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&#8217;,7&#8217;-dichlorodihydrofluoresin diacetate (DCFH2-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 &#38; 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

11.2K 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

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

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

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

全身纳米气溶胶吸入暴露

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

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

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

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 ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

用于斑马鱼胚胎毒性评估的 Startle 试验

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

作者聚焦：使用 Zebrafish 胚胎惊吓反应测定进行高通量毒性筛选

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

使用斑马鱼振动惊吓反应筛查系统评估化学品的毒性

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...