Name: assessment of the acute inhalation toxicity of airborne particles by exposing cultivated human lung cells at the air-liquid interface
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This work was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF, Germany (Grant 031A581, sub-project A-D)) and by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG,研究培训组 GRK 2338）。

注：一个暴露实验的协议为期三天。
第1天
1. 细胞的一般准备和培养
注：人类肺腺癌上皮细胞系A549用于暴露实验。细胞必须在无菌条件下处理。可使用其他适合在ALI种植的细胞系。
<ol><li>制备生长介质（Dulbeco的最小必需介质（DMEM），辅以10%胎儿牛血清（FBS）和5微克/mL金霉素）和接触介质（DMEM，辅以5微克/mL根霉素和最终HEPES浓度?...

<ol>
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近年来，为了获取有关可吸入颗粒物急性吸入危害的信息，并根据3R原理25减少和替换动物实验，开发了许多非动物吸入毒性试验模型。
在细胞培养模型方面，细胞的暴露可以在水下条件下或在ALI进行。在水下条件下暴露细胞可能会影响物理化学性质，从而影响测试物质12的毒性。然而，体外ALI吸入模型模拟了比水下暴露更高的生物和生理?...

吸入有毒的空气中颗粒是一个公共卫生问题，导致世界各地的多种健康风险，每年有数百万人死亡。气候变化、持续的工业发展以及对能源、农业和消费品的需求增加，使得近年来肺病的增多是3、4、5、6。对可吸入物质的急性吸入毒性的认识和评估为危害评估和风险管理提供了依据，但这类物质种类繁多，7、8仍然缺乏这方面的信息。自 2006 年以来，欧盟化学品立法 REACH（化学品注册、评估、授权和限制）要求现有和新引进的产品在投放市场之前，必须经过毒理学特征，包括吸入途径。因此，REACH侧重于替代和无动物的方法，实现"3R"原则（替换，细化和减少动物实验）和使用适当的体外模型9。近年来，为了评估空气中颗粒5、7、10、11的急性吸入毒性，已经开发出许多不同的和适当的非动物吸入毒性测试模型（例如体外细胞培养、片上肺模型、精密切肺切片（PCLS）。）。在体外细胞培养模型方面，培养的细胞可以在水下条件下或ALI（图1）中暴露。然而，在评价空气中化合物特别是颗粒的毒性方面，水下暴露研究的有效性是有限的。水下接触技术与人体体内情况不符;覆盖细胞的细胞培养基可能影响物理化学性质，从而影响测试物质12、13的毒性。ALI体外吸入模型允许细胞直接暴露于测试物质，不受细胞培养基与测试颗粒的干扰，从而模仿人类接触，比水下暴露12、14具有更高的生理和生物相似性。
然而，对于REACH等监管过程，急性吸入毒理学领域只有动物模型可用，因为迄今为止，还没有一种替代的体外方法得到充分的验证和正式接受。为此，测试模型必须根据欧盟动物试验替代实验室（EURL-ECVAM）原则的要求对测试有效性15进行验证。
以前的审前研究和最近完成的验证研究成功地证明了CULTEX RFS暴露系统的应用领域及其可转移性、稳定性和可重复性13。该暴露系统是一种基于体外细胞的暴露系统，由于其径向气溶胶分布概念和测试气溶胶在细胞16的连续流动中连续流动，使细胞能够均匀地暴露在ALI的气体、颗粒或复杂大气（如香烟烟雾）中。该径向流系统的基本模块包括入口适配器、带径向气溶胶分布的气溶胶导向模块、采样和插座模块以及带手轮的锁定模块（图 2）。生成的颗粒通过入口适配器和气溶胶引导模块到达细胞，并沉积在细胞培养插件上，这些入口位于采样模块的三个径向排列的暴露室中。气溶胶导向模块和取样模块可以通过连接到外部水浴17进行加热。
在这两项研究的框架内，A549细胞被用于所有暴露实验。细胞系A549是一种人类不朽的上皮细胞系，其特征非常好，在众多毒理学研究中已被用作II型阿尔韦拉上皮细胞的体外模型。细胞的特点是乳腺体，生产表面活性剂和一些炎症相关因素18。它们还显示支气管上皮细胞的特性，由于其粘液生产19。此外，可以在 ALI 进行培养。虽然这种细胞系缺乏建立细胞-细胞接触，这些细胞的培养是更方便，成本更低，其结果与原细胞20相比与供体无关。
A549细胞被播种在6孔细胞培养插件（PET膜，4.67厘米2，孔径0.4毫米），密度为3.0 x105细胞每插件，并在水下条件下培养24小时。然后，在三个独立的实验室中，细胞暴露于清洁空气和三种不同的暴露剂量（25，50和100微克/厘米2）的20个测试物质在ALI。暴露剂量与沉积时间相关，导致在15、30或60分钟后，细胞的恒定颗粒速率为25微克/cm 2，50μg/cm2和100μg/cm2。 然而，沉积的颗粒在沉积后没有被冲走，而是留在细胞上24小时。因此，粒子的沉积时间为15、30和60分钟，但细胞的暴露总共持续了24小时。试验物质的沉积率是在初步实验中根据以往方法确定的。
使用细胞活力测定测定，在颗粒沉积后24小时评估细胞作为毒性指标的可毒性性。特别注重清洁空气控制的质量、暴露协议的优化和改进、实验室内和实验室间的可重复性以及建立预测模型（PM）。导致细胞活力下降低于 50% 的物质 （PM 50%）或 75%（PM 75%）在三种接触剂量中的任何一次都被认为具有急性吸入危险。结果然后比较现有的体内数据（根据经合组织测试指南（TG）403或TG 43621，22至少一项可靠的研究），导致整体协调度为85%，特异性为83%，灵敏度为88%23。
除了测量细胞活力外，还可以评估其他终点，如细胞因子释放、通过LDH测定检查细胞解酶或膜完整性，但验证研究不需要。因此，接触系统（例如，CULTEX RFS）被证明是一种预测性筛选系统，用于对被测试的空气颗粒的急性吸入毒性进行定性评估，是动物试验的一种有前途的替代方法。建议使用此曝光系统对空气中的颗粒进行暴露实验，建议采用以下方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture medium and supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>FG 0415</td>
			<td>used as growth medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen, Darmstadt, Germany</td>
			<td>22320</td>
			<td>used as exposure medium</td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>S 0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin (10mg/mL)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A 2710</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Th. Geyer, Renningen, Germany</td>
			<td>L 0180</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (0.05%/0.02%)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 2143</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASY Cups</td>
			<td>Roche Diagnostic GmbH, Mannheim, Germany</td>
			<td>REF 05651794</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates</td>
			<td>Corning, Wiesbaden, Germany</td>
			<td>3516</td>
			<td>6&#173;-well plates</td>
		</tr>
		<tr>
			<td>Corning Transwell cell culture inserts</td>
			<td>Corning, Wiesbaden, Germany</td>
			<td>3450</td>
			<td>24mm inserts; 6-&#173;well plates; 0.4 &#181;m</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASYton</td>
			<td>Roche Diagnostic GmbH, Mannheim, Germany</td>
			<td>REF 05651808001</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed Air (DIN EN 12021)</td>
			<td>Linde Gas Therapeutics GmbH, Oberschlei&#223;heim, Germany</td>
			<td>2290152</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab155902</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments + equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASY Cell Counter</td>
			<td>Sch&#228;rfe System GmbH, Reutlingen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circulation thermostat</td>
			<td>LAUDA, Lauda-K&#246;nigshofen, Germany</td>
			<td>Ecoline RE 100</td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX HyP - Hydraulic Press</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX insert sleeve</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX RFS - Radial Flow System Type 2 (module for particle exposure)</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX RFS - Radial Flow System Type 2 (module for clean air exposure)</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX supply</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow controller 0-30 ml/min (IQ-Flow)</td>
			<td>Bronkhorst Deutschland Nord GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow controller 0-1,5 l/min (EL-Flow)</td>
			<td>Bronkhorst Deutschland Nord GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filters (large)</td>
			<td>Munktell &#38; Filtrak GmbH, Sachsen, Germany</td>
			<td>LP-050</td>
			<td>Munktell Sterile Filter; Particle retention efficiency &#62; 99,999%</td>
		</tr>
		<tr>
			<td>Filters (small)</td>
			<td>Parker Hannifin Corporation, Mainz, Germany</td>
			<td>9933-05-DQ</td>
			<td>Balston disposable filter</td>
		</tr>
		<tr>
			<td>Medium pump</td>
			<td>Cole-Parmer GmbH, Wertheim, Germany</td>
			<td>Ismatec IPC High Precision Multichannel Dispenser</td>
			<td>digital peristaltic pump</td>
		</tr>
		<tr>
			<td>Microplate Reader Infinite M200 Pro</td>
			<td>Tecan Deutschland GmbH, Crailsheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vakuum pump</td>
			<td>KNF, Freiburg, Germany</td>
			<td>N86 KT.18</td>
			<td></td>
		</tr>
		<tr>
			<td>V&#246;gtlin mass flow controller 0,2-10 l/min</td>
			<td>TrigasFI GmbH</td>
			<td>V&#246;gtlin red-y compact regulator, Typ-Nr.: GCR-C3SA-BA20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>LAUDA, Lauda-K&#246;nigshofen, Germany</td>
			<td>Ecoline Staredition RE 104</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of the acute inhalation toxicity of airborne particles by exposing cultivated human lung cells at the air-liquid interface

在这里，我们提出了一个专门设计的模块化体外暴露系统，使在ALI培养的人类肺细胞均匀地暴露于气体，颗粒或复杂的大气（如香烟烟雾），从而提供现实的生理人类阿尔皮拉尔区域的表层暴露在空气中。与采用线性气溶胶制导的连续曝光模型相比，径向流系统的模块化设计满足测试大气连续生成和输送到电池的所有要求，均匀分布和沉积粒子和大气的连续去除。这种接触方法主要设计用于细胞暴露于空气中的颗粒，但可以适应液体气溶胶和剧毒和腐蚀性气体的暴露，具体取决于气溶胶生成方法和接触模块的材料.
在最近完成的验证研究框架内，该暴露系统被证明是一种可转移、可重复和预测性筛选方法，用于对空气中颗粒的急性肺细胞毒性进行定性评估，从而可能减少或取代通常提供这种毒理学评估的动物实验。

CULTEX RFS 是专门设计的模块化体外暴露系统，可在 ALI 处实现细胞的直接和均质暴露。在前一项审前研究中，成功地证明了该暴露系统的总体适用性及其可转移性、稳定性和可重复性。在最近由德国联邦教育和研究部资助的一个研究项目中，该暴露系统被成功验证，并被建立为被测化合物急性吸入危害的预测模型（PM）。由于清洁空气控制的质量在验证前研究期间被证明是?...

Watch this Scientific Journal Video about Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface at JoVE.com

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

我们提供一个强大、可转移和预测性的体外暴露系统，通过在空气-液体界面（ALI）暴露培养的人类肺细胞来筛选和监测空气中的颗粒，其急性肺细胞毒性。

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 ...

利用这种体外暴露系统，通过暴露ALI的培养人类肺细胞，对有关急性肺细胞毒性的空气中颗粒进行筛选有助于减少动物实验。此暴露系统的主要优点是径向气溶胶分布概念，导致均匀分布和粒子在细胞上的位置。在播种细胞之前，在六井板的每个井中加入2.5毫升的回火生长介质。将无细胞培养插入物小心地放在孔内，并将一毫升的生长培养介质添加到每个细胞培养物的插入中。在37摄氏度和5%的二氧化碳下孵育板30分钟。细胞试青化后，在装满10毫升同位素溶液的杯子中稀释100微升细胞培养悬浮液。在不摇晃的情况下慢慢倾斜杯子。在细胞计数器上，根据 A549 细胞的细胞特定测量参数确定每毫升可行细胞的数量和细胞生存能力。回火后，吸气细胞培养中的培养物插入并播种一毫升的 A549 细胞，密度为每毫升细胞的三倍十至五细胞。通过轻轻的摇动和孵育24小时分配细胞悬浮液。通过压机正面的时间控制设置压榨时间。打开压缩空气阀的压缩空气供应。使用压机前侧的压力调节器，将压缩空气压力设置为大约两个柱。拉出抽屉，按下按下按钮，然后读取数字压力开关上的按压压力。在物质容器中填充少量测试物质。将柱塞插入物质容器中，然后稍微来回转动，将粉末均匀地分配到容器中。将物质容器（带柱塞）放在抽屉中，然后按下按下按钮。打开抽屉并拆下柱塞。关键步骤是压榨必须专门压缩到物质容器内的粉饼中的测试物质，以便实现稳定的颗粒暴露。从气溶胶导向模块中拆下进气适配器和冷凝水反射器。用插头关闭气溶胶引导模块中的三个气溶胶进气孔，并关闭带虚拟活门的采样模块中的介质供应连接。将真空管与气溶胶导向模块的管接头连接。使用手轮关闭模块并测量流量控制器的值。关闭几分钟后，值将减至每分钟 5 毫升以下。启动气溶胶发生器软件。如果物质刮刀不在最低位置，请按"定位模式"按钮。将物质容器与压榨的测试材料倒置在物质刮刀上。确保物质容器的玻璃朝前。将锁定板放在物质容器上的插槽中，然后拧紧黑色螺钉。将"进给"和"旋转"的值更改为所需的设置。使用向下箭头向下推滑，用物质容器，直到物质刮刀靠近压榨物质。通过轻点质量流量控制器打开气溶胶发生器的压缩空气供应，然后单击"开始"按钮启动气溶胶生成。将进给速率设置为每小时 15 到 20 毫米，以避免等待时间过长。通过观察细尘云，从下面放置一个小手电筒，在 Elutriator 的玻璃管后面，控制正确的颗粒生成。使用预热曝光介质启动介质电源，并填充采样模块，直到模块打开时覆盖下管。将盲细胞培养物插入曝光模块，向下泵送曝光介质，直到下管被介质覆盖，刀片的下侧与介质接触。启动气溶胶发生器，关闭暴露模块，然后将暴露模块连接到气溶胶发生器的暴露模块插座。交货时间后 30 分钟，用橡胶塞密封 Elutriator 的暴露模块插座，并拆下盲插拔。重新填充曝光介质，直到下管被介质覆盖。在钳子的帮助下，从六个井板中去除细胞培养插入物。将生长介质小心地从细胞培养物中倒掉，通过推翻插入物并使用移液器吸气并丢弃残留液体。将刀片放在曝光和清洁空气模块的暴露室中。关闭模块并启动暴露实验，将暴露模块连接到气溶胶发生器的暴露模块出口和清洁空气模块同时连接到载波气体供应。实验完成后，断开曝光和清洁空气模块并密封暴露模块出口。单击"停止"按钮停止压缩空气供应和气溶胶发生器。打开暴露和清洁空气模块，并使用钳子将细胞培养物插入到准备好的孵化后六孔板。在37摄氏度和空气液体界面上孵育6个井板以及未暴露的细胞培养物，24小时，5%的二氧化碳。曝光后24小时，将细胞培养物插入新准备好的6个井板中。向每个细胞培养插入中添加一毫升新鲜准备的 WST1 溶液。仔细摇动板，以便将溶液均匀地分布到细胞上。在37摄氏度和5%的二氧化碳下，用细胞培养物孵育6个井板，孵育一小时。之后，将 100 微升的上经液从每个六个井板转移到 96 井板。使用微孔板读卡器，测量参考波长为 650 纳米的 450 纳米的吸光度。CULTEX RFS 是一种专门设计的模块化体外暴露系统，使细胞能够直接和均匀地暴露在空气液体界面的空气中颗粒中。将细胞暴露在清洁空气中不会随着时间的推移降低细胞的生存能力。暴露系统已成功验证，并建立为被测试化合物急性吸入危害的预测模型。A549细胞暴露在无毒性、中等或强毒性的不同测试物质中。请确保您严格保留孵化时间、颗粒生成前期和颗粒沉积时间等时间框架。这种暴露方法主要设计用于细胞的颗粒暴露，但可以通过调整气溶胶生成方法扩展到液体气溶胶和气体的暴露。这种筛选方法能够对体外急性吸入毒性的可吸入颗粒物进行定性评估，从而减少通常提供这种毒理学评估的动物试验。不要忘记，处理有毒测试物质可能极其危险，在处理此类化合物时，应始终采取预防措施，如戴手套、戴口罩。

Research

JoVE Journal

Medicine

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

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

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Oral Health Assessment by Lay Personnel for Older Adults

老年人外行人员口腔健康评估

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...