一种用于气溶胶取样的新型便携式体外曝光盒
Summary
在这里, 我们提出了一个协议, 以执行便携式细胞气溶胶暴露和测量细胞反应。该方法使用细胞, 生长在空气液体界面,模仿体内生理。通过活性氧的产生和乳酸脱氢酶释放的细胞毒性, 观察到细胞对铜纳米颗粒气溶胶的反应为氧化应激。
Abstract
该协议引入了一种新的体外暴露系统, 能够磨损, 包括其特性和性能。体外接触系统中的空气-液体界面 (ali) 通常是大的和笨重的, 使在排放源或呼吸区内的场和操作运输变得困难。通过这些系统的小型化, 实验室可以被带到现场, 加快处理时间, 并提供更合适的曝光方法, 在接触细胞之前不改变气溶胶。便携式体外曝光盒 (pivec) 采用37毫米滤镜盒, 允许在传统实验室环境之外进行体外毒性测试。利用三种尺寸的铜纳米粒子对 pivec 进行了表征, 在重量和颗粒数量浓度分析的基础上确定了沉积效率。对暴露的肺细胞进行了初步的细胞毒性实验, 以确定该系统在保持细胞活力的同时沉积颗粒的能力。与体外接触设备中可用的垂直流动相比, pivec 提供了类似或更高的沉积效率。尽管样品吞吐量较低, 但小尺寸为目前的体外 ali暴露系统提供了一些优势。其中包括个人监测所需佩戴的能力、从实验室到排放源的流动性, 以及在保持较低用户成本的同时设置多个空间分辨率系统的选项。pivec 是一个系统, 能够收集气溶胶在现场和呼吸区到一个空气接口,体外模型。
Introduction
使用体外技术进行个人取样可以提供有关气溶胶在工作场所的生物效应的全面信息。1接触空气中的污染物包括在将气体引入电池悬浮液的淹没条件下接触到化学品本身、接触采集到的空气样本、使用摇杆等装置进行间歇性接触或直接接触在空气-液体界面 (ali) 处曝光。2其中许多技术是在悬浮液中生长的细胞或在接触前收集样品的情况下进行的, 由于气溶胶的潜在变化, 每一种技术都会影响毒理学研究。3为避免这些变化, 可以使用文献中使用的几个体外ali 培养暴露系统将实验室带到现场, 这些系统在文献中使用, 4,5,6, 7,然而,8、9、10、11、12、13很少有商业产品可供选择。8,9,12这些系统通常体积庞大, 特别是在包括调节细胞环境的温度和湿度以及样品气溶胶流量的仪器时。通过使用 pivec, 气溶胶暴露可以在传统实验室环境之外进行, 也可以在呼吸区内进行, 同时模拟吸入条件。
体外气溶胶沉积的测定对研究吸入对健康的影响具有重要意义。呼吸区, 距离口腔和鼻子不到 3 0 厘米的区域,1 4对于了解纳米颗粒的暴露程度和与肺部的生物效应联系起来至关重要。2通常, 细胞上的沉积被定义为沉积效率, 即沉积在细胞上并由被给系统的粒子 6、15 或在相同数量的质量基础上吸收的颗粒。4 个,16目前测量呼吸区气溶胶的方法是以过滤器为基础的, 在给定的采样期间捕获粒子, 并使用过滤器进行进一步的测试。17个人监控需要一个小系统, 附带较少样本的权衡。
有许多方法可以确定暴露于气溶胶对健康的影响。ali 模型允许气雾剂通过空气直接给细胞, 就像在实际暴露情况下一样, 但它比体内研究更具成本效益, 时间密集程度更低, 同时模仿空气液体屏障, 如眼睛,皮肤和肺。在 ali 生长的肺细胞具有产生偏振屏障层的能力, 18-19,产生类似于体内肺上皮的生理特征, 包括特定的粘液和表面活性剂的产生支气管或肺泡细胞系, 纤毛跳动,19个紧密连接,19个,20个和细胞极化。18这样的变化会影响毒性研究中测量的细胞反应。21此外, ali体外模型结果往往比通过悬浮液模型22暴露的细胞更敏感, 能够在体内模拟急性吸入毒性。23,24因此, 能够在呼吸区内进行测量的 ali 暴露系统是自然的下一步。
通过将细胞直接暴露在排放源处的气溶胶中, 就会对混合物中的所有气体、半挥发性化合物和颗粒的影响进行调查。当混合物收集在过滤器上时, 气体和挥发性化合物不会被捕获, 也无法对整个混合物进行调查。此外, 将颗粒重组为粉末或液体悬浮液会导致液体悬浮液中的聚集或颗粒-流体相互作用, 如溶解。25,26当气溶胶颗粒被添加到液体中时, 聚集的可能性较高,25, 27 形成蛋白质电晕, 28 或与液体中的化合物相互作用,这可能会影响沉积和影响生物反应。29,30
ali 的暴露是基于三个主要的气溶胶剖面, 云沉降, 平行流动, 和垂直流动。云沉降, 空气-液界面细胞暴露 (alice) 使用,4是一个批处理系统, 其中粒子沉积通过重力和扩散沉降作为气溶胶作为一个单位处理。平行流动, 由静电气溶胶体外暴露系统 (eaves)5和多文化曝光室 (mec) ii, 6 允许沉积通过增加布朗运动通过流动剖面。垂直流量, 由一个微喷雾器,7纳米气溶胶室用于体外毒性 (nacivt),11和商业 ali 系统8,9,10, 12,增加了冲击沉积区域内的颗粒。其中许多暴露系统体积大、体积大, 需要过多的气雾剂预处理系统、流量泵, 甚至是细胞孵化加热室。这种较大的尺寸降低了系统的可移植性。而不是直接在排放源取样, 这些系统往往有样品带到实验室或模型气溶胶产生进行分析。从现场到实验室的转换可能会降低发射气溶胶的复杂性。pivec 比当前系统更小, 外部表面积约为460厘米2 , 重量仅为60克, 并在系统中集成了热和湿度控制, 可实现高度便携的设备。尺寸和重量的减少使系统能够磨损或带到接触源, 从而允许直接取样。
目前暴露系统的巨大规模也降低了进行取样以调查浓度空间梯度的能力。在确定许多潜在环境和职业危害的毒理影响时, 该决议是关键, 例如发生雾化的车辆排气颗粒物或工作场所活动。发射后, 粒子浓度立即出现空间方差。这随着粒子在整个大气中的扩散而随时间增长, 这些影响可能会根据温度、压力、风和太阳等环境条件而改变。颗粒一旦发出31、32,就会开始老化和氧化, 扩散率也会受到地形的影响;在峡谷和隧道中会发现浓度较高, 那里的分散效应会变慢, 在分散面积较大的地方可以发现浓度较低。33 . 分散率的这些变化可对人类健康产生重大影响, 在比较生活在城市和农村的哮喘成年人人数时可以看到这些变化。34 . 虽然许多曝光系统同时提供多个样本, 但需要多个系统, 并配备大量大型设备来执行空间分辨率。
通过将实验室带到现场, 可以通过使用整个细胞作为传感器来减少分析时间。遵循已知的生物机制和终点可以帮助确定气溶胶的组成和大小。由于缓慢的清除方法, 包括黏膜清除, 吞噬, 和移位, 这些粒子往往与细胞相互作用约几天到第3周产生氧化应激, 炎症, 甚至细胞死亡。这些生物终点可能是心血管疾病或慢性阻塞性肺病不良结局途径的起点。此外, wiemenn等人还进行了一系列体外检测, 以便与短期体内吸入毒性的文献值进行比较。35通过乳酸脱氢酶释放、谷胱甘肽还原和过氧化氢的形成和释放以及炎症潜能检测细胞毒性的四个阳性结果中的两个预测了体内反应。肿瘤坏死因子α基因。在10种纳米金属氧化物测试中, 6 种使用体外暴露进行活性测试 (氧化钛、氧化锌和4种不同的氧化锆),并在体内确认。
为了研究气溶胶在职业环境中的影响, 我们的实验室开发了用于现场曝光的 pivec。此外, 可以佩戴 pivec 进行个人取样, 以监测和调查吸入暴露情况, 如37毫米滤芯盒36或多个系统可用于在给定区域内实现空间分辨率。在该协议中, 讨论了 pisec 的特性和使用。接触后, 通过细胞毒性检测观察生物效应。
Protocol
Representative Results
Discussion
滤芯提供了一种简单、廉价的方法来收集呼吸区的气溶胶;然而, 从过滤器中提取的气溶胶样本并不代表整个气溶胶 (即气体、挥发物和微粒), 因此限制了对相关生物效应的评估。pivec 采用37毫米滤芯盒的初步设计, 旨在保持可移植性, 并模拟吸入颗粒的体内沉积。pivec 明显小于当前的 ali 曝光系统, 大约与包含温度和湿度控制的纸巾盒的大小相当。大小类似于个人级联撞击器, 同时提供有关细?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者要感谢鲍里斯·索洛莫诺夫和弗吉尼亚联邦创新机器商店对该设备的快速原型设计。作者还要感谢 lewinski 集团的克里斯蒂安·罗梅罗-富恩特斯、维塔利·阿夫鲁廷博士、德米特里·佩斯托夫博士和弗吉尼亚联邦纳米材料核心表征设施在粒子表征方面提供的帮助。这项工作得到了弗吉尼亚联邦大学工程学院向 leeinski 博士提供的创业资金的支持。
Materials
| Scanning mobility particle sizer (SMPS) | TSI, Inc. | 3910 | NanoSMPS |
| Optical particle sizer (OPS) | TSI, Inc. | 3330 | |
| Stainless Steel Pipe, 4" Long | McMaster-Carr | 4830K116 | Standard-Wall 304/304L, Threaded on Both Ends, 1/8 Pipe Size |
| Brass Ball Valve with Lever Handle | McMaster-Carr | 4112T12 | Compact High-Pressure Rating, 1/8 NPT Female |
| Steel Pipe, 2" Long | McMaster-Carr | 7753K121 | Standard Wall, Threaded on One End, 1/8 Pipe Size |
| HEPA filter | GE Healthcare | 09-744-12 | HEPA-Cap Disposable Air Filtration Capsule |
| Vacuum Generator | PISCO USA | VCH10-018C | |
| PIVEC | VCU | For design please contact authors | |
| Resistive heater | |||
| 1/4" barbed connectors | Zefon International, Inc. | 459743 | |
| Porous tubing | Scientific Commodities, Inc. | BB2062-1814A | Hydrophilic 10 um pores |
| Battery power bank | |||
| Cell culture insert | Fisherbrand | 353095 | 24 well plate insert |
| Filter Forceps | Fisherbrand | 09-753-50 | |
| Transfer Pipette | ThermoScientific | 13-711-27 | |
| Glass Fiber Filters | SKC | 225-7 | Binder-Free Type AE Filter 37 MM 1.00 um pore |
| Ultra Micro Balance | A&D | BM-22 | Housed in environmental chamber |
| 37 mm filter cassette | SKC | 225-3250 | Filter Cassette Blank, 37 mm, Clear Styrene |
| Variable flow vacuum pump | SKC | 220-5000TC | AirChek TOUCH, 5 to 5000 mL/min |
| Copper Particles | U.S. Research Materials, Inc. | US1090 | 40 nm |
| Copper Particles | U.S. Research Materials, Inc. | US1088 | 100 nm |
| Copper Particles | U.S. Research Materials, Inc. | US1117M | 800 nm |
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