸入颗粒）。 </li><li>重悬沉淀在1ml BEGM用10微升的100×DNA酶1。 </li><li>在室温下孵育20分钟。 </li><li>离心（4℃，400×g离心5分钟），不要吸出上清液！ </li></ol> 3。 SEED对塑料的细胞（0天） <ol><li>从板块中删除PureCol和冲洗用HBSS（参见步骤1.2）。 </li><li>加BEGM + +介质的最小体积（80-100微升，直到介质的弯月面会出现在井的中心）到1-4孔中的涂板（取决于活检大小）。 </li><li>使用P-1000移液管小心地从管中取出活检组织的粒料，不吸过多介质。 </li><li>转移球团入井的中间，保持活检一起作为小区的集群，而不要打散，使单细胞悬液。一个好的切片产量可达4口井，可播种。将板放入组织培养箱（37℃，5％CO 2，&gt; 90％相对湿度）。 </li><li>检查后两小时，以保证有足够的介质（不干扰弯月面）。 </li><li>第1天，24小时后播种：收集所有井的所有非贴壁细胞（倾斜板，并收集在一个P-1000，C与细胞介质在1.5ml的离心管中ollect所有孔中）。 </li><li>离心（4℃，400×g离心5分钟）。 </li><li>而离心细胞悬浮液：向接种在第0天的细胞中加入约250μl的BEGM + +和250微升的BEGM +介质的混合物。 </li><li>重复步骤3.1-3.5的非贴壁细胞。 </li><li>一天2-5：每日细胞变化的媒体;加入500μl的媒体，每孔，减少BEGM +媒体逐步（2天量播种后：125微升BEGM +加375μLBEGM +，播种后第3天及以下天数：73微升BEGM +加427μLBEGM +）。 </li></ol> 4。扩大细胞在烧瓶<ol><li>每天监测细胞，一旦他们达到80-90％汇合（通常在活检后5-7天，如果他们需要更长的时间，他们不会成功发展）电梯细胞如下：小心吸介质;加入500微升0.25％胰蛋白酶每口井，让他们在37°C，直到细胞被分离（通常需要2-3分钟）。 </li><li>准备需要大豆胰蛋白酶抑制剂（SBTI）（每1ml胰蛋白酶750微升）在15毫升管版体积。 </li><li>通过反复上下吹打的胰蛋白酶溶液移去细胞;转移分离的细胞中的15毫升锥形管与SBTI。 </li><li>冲洗所有井，共有1毫升的HBSS，加入HBSS到管细胞。 </li><li>离心沉淀（4℃，400×g离心5分钟），吸1毫升BEGM +媒体的媒体，重悬，涡旋管。 </li><li>这取决于颗粒的大小扩大在T25或T75瓶中的细胞（这是P1，大约两汇合口井去到一个T75瓶中，一是融合以及足够一个T25，一般2汇合口井产生一个T75）。 <ol><li>加BEGM +向烧瓶（每次5毫升一T75，将3ml每一个T25）。 </li><li>添加细胞悬浮液的烧瓶中。转移到烧瓶组织培养箱。 </li></ol></li><li> 24小时后瓶中接种：添加额外的BEGM +媒体向烧瓶（3毫升到一个T25，将10毫升至T75）。 </li><li><stroNG&gt;维持细胞在培养瓶中：移除介质（分泌物从眼角），并添加BEGM +媒体（5 ml/T25，20 ml/T75）。媒体需要改变每2-3天。维持细胞在烧瓶中，直至其大致80％融合（融合未必甚至在整个烧瓶，通常这需要7-10天之间，如果他们在10天后都没有汇合他们不会成长成功）。 </li></ol> 5。种子的组织培养插入单元格<ol><li>从烧瓶中取出介质，并加入胰蛋白酶（3毫升的T25 4毫升T75瓶）。 </li><li>孵育在37℃，直至细胞被分离（约2-3分钟）。 </li><li>准备SBTI（每1ml胰蛋白酶750微升&gt;2.25毫升为T25，将3ml于T75）在一个15毫升管。 </li><li>用胶带将烧瓶和上下吹打移去细胞，并转移到锥形管与SBTI。 </li><li>冲洗烧瓶用HBSS（1毫升T25 2毫升T75），添加HBSS到15ml试管中。 </li><li>离心分离机沉淀（4℃，400×g离心5分钟），小心取出上清液。 </li><li>准备板：决定多少板将要去籽，用标记示例代码和号码，通道数（ 即 P2）和日期的板块。加入700μlBEGM +介质基底室。 </li><li>通过涡旋管重悬细胞沉淀在1ml BEGM ALI媒体。 </li><li>计数细胞，用血细胞计数器（一个T25通常会产生约4百万个细胞，一T75可以有多达20亿个细胞依赖于胰岛，使用1-2万细胞一12孔板）和稀释细胞悬液，用BEGM ALI媒体所需的板应接种（1.2×10 6细胞每盘2.5毫升媒体）的金额总量（注：如果细胞的一部分要被冻结，标示管和去除细胞在这里：我们通常冻结2×10 6个细胞在2毫升冷冻介质） </li><li>添加200μl的细胞悬浮液以每个未包衣康宁12well细胞的顶侧插入（&gt;这将约80,000-150,000个细胞/插入物;12毫米转孔用0.4微米孔径的聚酯（聚对苯二甲酸乙二醇酯（PET）等）的膜插入物）;转移到组织培养箱中。 </li><li> 24小时后，改变心尖媒体（200微升膨胀介质）。 </li><li> 维护细胞培养：更改介质（BEGM ALI媒体; 700微升基底和200微升顶）隔日一次。刀片必须保持淹没，直到细胞完全融合（单层无孔可见）。根据细胞数量，这通常需要2-6天之间，如果需要更长的电池将最有可能是不可行的。 </li></ol> 6。建立空液界面一旦细胞被完全合流（当在转孔的细胞是完全合流） <ol><li>一旦细胞完全融合与补充500NM维甲酸48小时BEGM ALI媒体更换两个心尖和基底介质。 </li><li>加入维甲酸后48小时补充BEGM ALI媒体：准备PneumaCult-ALI 维护中（以下制造商的说明）：计算所需介质的体积（一板通常8.5毫升为基底室），每1 ml加入PneumaCult完整的基础培养基： 10微升PneumaCult 100X维护补充 2微升肝素（的2毫克/毫升储液） 5微升氢化可的松（一个96微升/毫升的储备溶液）。 </li><li>取出所有介质，加入700μlPneumaCult-ALI维护媒体的基底侧只（在顶面上无介质）。 </li><li>维持细胞培养4周的ALI： <ol><li>每隔一天（周一，周三和周五的日程安排很适合我们）改变媒体。 </li><li> 1周在ALI后，一个星期（星期三），一旦顶表面清洗：加入200μl的HBSS到心尖的一面，让他们在孵化10分钟，小心吸出HBSS（使用玻璃吸管9安装在真空陷阱中的生物安全柜，用200微升技巧上的吸管尖抽吸掉HBSS）。 </li></ol></li></ol>注：板块之间切换的提示，以及从根尖表面会与基底膜改变时的提示。约2周后在急性肺损伤，细胞开始分化和纤毛出现。细胞培养后约4周在ALI达到完全分化。

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C., Jaspers, I., Dailey, L., Diaz-Sanchez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+microRNA+expression+in+human+airway+cells+by+diesel+exhaust+particles+is+linked+to+tumorigenesis-associated+pathways.">Disruption of microRNA expression in human airway cells by diesel exhaust particles is linked to tumorigenesis-associated pathways.</a> Environ. Health Perspect. 117, 1745-1751 (2009).</li><li>Horvath, K. M., Brighton, L. E., Herbst, M., Noah, T. L., 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=Live+Attenuated+Influenza+Virus+(LAIV)+induces+different+mucosal+T+cell+function+in+nonsmokers+and+smokers.">Live Attenuated Influenza Virus (LAIV) induces different mucosal T cell function in nonsmokers and smokers.</a> Clin. Immunol. 142, 232-236 (2012).</li><li>Horvath, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+lavage+natural+killer+cell+function+is+suppressed+in+smokers+after+live+attenuated+influenza+virus.">Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus.</a> Respir. Res. 12, 102 (2011).</li><li>Noah, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+exposure+and+altered+nasal+responses+to+live+attenuated+influenza+virus.">Tobacco smoke exposure and altered nasal responses to live attenuated influenza virus.</a> Environ. Health Perspect. 119, 78-83 (2011).</li></ol>

作者什么都没有透露。

呼吸道内皮细胞不仅提供了一个物理屏障，以保护人体从外源性刺激20，而且还充当交换机通过可溶性介质或直接的受体-配体细胞间的相互作用的分泌，启动和调节呼吸道的免疫防御反应1-3。为了进一步研究内皮细胞在免疫应答中的作用，研究从患病人群内皮细胞之间的电势差，或者以研究对内皮细胞的外源应激的影响，它概括了在体内的情况是很重要的。人类呼吸道含有...

该技术的目的在气道上皮细胞（EC），其中的第一个位点被直接暴露于吸入的环境压力，如病原体，变应原或污染物。内皮细胞多于一个结构性障碍：他们作为一个交换机通过可溶性介质4-6和配体/受体对他们的表面活性7-9表达式的释放来启动和调节呼吸系统的免疫反应1-3。虽然永生化的细胞系可作为模型来研究呼吸内皮细胞在体外 ，它们不能分化成偏振纤毛和黏液分泌细胞，类似的是， 在体内观察到的由异质细胞群。扼要重述在体内观察到的呼吸道上皮细胞的重要特征，主气道的EC可以被使用。因此，我们的目标是优化我们利用鼻内皮细胞（邻舍）方法从人类志愿者获得。在NEC的是在体外扩增和培养，得到分化邻舍的细胞培养模型模仿许多的鼻上皮见于体内的功能。 合理使用体外模型与人类原发性内皮模仿体内的情况-如本研究中所描述-赋予独特的可能性，研究内皮细胞的疾病的具体差异，与气道上皮细胞相关生理参数，或内皮细胞和其他细胞类型之间的相互作用，如树突细胞10，自然杀伤细胞11或巨噬细胞。现有的几种方法利用支气管内皮细胞，它可以侵入通过刷活检支气管镜检查过程中获得，或者从其他废弃肺组织12-17。此外，商业来源获得完全分化的人类呼吸道上皮细胞培养从MatTek，阿什兰，马萨诸塞，Clonetics存在（EpiAirway模型从龙沙，瑞士巴塞尔，MucilAir从Epithelix非典，瑞士日内瓦），其中研究人员可以从不同的捐赠者选择。因为市售的上皮细胞，限制或规定的一套参数的有限池然而，与市场上取得初级人类呼吸道内皮细胞相关的成本，以及有限的访问丢弃肺组织或刚获得的人支气管活检组织，禁止许多研究者从开展研究中完全分化的人类呼吸道内皮细胞。因此，我们着手开发，将1）利用非侵入性得到人类邻舍，2）提供了一个协议产生的分化的人类呼吸道上皮细胞培养，以及3）与现有组织培养的基础设施大部分实验室的设置可再现的技术。 优于现有的技术/型号获得新鲜的邻舍，较支气管或者小气道的精英，很多创伤小，个别S可活检多次无显著副作用，而且肤浅的鼻刮活检可以，否则将不符合研究相关的支气管镜检查患病人群进行。非侵入性的浅表刮活检以获得长者邻舍中心允许研究者设置捐助规范是先验的 ，包括年龄，性别，疾病状况等按照要完成的研究目标。因此，在设计调查研究时，研究者没有被临床上可用的组织/上皮标本的限制，购买自商业供应商，或设计侵袭性纤维支气管镜研究昂贵的分化细胞培养模型。此外，易于获得NEC的增加，从不同供体进行的实验中的细胞，从而提高观察结果的统计验证的能力。最后，鼻子是第一的网站暴露于任何种类的空气传播的压力之一。因此，生成的器官体外培养系统模仿鼻腔上皮细胞是研究吸入病毒颗粒，污染物，过敏原，或气体，这可以通过使用这种细胞培养系统很容易实现的是有用的。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Nasal speculum</td>
 <td>Welch Allyn</td>
 <td>Model 22009</td>
 <td>9 mm, reusable polyproylene</td>
 </tr>
 <tr>
 <td>Operating otoscope</td>
 <td>Welch Allyn</td>
 <td>Model 21700</td>
 <td></td>
 </tr>
 <tr>
 <td>Rhino probe cell cuvette</td>
 <td>Arlington Scientific</td>
 <td>SY-96-092</td>
 <td>bend the head of the cuvette a little more</td>
 </tr>
 <tr>
 <td>12-well culture plates</td>
 <td>Corning</td>
 <td>3512</td>
 <td></td>
 </tr>
 <tr>
 <td>Transwell membrane cell culture inserts</td>
 <td>Corning</td>
 <td>3460</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture flask </td>
 <td>Corning</td>
 <td>430639 and 430641</td>
 <td>T25 and T75 vented flask</td>
 </tr>
 <tr>
 <td>RPMI 1640 media (with L-Glutamine)</td>
 <td>Gibco</td>
 <td>11875</td>
 <td></td>
 </tr>
 <tr>
 <td>Bronchial/Tracheal Epithelial Cell Growth Medium</td>
 <td>Cell applications</td>
 <td>511-500</td>
 <td></td>
 </tr>
 <tr>
 <td>BEGM Bronchial Epithelial Cell Growth Medium</td>
 <td>Lonza</td>
 <td>CC-3170</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements.</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate 7.5% solution</td>
 <td>Gibco</td>
 <td>25080</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>NuSerum</td>
 <td>BD Biosciences</td>
 <td>355104</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>BAMBANKER freezing media</td>
 <td>BAMBANKER</td>
 <td>302-14681</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>CoolCell </td>
 <td>Biocision</td>
 <td>HTBCS-136</td>
 <td>(CryoPrep alcohol-free cell freezing container)</td>
 </tr>
 <tr>
 <td>PureCol</td>
 <td>AdvancedBioMatrix</td>
 <td>5005-B</td>
 <td>dilute 1:100 with sterile water (should be roughly 0.03 mg/ml)use 500 &mu;l/well of a 12-well plate</td>
 </tr>
 <tr>
 <td>HBSS with Ca2 and Mg2+</td>
 <td>Gibco</td>
 <td>14025</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse 1</td>
 <td>Sigma</td>
 <td>DN25</td>
 <td>Dissolve at 1.5 mg/ml, which is a 100x solution, add 10 &mu;l to each ml of media</td>
 </tr>
 <tr>
 <td>Trypsin, 0.25%, 1x, with phenol red</td>
 <td>Gibco</td>
 <td>25200-056</td>
 <td>Use at it is</td>
 </tr>
 <tr>
 <td>Soy Bean Trypsin Inhibitor (SBTI)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td>Use dissolved in HBSS at a concentration of 1 mg/ml</td>
 </tr>
 <tr>
 <td>Retinoic acid</td>
 <td>Sigma</td>
 <td>R7632</td>
 <td>Make it up 100 &mu;M stock solution (see below), dilute to 10 &mu;M with absolute alcohol, take 5 &mu;l of this and add it to 1 ml BEGM media (our working solution). 
 
 All-trans Retinol has a coefficient of extinction of 52,480 in ethanol at 325 nm. Dissolve 25 mg in 50 ml absolute ethanol. Serial dilutions need to be made, dilute 50 ml of stock in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, and again take 50 ml of this, dilute in 450 ml absolute Ethanol, take 100 ml of this and dilute in 900 ml absolute ethanol and read in the spectrophotometer at 325 nm, this number is then multiplied by its' dilution factor, then divided by the extinction coefficient of 52,480, this will give a number in mM. Make a 100 mM stock (store at -20 &deg;C) and from this stock a 10 mM aliquot is made, the daily retinoic acid needed would be 5 ml of this 10 mM aliquot in 995 ml ALI media.
 Example: 325nm reading is 1.15. 1.15 x 10,000 (dilution factor) / 52,480 = 0.219 = 219 mM, make a 100 mM stock from this, in absolute ethanol.</td>
 </tr>
 <tr>
 <td>DMEM high glucose (1x), liquid, with L-glutamine and sodium pyruvate</td>
 <td>Gibco</td>
 <td>11995</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Pituitary Extract</td>
 <td>Gibco</td>
 <td>13028-014</td>
 <td>Used for BEGM ALI media.</td>
 </tr>
 <tr>
 <td>Nystatin</td>
 <td>Sigma</td>
 <td>N4014-50 mg</td>
 <td>Make up a solution of 3.3 mg/ml.</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td>Make up a solution of 10 mg/ml solution in milliQ water.</td>
 </tr>
 <tr>
 <td>PneumaCult-ALI media</td>
 <td>StemCell</td>
 <td>5001</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements. For details see below.</td>
 </tr>
 <tr>
 <td>Hydrocortisone </td>
 <td>StemCell</td>
 <td>7904</td>
 <td>Dissolve 2.4 mg hydrocortisone in 1 ml ddH2O (used to make the 200x Hydrocortisone stock solution)</td>
 </tr>
 <tr>
 <td>Heparin Sodium Salt in PBS (2 mg/ml)</td>
 <td>StemCell</td>
 <td>7980</td>
 <td>Use at it is. </td>
 </tr>
 <tr>
 <td>Filter flasks (500 ml)</td>
 <td>Corning</td>
 <td>431097</td>
 <td>0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Filter tubes (50 ml)</td>
 <td>Millipore</td>
 <td>SCGP00525</td>
 <td>Steriflip, 0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Pipette tips - ART Barrier Tips</td>
 <td>Molecular Bioproducts</td>
 <td>2139RI, 2069RI, 2179RI</td>
 <td></td>
 </tr>
 <tr>
 <td>Conical tubes, sterile, 15 ml and 50 ml</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ddH2O, absolute ethanol, ice</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>refrigerated centrifuge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>biological safety cabinet</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>pipettes (p2, p200, p1000), 9 in glass pipettes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>gloves</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lab coat with tight cuffs</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Media and solutions used:</td>
 <td>Recipe</td>
 <td>Used for</td>
 </tr>
 <tr>
 <td colspan="3">General remark: always warm the media to room temperature</td>
 </tr>
 <tr>
 <td rowspan="2">BEGM media</td>
 <td><ul><li> 500 ml BEGM Cell Application media with one aliquot of retinoic acid</li></ul> </td>
 <td>(mix them half-half and filter )</td>
 </tr>
 <tr>
 <td><ul><li> 500 ml BEGM Lonza media with one aliquot of single quot supplements </li></ul></td>
 <td><ul><li> digestion of the biopsy</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="3">BEGM+ media</td>
 <td><ul><li> 50 ml BEGM media </li></ul></td>
 <td><ul><li> maintaining cells on plastic in the plates (passage0) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l retinoic acid (working solution)</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td></td>
 <td><ul><li> maintain the cells in the flask </li></ul></td>
 </tr>
 <tr>
 <td rowspan="3">BEGM++ media</td>
 <td><ul><li> 50 ml Lonza's BEGM media with supplements</li></ul> </td>
 <td><ul><li> seeding fresh cells in 12-well plate on plastic</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Sodium BiCarb</li></ul> </td>
 <td><ul><li> maintaining cells in the plates on plastic (passage0; only partially)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2.5 ml NuSerum</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">BEGM ALI media</td>
 <td><ul><li> 250 ml DMEM-H </li></ul></td>
 <td rowspan="5"><ul><li> Maintaining cells on the transwell before they go ALI</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 250 ml BEGM including supplements</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2 ml Bovine Pituitary Extract (12.5 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Nystatin (3.3 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 75 &mu;l BSA (10 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">200X Hydrocortisone Stock Solution (96 &mu;l/ml solution)</td>
 <td><ul><li> 200 &mu;l dissolved hydrocortisone</li></ul> </td>
 <td rowspan="5"><ul><li> As a supplement to the PneumaCult-ALI Complete Base Medium for the last day in the flask and when cells are on inserts</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 4250 &mu;l dissolved sodium chloride </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 540 &mu;l ddH2O</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l absolute ethanol</li></ul> </td>
 </tr>
 <tr>
 <td>Filter solution through 0.22 &mu;m filter, aliquot solution and store at -20 &deg;C, avoid additional freeze/thaw cycles</td>
 </tr>
 <tr>
 <td rowspan="3">PneumaCult-ALI Complete Base Media</td>
 <td><ul><li> 450 ml PneumaCult-ALI Basal Medium</li></ul> </td>
 <td><ul><li> Base medium for all three PneumaCult-ALI media </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 50 ml PneumaCult 10x Supplement </li></ul></td>
 <td><ul><li> The PneumaCult media is light sensitive, thus always keep it in the dark</li></ul> </td>
 </tr>
 <tr>
 <td>(this is stable for 2 weeks at 2-8 &deg;C or for up to 6 months at -20 &deg;C)</td>
 <td></td>
 </tr>
 <tr>
 <td rowspan="4">PneumaCult-ALI Maintenance Medium</td>
 <td>Per 1 ml of PneumaCult-ALI Complete Base Medium add:</td>
 <td rowspan="4">ALI culture on inserts</td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l PneumaCult 100x Maintenance Supplement </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 2 &mu;l Heparin (of a 2 mg/ml stock solution) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 5 &mu;l Hydrocortisone (of a 96 &mu;l/ml stock solution) </li></ul></td>
 </tr>
 </tbody>
</table>
 </td>
 </tr>
 </tbody>
</table>

culturing of human nasal epithelial cells at the air liquid interface

在使用体外模型的人原代上皮细胞是在理解微生物感染或吸入剂的情况下的呼吸道上皮关键功能是必不可少的。细胞的患病人群中获得的直接比较让我们来描述不同表型和解剖调解变化上皮细胞功能的基本机制。培养从人支气管区域的上皮细胞已被很好的记载，但通过与获得的支气管刷活组织检查相关的人类肺组织或侵袭的可用性的限制。鼻粘膜上皮细胞通过较少侵入性的浅表鼻刮活检获得，主体可以活检多次无显著副作用。此外，鼻子的入口点的呼吸系统和因此的第一站点被暴露于任何种类的空气传播的应激物，如微生物剂1，污染物，或过敏原。 简要地说，从人类志愿者获得的鼻上皮细胞上涂覆的组织培养板中膨胀，然后转移到细胞培养插入。在达到汇合，将细胞继续在气 - 液界面（ALI），以进行培养，持续数周，从而产生更多的生理相关的条件。 ALI的培养条件下用确定的培养基导致了分化的上皮细胞表现出形态和功能特征类似于人的鼻上皮，既纤毛和黏液分泌细胞。组织培养插入分化鼻上皮细胞可在各种不同的方式来操作取决于研究问题（治疗用药物制剂，转导与慢病毒载体，暴露于气体或感染微生物剂），并为许多不同的端点，从分析细胞和分子通路，通道功能安格斯，形态等。 在分化的人类鼻黏膜上皮细胞的体外模型将使研究者通过实验器官模型，主要模拟体内鼻腔上皮细胞，以解决新的和重要的研究问题。

NEC的培养继此处描述的协议重新分化成内皮细胞的异质层纤毛和非纤毛细胞，相当于本体内的情况（ 图1）构成的。有些区别的长者邻舍中心是粘液产生（细胞蓝染色使用AB / PAS， 图1B）。尽管这里介绍的协议进行了优化，分 ​​化可以与问候的整体细胞群（：粘液产生细胞： 即不同比例纤毛细胞的非纤毛细胞）的分布有所不同，甚至得到更多的鳞状上皮?...

Watch this Scientific Journal Video about Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface at JoVE.com

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

鼻粘膜上皮细胞，通过志愿者的浅表刮活检，是扩大和转移到组织培养插入。一旦达到融合，细胞生长在空气液体界面，产生纤毛和非纤毛细胞的培养。分化鼻粘膜上皮细胞培养提供可行的实验模型为研究呼吸道黏膜。

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 ...

culturing-of-human-nasal-epithelial-cells-at-the-air-liquid-interface

Research

JoVE Journal

Biology

36.7K 次观看.  The University of North Carolina at Chapel Hill. 鼻粘膜上皮细胞，通过志愿者的浅表刮活检，是扩大和转移到组织培养插入。一旦达到融合，细胞生长在空气液体界面，产生纤毛和非纤毛细胞的培养。分化鼻粘膜上皮细胞培养提供可行的实验模型为研究呼吸道黏膜。 

视频: Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

科研

生物学

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

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 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

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

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 in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

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 ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

从人牙囊中分离上皮细胞

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...