这项工作由欧盟项目巡逻队（现实纳米材料危害的生理锚定工具）和荷兰卫生、福利和体育部（V/050012项目）资助。我们要感谢伊冯娜·斯塔尔博士和扬·范本瑟姆博士对手稿的批判性审查。

1. 准备细胞培养介质（CCM）
<ol><li>准备一瓶500 mL的最低基本介质（MEM），辅以谷氨酰胺。</li>
	<li>添加5 mL青霉素链霉素（即100U/mL青霉素和100μg/mL链霉素）。</li>
	<li>加入5 mL的非必需氨基酸（NEAA）溶液。</li>
	<li>添加 10 mL 的安培素 B（可选）。</li>
	<li>添加 50 mL 的 FBS（热灭活，请按照 ATCC 协议进行热灭活;（https://www.atcc.org/~/media/PDFs/Culture%20 指南/AnimCellCulture_Guide.ashx，第 19 页）。</li>
</ol>2. Calu-3细胞的亚培养
注:Calu-3细胞在T75或T175细胞培养瓶中培养在37°C和5%CO2中。细胞每7天通过60-80%的汇合，CCM每2-3天更新一次。CCM 被倒入烧瓶中，将新鲜的 CCM（T25 = 5 mL、T75 = 15 mL 和 T175 = 25 mL）吹回烧瓶中，将烧瓶放回孵化器中。细胞在解冻后、在实验中使用之前或冷冻前应至少通过2次，它们总共应不超过25次。
<ol><li>通过在光显微镜下检查，确认烧瓶是否为 60-80% 的汇合。</li>
	<li>从烧瓶中倒出 CCM。</li>
	<li>用 5 mL 的 1x 汉克斯平衡盐溶液 （HBSS） 清洗细胞 2x， 没有钙， 没有镁。每次清洗后丢弃 HBS。HBSS 去除血清，抑制尝试。</li>
	<li>为 T75 添加 3 mL 的尝试-EDTA（T175 的 4 mL），并将烧瓶以 37 °C 和 5% CO2 放入孵化器中，10-15 分钟。10分钟后检查，确保细胞脱离烧瓶表面。如果细胞生长到 +80% 的汇合度，它们不会使用尝试素 0.05% 分离，并且可以使用尝试蛋白 0.25%。</li>
	<li>将 6 mL（即最初添加的试金石-EDTA 体积增加一倍）添加到烧瓶中，轻轻摇动烧瓶以确保正确混合。这是为了确保在CCM中，FBS已经中和了尝试素，并且它在细胞上的活性停止了。如果允许尝试与细胞保持接触太久，当放入新的细胞培养瓶时，它们不会重新连接。</li>
	<li>将烧瓶的全部内容倒入 50 mL 离心管中。</li>
	<li>在130 x g下将电池离心5分钟，确保离心机正确平衡。</li>
	<li>将含有细胞的瓶返回无菌状态，轻轻取出超纳坦，而不会干扰颗粒。超纳坦可以倒掉，其余的管道关闭，确保颗粒不受干扰。</li>
	<li>通过上下管道将细胞颗粒重新悬浮在 1 mL 中，直到所有细胞暂停（即未观察到颗粒或细胞聚集物）。可以添加额外的 CCM 以稀释细胞悬架。</li>
	<li>使用血细胞计计算CCM1mL中的细胞，无论是死细胞还是活细胞。如果需要正确计数，在 3 或 4 mL 中稀释细胞。</li>
	<li>将细胞暂停到所需的 CCM 体积中，并将细胞悬浮物添加到每个烧瓶中。要在一周内实现约 80% 的汇合，T75 中的种子 2 x10 6 细胞或 T175 中的 6 x10 6 细胞。</li>
	<li>轻轻摇动烧瓶，然后将其放回37°C和5%CO2的孵化器中。</li>
	<li>每2-3天更换一次新鲜的CCM，当细胞达到60-80%的结合时，亚文化。</li>
</ol>3. 将 Calu-3 细胞播种到培养插入物上
注:插入物具有不同的毛孔尺寸。小孔径（例如，0.4 μm）具有细胞更容易生长的优势，并且能够在水下条件下培养5天后达到良好的屏障，通过跨上皮电阻电 （TEER） 测量。然而，当对粒子转移感兴趣时，这些毛孔太小，会捕获颗粒。因此，通常选择较大的孔径（例如 3 μm）来测试颗粒。当使用更大的毛孔尺寸时，细胞需要较长的时间周期（例如，在水下条件下进行 7-10 天的培养），才能实现良好的 TEER。
<ol><li>按照步骤 2.1-2.10 准备已知浓度的细胞悬架。</li>
	<li>将细胞稀释至预热CCM中浓度为5 x 105 细胞/mL，用于6口井插入，或将2.5 x 105 细胞/mL稀释为12口井插入。</li>
	<li>取一个带插入物的细胞培养板，并在无菌条件下放置。</li>
	<li>用 2 mL 预热 CCM 填充基底侧，用于 6 口井插入，或 1 mL 填充 12 口井插入。在添加培养基时，使用钳子将插入物取出。</li>
	<li>小心地混合细胞悬架，上下管道。Pipette 1.0 mL 代表 6 口井插入物，500μL 代表 12 口井插入（相当于 100，000 个细胞/cm2）位于细胞培养插入膜顶部，毛孔为 0.4 μm。对于 3.0 μm 毛孔，将细胞密度增加到 128，000 细胞/厘米2。</li>
	<li>盖住细胞培养板，在37°C和5%CO2下孵育。</li>
	<li>每2-3天更换一次CCM。</li>
	<li>让细胞在水下条件下聚集7天，然后继续在ALI培养。</li>
	<li>测量泰尔。<ol>
<li>服用以筷子电极集为补充的上皮伏托姆计，并在一夜之间为电池系统充电。</li>
		<li>断开充电器的伏托姆计，连接筷子电极。</li>
		<li>用 70% 乙醇清洁电极。</li>
		<li>将电极放置在 CCM 中，将较长的电极放入外部培养介质中，直到它触及盘底，并将较短的电极放入介质中而不接触膜。</li>
		<li>从没有单元格的空插入开始。等到测量稳定下来，并写下欧姆斯的阻力。此测量是没有任何细胞的插入膜的电阻（即空白电阻）。</li>
		<li>重复每个插入的测量，并减去空白电阻以获得真正的电阻。</li>
		<li>对于数据分析，请将插入物的表面积的电阻值乘以Ω x cm2。对于 6 井插入，表面积为 4.67 厘米2。因此，如果测量到 600 Ohm 的电阻，背景为 120 Ohm，则电阻为 480 Ohm，然后乘以 4.67 厘米2 的表面积，总计 2，241.6 Ohm x cm2。TEER 应 =1，000 Ω x cm2 继续。</li>
	</ol>
</li>
	<li>从插入物的 apical 侧移除 CCM。</li>
	<li>添加 2 mL 预热 CCM 为 6 井和 1 mL 为 12 井插入到井的基底侧（即在细胞培养插入下）。CCM 应从底部触摸膜，但不要泄漏到插入物的顶部。</li>
	<li>此时，细胞在空气中暴露，这被称为ALI的培养。</li>
	<li>在37°C和5%CO2 的孵化器中的ALI培养细胞在暴露前7天。</li>
	<li>每2-3天更换一次基础CCM。这些细胞可以在ALI使用6周。</li>
</ol>4. 准备曝光设置
注:第 5-7 节描述了使用自动曝光站（AES，见 材料表）对粒子暴露的准备情况。粒子星云和特征设置也与其他制造商的其他 ALI 暴露系统兼容。例如，下面描述了对粒子的暴露。此类系统也可用于其他暴露，如敏化剂、香烟烟雾和柴油排气。 图 1 显示了 AES 和曝光模块。 图 2 显示了曝光设置的示意图，包括所有其他仪器。
<ol><li>在使用 AES 开始暴露之前，将系统连接到多个仪器以测量气溶胶特性:这些是在气溶胶进入机柜之前用侧流测量的。 注:流分流器用于将侧流连接到曝光特征描述设备。一般使用以下设备:扫描移动粒子测集器 （SMPS）、光学粒子测集器 （OPS）、冷凝粒子计数器 （CPC）、锥形元素振荡微平衡 （TEOM）。SMPS 和 OPS 测量每小时执行 1 次，并使用流分流器中的相同管子。CPC 和 TEOM 执行连续测量，两者的数据都记录在松鼠模型 2020 数据记录器上。此外，在受控相对湿度（40-70%） 中，利用微平衡确定重力质量浓度和温度（21-23°C）条件。每次接触前后都会对特氟龙过滤器进行称重，以确认暴露浓度。为了捕获排气，使用 HEPA 过滤器。包括工程纳米材料 （ENM） 悬架和雾化器在内的设置都放置在流程柜中，以防止任何人接触。AES 可用于测试许多不同类型的 ENM，包括金属和金属氧化物。</li>
	<li>在曝光前不久准备纳米材料悬架。通常，1% 的暂停是作为股票解决方案准备的。例如，在 10 mL 纯水中暂停 100 毫克纳米材料。 注:对于DQ12暴露，300毫克用于实现约2μg/cm2。此量可用于单次曝光或重复曝光（例如，300 毫克在 30 mL 中暂停一次曝光，或 21.5 mg 每天在 2.15 mL 中悬浮 3 周的重复曝光）。悬挂是在每个暴露日新鲜准备的。粒子悬架使用探针声波进行16分钟的声波化。通过添加纯净水，将 1% 库存溶液的体积调整为总容量 100 mL。</li>
	<li>将 ENM 悬架放入带盖子和磁搅拌器的小瓶子中，以防止颗粒沉降。通过小管将瓶子连接到腹水泵，并将流量调整到 25 mL/h。</li>
	<li>将静态泵连接到喷嘴，并调整设置以允许连续气溶胶化。</li>
	<li>喷嘴安装在一个60厘米长的铝管（搅拌室，直径15厘米，加热到60°C）。该设置通过一个1.5米长的铜管（直径15厘米）连接到AES。在 AES 顶部，冲击器可以去除所有大于 2.5 μm 的气溶胶。</li>
	<li>通过两个质量流量控制器 （MFC） 将喷嘴连接到 3 条压缩空气，以便悬架的雾化。一个流量为 14 L/min 用于喷雾喷嘴，另一个流用于混合管中的空气。</li>
	<li>曝光前一天，打开 AES，使机柜温度达到 37 °C。</li>
	<li>在开始暴露前2小时打开机柜中的气流和湿度，达到85%的湿度。打开将插入细胞的暴露室的加热。</li>
	<li>打开石英微平衡 （QCM），使用软件将初始值设置为 0。开始记录。每10s的质量被测量和表达为ng/cm2。</li>
	<li>在水浴中将细胞培养介质加热至37°C（~20-30分钟）。</li>
</ol>5. 准备卡鲁-3细胞暴露
注:对于使用 AES 的典型 ALI 暴露，需要 15-20 个具有汇合细胞层的插入。其中包括 3 个清洁空气控制装置、3 个与 AES 中未暴露的其他插入物类似的孵化器控制、6-8 个气溶胶暴露插件（取决于 0、1 或 2 微平衡的使用）、1-3 个用于控制测量的插件（如最大 LDH 释放）以及 3 个备用插入器，以防某些插入物的 TEER 不够。细胞应具有 +1，000 Ω x cm2 的 TEER 才能继续。
<ol><li>在暴露的第一天，用CCM清洗细胞1倍，检查细胞形态，并使用伏托姆计测量细胞模型的TER。细胞应形成一个没有缝隙的紧密单层。</li>
	<li>将 2 mL/1 mL 的 HEPES 缓冲 CCM（无 FCS）放在 6 井/12 井板的基底侧，并将带细胞的培养插入物转移到新板上。 注:暴露期间，AES 中不存在 CO2。 因此，在运输和暴露过程中使用 HEPES 缓冲培养介质（25 mM HEPES）。这种介质既用于 AES 中的暴露细胞，也用于孵化器控制细胞。</li>
	<li>如果将细胞培养物传输到 AES 的时间超过 5 分钟，请在传输过程中将细胞放入 37 °C 的便携式孵化器中。</li>
</ol>6. 暴露期间处理 AES
<ol><li>在 AES，在没有胎儿小腿血清 （FCS） 的情况下，将 HEPES 缓冲的 CCM 填充暴露模块。CCM 的量取决于所使用的单位和细胞培养插入的类型。请记住，要保持 ALI 中的细胞，介质应到达膜的底部，但不应在膜顶部泄漏。使用 6 口井插入时，在曝光模块中添加 6 mL 的 HEPES 缓冲 CCM。</li>
	<li>使用无菌钳子将带细胞的插入物从板转移到暴露模块。检查细胞的基底侧是否有气泡，并取出插入器的上皮侧的任何CCM。万一玄武岩侧有一些气泡，使用无菌钳子轻轻转动插入物，直到它们被移除。将含有CCM的板放在孵化器中，在暴露后进行转移。</li>
	<li>使用触摸屏显示屏选择曝光时间、气流速率和静电沉积增强。该显示屏还可用于检查湿度和温度。通常，选择 4 小时的曝光持续时间，插入物的流速为 50 mL/min，湿度为 37 °C 和 85%。 注:第一层的模块（图1）用于清洁空气暴露:此级别中的插入物用作清洁空气暴露控制。第二级和第三级的其他模块可用于气溶胶暴露，包括石英晶体微平衡 （QCM） 的两个模块，用于在线测量沉积。</li>
	<li>泄漏测试应在开始暴露之前进行。泄漏需要小于 5 mL/分钟。按照 AES 显示屏上的说明操作。泄漏测试完成后，可以开始暴露。如果发生泄漏，应检查管道。</li>
	<li>曝光结束时，打开 AES 模块的门，打开曝光模块，将细胞培养插入回细胞培养板，并将板转移到便携式孵化器。</li>
	<li>从模块（即暴露的样品）和板材（即孵化器控制）收集介质，以供以后分析，如乳酸脱水酶 （LDH） 测量。</li>
	<li>回到细胞培养实验室，将细胞培养插入物转移到充满新鲜标准CCM的盘子中。将细胞培养板放入孵化器中，直到下一次暴露或分析。</li>
	<li>最后曝光后，将插件放入孵化器直到第二天。</li>
	<li>在最后曝光后的第二天，将 CCM 添加到 apical 侧，使用伏托姆计测量 TEER。分别收集猿类和基质CCM，用于细胞因子的分析。</li>
	<li>删除所有 CCM，并通过向 apical 侧添加增殖试剂来执行细胞生存能力测试。</li>
</ol>7. 清洁 AES
<ol><li>将暴露模块装满水，等待 1 分钟，并取出水。接下来，在模块中填充 70% 的乙醇，离开 10 分钟，然后取出乙醇。用 70% 的乙醇清洁暴露喇叭。</li>
	<li>停止 85% 的湿度控制，但将机柜温度保持在 37 °C 以进行下一个实验。</li>
</ol>

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作者没有什么可透露的。

本文描述了一种在ALI下培养人类支气管上皮细胞的方法，并将这种支气管模型暴露在气溶胶或气体中。使用 Calu-3 细胞的优点是它们形成紧密的交汇点，保持单层，能够承受气流，并且可以在 ALI 培养数周，与许多其他细胞类型不同（例如，16HBE、H292 和 BEAS-2B）。使用 VITROCELL® 自动曝光站 （AES） 具有在现实和相关条件下暴露细胞的优点，因为低浓度可以使用连续气流进行应用。
与使用云系统3、32相比，持续流系统（如 AES）具有优势，云系统使用单个悬架的模糊化。连续流更逼真，并控制许多变量，如流速、湿度和温度。此外，还可以使用电场增强沉积。最后，在线监控气溶胶特征，如体型、数字浓度和质量。缺点是，与云系统相比，持续流系统更为复杂。因此，重要的是要进行预备性实验，重点是气溶胶的粒子特性和插入物上的输送剂量。然后，可以调整粒子的初始启动浓度和 AES 设置，以达到33细胞上所需的剂量。根据测试的粒子类型，气溶胶生成方法可能有所不同。静电沉积的使用取决于颗粒类型，最适合金属颗粒。对于表面电荷为正的粒子，应应用负静电场，反之亦然。
对于任何气液暴露实验来说，选择暴露浓度都可能很困难。对于 DQ12 暴露，目标是在 3 周暴露后，每周 5 天，每天 4 小时，实现 1μg/cm2的总累积剂量。这种剂量类似于在体内21，25，27，32，33引起效果的剂量。在执行暴露时，不同暴露天之间存在一些差异。虽然实际沉积剂量为 1.6 μg/cm2高于目标的 1 μg/cm2，但剂量可能太低，无法观察 Calu-3 模型的影响。在清洁空气暴露和DQ12暴露之间只观察到TEER、生存能力和细胞因子反应的细微差异，这些差异在统计学上没有显著性。对DQ12暴露3周没有在Calu-3细胞中引起显著影响的观察的一个解释是，Calu-3模型缺乏巨噬细胞。可能，在DQ12吸收巨噬细胞后产生可能影响Calu-3细胞的前炎细胞因子。另一个解释是，用于实验的DQ12批次没有预期的那样被动。当使用LPS作为阳性控制物质时，Calu-3确实表现出一种反应，其衡量标准是LDH释放量的增加和TNF-α释放量的增加。这表明该模型可以检测毒性。
正如结果部分所讨论的，Calu-3细胞模型有许多优点。此外，当在ALI培养更长的时间，Calu-3细胞可以生长西莉亚/西莉亚状结构13和产生粘液11，12，13。尽管有这些优点，该模型在生理相关性方面也有局限性。Calu-3细胞系源自腺癌，而16HBE和BEAS-2B来自健康组织。不幸的是，后两个不适合重复的ALI暴露，因为它们不保持一个稳定的单层随着时间的推移。Calu-3模型的另一个局限性是，它只代表单个细胞类型。在人类肺中，存在多种细胞类型，它们与暴露不同的相互作用和反应。吸入的颗粒会沉积在肺部的不同区域，这取决于它们的空气动力学大小。这是粒子与上皮细胞屏障接触的地方，正如 Calu-3 模型所模仿的那样。在人类肺中，肺活体巨噬细胞被粒子吸引，吞没它们，并清除它们从肺部。巨噬细胞在颗粒暴露的炎症反应中也起着至关重要的作用。因此，正在努力扩展Calu-3模型，增加主要的巨噬细胞，以更密切地模仿肺屏障。巨噬细胞的缺点是，当在ALI的Cau-3细胞之上培养时，它们只能存活7天左右。因此，应每周阅读巨噬细胞，将当前的 Calu-3 模型转换为 co 文化模型。目前正在优化 co 文化协议。
鉴于上述情况，Calu-3支气管模型是反复接触部分可溶性物质（如香烟烟雾和LPS中的化学物质）气溶胶的合适模型。这些可溶性物质诱发卡鲁-3细胞细胞因子反应显著增加。为了测试不溶性颗粒，如柴油排气和DQ12，需要一个培养模型，因为巨噬细胞在粒子暴露效应的诱导中起着至关重要的作用。
对于描述的暴露，使用 3.0 μm 孔隙插入膜。选择这种类型的插入的主要原因是可以测试纳米材料的易位性。当使用较小的 0.4 μm 孔径时，粒子聚集物将无法穿过插入膜。使用大孔径的缺点是细胞需要更长的时间来生长汇合体，并且使用光显微镜更难想象细胞的形态。要检查细胞是否确实形成汇合单层，TEER 在开始暴露之前应 =1，000 Ω x cm2。
综合起来，这里介绍的 Calu-3 支气管模型适合反复暴露在气溶胶下，至少长达 3 周。该模型可以承受通过连续气流培养和暴露，并能够检测到支气管上皮的毒性。

肺部容易吸入空气中的颗粒。为了评估空气中粒子的潜在毒性，在开发空气-液体接口（ALI）暴露系统1、2、3、4、5方面取得了进展。ALI暴露系统允许更相关和现实的暴露模型相比，传统的水下暴露通过培养基，改变粒子的特征和动能6。ALI暴露系统对细胞培养模型提出了具体要求，因为模型缺乏培养基，因此在apical方面缺乏营养物质。许多细胞模型受到培养和暴露在空气中的负面影响（例如干燥），并且只存活几天。这限制了这些模型中可用于的暴露条件:通常相对较高的浓度在短时间内作为云（即含有粒子的液滴，这些颗粒迅速沉降）。这种实验条件并不反映长期暴露于低浓度颗粒的现实情况:因此，结果的相关性是可以质疑的。为了克服这些限制，优化了由人类支气管上皮细胞系Calu-3 7组成的支气管模型的培养和暴露方案。
大多数用于ALI暴露的体外肺模型包含其他细胞系，如A549、BEAS-2B和16HBE14o-（16HBE）或原细胞作为基础8。这些细胞系的缺点是，在ALI培养时，它们只能存活几天。此外，其中一些细胞系在培养超过5天时过度生长。最后，A549细胞错过功能紧密的交汇点，因此不能形成一个紧密的屏障，需要模仿肺9，10。原发性上皮细胞可能是ALI暴露的一个很好的选择，因为它们可以在ALI培养数周。然而，原细胞因批次而异，难以维持，而且与细胞系相比成本更高，因此不太适合毒性测试和筛选。在比较不同的人类支气管上皮细胞系（16HBE、Calu-3、H292 和 BEAS-2B）时，只有 Calu-3 细胞符合现实、重复 ALI 暴露所需的所有标准:它们在 ALI 培养时保持数周存活，提供高屏障完整性，不过度生长，并且易于培养和维护。Calu-3细胞起源于腺癌，能够产生粘液11，12。细胞能否发育成西莉亚11，13，存在不一致之处。Calu-3细胞也是研究呼吸道同步病毒（RSV）感染感染气道上皮细胞14的合适模型。
除了细胞模型，自动曝光系统（AES）还用于气溶胶15，16的气液暴露。AES 的优势是它使用连续气流将细胞模型暴露在气溶胶下。这与其他空气-液体暴露系统形成鲜明对比，这些系统通常在短时间内使用相对较高的浓度作为云（即含有快速沉降的颗粒的液滴）17、18、19。这些云系统并不反映长期暴露于低浓度粒子的现实。通过使用 AES 应用连续气流，细胞模型可以在较长时间内暴露在低浓度的颗粒中，从而反映现实的暴露条件。与云系统的另一个优势是，AES 可以选择连接粒子表征仪器，从而能够测量粒子大小、数字浓度和质量。AES 的一个限制是，它使用的气流相对较高，在 10 mL/min 和 100 mL/min 之间。

<table><tbody><tr><td>0.01 M NaOH</td><td>Sigma Aldrich</td><td>S5881</td><td></td></tr><tr><td>0.1M (x10) PBS</td><td>Gibco</td><td>14200-059</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorodihydrofluoresin diacetate (DCFH&lt;sub&gt;2&lt;/sub&gt;-DA)</td><td>Sigma Aldrich</td><td>D6883</td><td></td></tr><tr><td>3-Morpholinosydnonimine hydrochloride (SIN-1 hydrochloride)</td><td>Abcam</td><td>ab141525</td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo Fisher Scientific Inc.</td><td>15290</td><td></td></tr><tr><td>Automated exposure station</td><td>Vitrocell</td><td></td><td></td></tr><tr><td>Cell proliferation reagent WST-1</td><td>Roche</td><td>11644807001</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>CPC</td><td>TSI inc., St Paul MN, USA</td><td>3022</td><td></td></tr><tr><td>Cytotoxicity detection kit LDH</td><td>Roche</td><td>11644793001</td><td></td></tr><tr><td>DQ12</td><td>IOM</td><td></td><td>nanomaterials</td></tr><tr><td>ELISA Ready-SET-Go</td><td>Fischer Scientific</td><td>88-8086-86, 88-7399-88</td><td></td></tr><tr><td>EVOM2</td><td>World Precision Instruments Inc., FL, USA</td><td>EVOM2-STX2</td><td></td></tr><tr><td>FBS</td><td>Greiner bio-one</td><td>758093</td><td></td></tr><tr><td>Flow splitter</td><td>TSI inc., St Paul MN, USA</td><td>model 3708</td><td></td></tr><tr><td>Fluorescein diacetate (F-DA)</td><td>Sigma Aldrich</td><td>F7378</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific Inc.</td><td>14175</td><td></td></tr><tr><td>Light microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>Mass flow controllers</td><td>MFC, Bronkhorst, the Netherlands</td><td></td><td></td></tr><tr><td>Methanol (analytical grade)</td><td>Sigma Aldrich</td><td>34860</td><td></td></tr><tr><td>Microbalance</td><td>Sartorius, Goettingen, Germany</td><td>ME-5</td><td></td></tr><tr><td>Minimum essential medium (MEM) + GlutaMAX</td><td>Thermo Fisher Scientific Inc.</td><td>41090</td><td></td></tr><tr><td>NEAA</td><td>Thermo Fisher Scientific Inc.</td><td>11140</td><td></td></tr><tr><td>OPS</td><td>TSI inc., St Paul MN, USA</td><td>3339</td><td></td></tr><tr><td>Pen/Strep</td><td>Thermo Fisher Scientific Inc.</td><td>15140</td><td></td></tr><tr><td>Phenol red free MEM</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Pure water</td><td>Merck</td><td>MilliQ</td><td></td></tr><tr><td>SMPS</td><td>TSI inc., St Paul MN, USA</td><td>3936</td><td></td></tr><tr><td>Spray nozzle</td><td>Schlick, Coburg, Germany</td><td></td><td></td></tr><tr><td>Teflon filters</td><td>Pall</td><td>R2PJ46</td><td></td></tr><tr><td>TEOM</td><td>Rupprecht &amp;amp; Patashnick NY, USA</td><td>series 1400</td><td></td></tr><tr><td>Tissue culture flask</td><td>Thermo Fisher Scientific Inc.</td><td>690175, 658175, 660175</td><td></td></tr><tr><td>Transwell inserts</td><td>Corning</td><td>3460, 3462</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific Inc.</td><td>25300</td><td></td></tr><tr><td>Tryptan Blue</td><td>Sigma Aldrich</td><td>T8154</td><td></td></tr></tbody></table>

an air-liquid interface bronchial epithelial model for realistic, repeated inhalation exposure to airborne particles for toxicity testing

为了对空气中的颗粒进行毒性测试，已经开发了空气-液体接口（ALI）暴露系统进行体外测试，以模拟逼真的暴露条件。这就对细胞培养模型提出了具体要求。许多细胞类型受到空气暴露的负面影响（例如干燥），并且只存活几天。这限制了这些模型中可用于的暴露条件:通常相对较高的浓度在短时间内用作云（即含有粒子的液滴，它们迅速沉降）。这种实验条件并不反映长期暴露于低浓度粒子的现实情况。为了克服这些限制，使用人类支气管上皮细胞线，Calu-3进行了调查。这些细胞可以在ALI条件下培养几个星期，同时保持健康的形态和稳定的单层与紧密的交汇点。此外，此支气管模型还适用于使用 ALI 暴露系统测试反复暴露在低、逼真的空气中颗粒浓度的影响。此系统使用连续气流，与其他 ALI 暴露系统相比，该系统使用生成云的单个星云。因此，连续流系统适用于反复和长时间暴露在空气中的颗粒，同时持续监测粒子特性、暴露浓度和输送剂量。综合起来，这种支气管模型，结合连续流动暴露系统，能够模拟现实，重复吸入暴露条件，可用于毒性测试。

本文提供了一种在 ALI 中培养和暴露人类支气管上皮细胞的方法，该方法模拟了可用于毒性测试的逼真的重复吸入暴露条件。细胞模型和暴露系统的特征对于实现可用于重复暴露的逼真的吸入暴露模型至关重要。这些特征的结果如下。
细胞模型要求和选择
在选择合适的单元格模型时，必须考虑以下特征:
<ol><li>细胞模型应该能够形成一个汇合单层与功能紧密的交汇点，以模仿肺屏障。</li>
<li>当反复暴露在有条件的（温度和湿度）空气中时，细胞模型应显示最佳性能。</li>
<li>细胞模型应响应暴露。</li>
</ol>这项研究开始于四个不同的人类支气管上皮细胞系:16HBE、Calu-3、H292和BEAS-2B。这些都广泛用于纳米材料和化学品的毒性测试。在四个细胞系中，只有 Calu-3 细胞满足上述所有要求。细胞形成一个单层，紧密的结（图3），在TER测得的一段时间内保持稳定的屏障，而其他细胞线在ALI培养时没有形成屏障或显示出屏障功能下降（图4）。此外，H292 和 BEAS-2B 在培养较长时间内往往会过度生长成多个细胞层。传统的水下细胞培养和ALI培养有很大的不同，因为ALI营养物质只能从基索拉侧获得，细胞暴露在呼吸道一侧的干燥条件下。这些条件可能会对细胞模型造成压力，可以通过测量细胞的生存能力来观察。细胞系 16HBE、H292 和 BEAS-2B 在 ALI 培养时都显示 LDH 释放增加，而 Calu-3 细胞仅显示轻微的 LDH 释放（图 5）。
接下来，对 Calu-3 模型对物质的反应进行了测试。作为一种积极的控制物质，LPS是通过对模型的 apical 侧的模糊处理。沉积剂量为0.25μg/cm2。Calu-3细胞表现出对脂多糖（LPS）的反应，增加LDH释放和肿瘤坏死因（TNF-α）释放（图6）。
最后，Calu-3单层接触到石英二氧化硅（DQ12）纳米材料（IOM，爱丁堡）。晶体二氧化硅可以诱发硅化，也可能导致肺肿瘤。因此，国际癌症研究机构（IARC）已将晶体二氧化硅列为第一类人类致癌物质20。晶体二氧化硅的作用机制被认为是通过诱导持续炎症引起的反应表面21，22，23。大鼠和小鼠的一些体内研究报告炎症和组织病理学的变化，包括肿瘤和纤维化，吸入后接触晶体二氧化硅24，25，26，27，28，29。这些影响都是在反复曝光和/或长期随访后观察到的。Calu-3模型用于研究体内研究的观测结果是否可以使用体外模型进行模仿，该模型可以在ALI反复暴露。
Calu-3细胞连续3周暴露，每周5天，DQ12每天4小时。沉积剂量使用 QCM 测量。平均沉积剂量为每天120 ng/cm2， 累计剂量为1.6 μg/cm2，类似于在体内诱导效应的剂量。 表 1中显示了其他粒子特性。与清洁空气控制（图7）相比，DQ12在3周的暴露后，在TEER、细胞生存能力和单细胞化疗蛋白1（MCP-1）释放中没有引起显著影响。由于根据体内数据预计DQ12的毒性会更大，因此根据欧盟项目GRAYCIOUS（可交付5.3）优化的协议，使用细胞检测来检查颗粒的活性。DQ12批次的响应率低于预期（图8），比正对照粒子炭黑（CB）低数量级。这种缺乏反应性可能是卡鲁-3模型中缺乏毒性反应的原因。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61210/61210fig01.jpg"> 图1:自动曝光站（AES）。 左图显示机柜外侧与触摸屏。AES 具有三个带暴露模块的水平:清洁空气暴露的顶层和气溶胶暴露的中低层。右图显示放置带单元的插入物的暴露模块。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61210/61210fig02.jpg"> 图2:曝光设置的示意图表示。 从左到右:1） ENM 悬架通过永久泵连接到喷嘴:2） 使用压缩空气，喷嘴使 ENM 悬架发云，并通过混合室将气溶胶引向 AES:3） 在进入 AES 之前，气溶胶表征仪已连接:SMPS、OPS、CPC 和 TEOM。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61210/61210fig03.jpg"> 图3:Calu-3细胞在气液接口（ALI）培养10天后具有代表性的图像。 在 ALI 培养 10 天后，Calu-3 细胞的荧光显微镜图像。紧密结蛋白ZO-1染成绿色，细胞核染成蓝色。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61210/61210fig04.jpg"> 图4:在21天的培养期内，四条不同细胞系的外皮电阻（TEER）。 16HBE、Calu-3、H292 和 BEAS-2B 的 TEER 值在培养了 21 天时:前 7 天被淹没，随后在 ALI 进行了 14 天。为插入物的背景电阻校正了 TEER 值，并乘以插入物的表面积。符号和错误条表示六个插入的平均值和标准偏差。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61210/61210fig05.jpg"> 图5:LDH在21天的培养期内释放出4条不同的细胞系。 Ldh 释放 16hbe， Calu - 3， H292 和 Beas - 2b 时， 培养了 21 天: 7 天淹没， 然后 14 天在阿里。显示的 LDH 值相对于每个细胞类型的最大 LDH 释放量。符号和错误条表示五个插入的平均值和标准偏差。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig05large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61210/61210fig06.jpg"> 图6:暴露在LPS下的 Calu-3 细胞中的细胞效应。 Calu-3细胞通过云云云化暴露在0.25μg/cm2 LPS中。（A） WST-1转换。（B） LDH 释放。（C） TNF-α LPS曝光后发布。符号和错误条表示三个插入的平均值和标准偏差。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig06large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61210/61210fig07.jpg"> 图7:暴露在DQ12纳米材料中的卡鲁-3细胞的细胞效应。 Calu-3细胞暴露在DQ12纳米材料中3周（每天4小时，每周5天），每天约120克/厘米2， 累计剂量为1.6微克/厘米2。（A） 特勒值。（B） LDH 释放。（C） MCP-1 在 DQ12 曝光后发布。所有符号和错误条表示控制的三个插入和 DQ12 曝光的六个插入的平均值和标准偏差。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig07large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61210/61210fig08.jpg"> 图8:DQ12的细胞反应。 DQ12用2ʹ，7ʹ-二氯氟酸二乙酸酯（DCFH-DA）探针孵化，以检测其表面反应能力。作为积极的控制，碳黑色 （CB） 颗粒被包括在内。与 CB 相比，DQ12 的表面反应能力非常低。 <a href="https://www.jove.com/files/ftp_upload/61210/61210fig08large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>粒子质量 （μg/m3）</td>
			<td>粒子数 （#/厘米3）</td>
			<td>移动粒子大小 （nm）</td>
			<td>几何标准偏差</td>
			<td>光学粒子大小 （μm）</td>
		</tr>
<tr>
<td>2969 (418)</td>
			<td>83983 (10215)</td>
			<td>66.6</td>
			<td>2.5</td>
			<td>1.1 (1.3)</td>
		</tr>
</tbody></table>表1:DQ12暴露特征。 值显示为平均值，括号中的标准偏差。

Watch this Scientific Journal Video about An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing at JoVE.com

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

描述的是体外支气管模型的细胞培养和暴露方法，用于逼真、反复吸入颗粒以进行毒性测试。

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 ...

an-air-liquid-interface-bronchial-epithelial-model-for-realistic

Research

JoVE Journal

Biology

11.3K 次观看.  National Institute for Public Health and the Environment (RIVM). 描述的是体外支气管模型的细胞培养和暴露方法，用于逼真、反复吸入颗粒以进行毒性测试。

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

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

科研

医学

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

一种用于气溶胶取样的新型便携式体外曝光盒

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

环境科学

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

生物工程

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

免疫与感染

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent models continue to be an essential tool for studying human silicosis. For silicosis, the classic pathological process needs to be inducible via the inhalation of freshly generated quartz particles, which means specifically inducing human occupational disease. This study described a technique to establish and effectively mimic the pathological dynamic evolution process of silicosis. Further, the technique had good repeatability with no surgery involved. The inhalation exposure system was fabricated, validated, and used for toxicology studies on respirable particle inhalation. The critical components were as follows: (1) bulk dry SiO2 powder generator adjusted with an air-flow controller; (2) 0.3 m3 whole-body inhalation exposure chamber accommodating up to 3&#160;adult rats; (3) a monitoring and control system for regulating oxygen concentration, temperature, humidity, and pressure in real-time; and (4) a barrier and waste disposal system for protecting laboratory technicians and the environment. In summary, the present protocol reports the inhalation via the whole body, and the inhalation chamber created a reliable, reasonable, and repeatable rat silicotic model with low mortality, less injury, and more protection.

This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.

通过全身暴露于可吸入二氧化硅来建立矽肺大鼠模型

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent ...

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

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

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 ...

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

摘要

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此视频中的章节

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

一种用于气溶胶取样的新型便携式体外曝光盒

对于中吸入有毒化学测试体外细胞培养模型

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

通过全身暴露于可吸入二氧化硅来建立矽肺大鼠模型

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

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

摘要

探索更多视频

此视频中的章节

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

一种用于气溶胶取样的新型便携式体外曝光盒

对于中吸入有毒化学测试体外细胞培养模型

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

通过全身暴露于可吸入二氧化硅来建立矽肺大鼠模型

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

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估