Bannuscher Anne, Schmid Otmar, Drasler Barbara, Rohrbasser Alain, Braakhuis Hedwig M, Meldrum Kirsty, Zwart Edwin P, Gremmer Eric R, Birk Barbara, Rissel Manuel, Landsiedel Robert, Moschini Elisa, Evans Stephen J, Kumar Pramod, Orak Sezer, Doryab Ali, Erdem Johanna Samulin, Serchi Tommaso, Vandebriel Rob J, Cassee Flemming R, Doak Shareen H, Petri-Fink Alke, Zienolddiny Shanbeh, Clift Martin J D, Rothen-Rutishauser Barbara
Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland.
Comprehensive Pneumology Center (CPC-M), Helmholtz Zentrum München - Member of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany; Institute of Lung Health and Immunity, Helmholtz Zentrum München - German Research Center for Environmental Health, 85764 Neuherberg, Germany.
NanoImpact. 2022 Oct;28:100439. doi: 10.1016/j.impact.2022.100439. Epub 2022 Nov 17.
Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ) and titanium dioxide nanoparticles (TiO NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ and TiO NM-105 particles showed good linearity (R > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO NM-105 (0.54 (41%)), which was likely due to partial retention of TiO NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.
在可渗透的Transwell小室上培养的气液界面（ALI）肺细胞模型越来越多地用于呼吸危害评估，这需要将任何物质以可控的方式雾化并沉积在ALI细胞上。本文介绍的方法旨在使用VITROCELL® Cloud12系统（一种市售的气溶胶-细胞暴露系统）评估通过Transwell小室递送的雾化物质剂量。与七个对VITROCELL® Cloud12有不同经验水平的欧洲合作伙伴进行了一项实验室间比较研究。所有合作伙伴都制定并应用了一种标准操作程序（SOP）来雾化递送材料，即一种水溶性分子物质（荧光加标盐）和两种难溶性颗粒，结晶二氧化硅石英（DQ）和二氧化钛纳米颗粒（TiO NM-105）。通过荧光分光光度法（荧光素）和VITROCELL® Cloud12系统中集成的石英晶体微天平（QCM）对递送至Transwell小室的材料剂量进行定量。用透射电子显微镜（TEM）确认沉积颗粒的形状和团聚状态。分两步进行了特定设备性能的实验室间比较，首先针对分子物质（荧光素加标盐），然后针对颗粒。VITROCELL® Cloud12系统在气溶胶沉积方面的设备和/或操作特定差异通过所谓的沉积因子（DF）来表征，该因子可根据雾化悬浮液中的颗粒浓度预测Transwell小室沉积的颗粒剂量。尽管不同实验室之间的DF在0.39至0.87之间变化（平均值（变异系数（CV））：0.64（28%）），但每个VITROCELL® Cloud 12系统的QCM都准确测量了各自Transwell小室沉积的剂量。DQ和TiO NM-105颗粒的雾化递送在雾化悬浮液的颗粒浓度与QCM测定的小室递送颗粒剂量之间显示出良好的线性关系（R > 0.95）。VITROCELL® Cloud 12对DQ颗粒的性能与荧光素加标盐的性能相同，即测量剂量与盐预测剂量的比值为1.0（29%）。另一方面，观察到TiO NM-105的剂量降低了约2倍（0.54（41%）），这可能是由于TiO NM-105团聚体部分保留在VITROCELL® Cloud12的振动筛网雾化器中。这项实验室间比较表明，VITROCELL® Cloud 12中集成的QCM是一种可靠的剂量测定工具，它考虑了由于设备、操作和/或材料特定效应导致的Transwell小室递送剂量的潜在变化。通过本文介绍的详细方案，所有七个合作伙伴实验室都能够使用VITROCELL® Cloud12暴露系统在与观察体外危害反应相关的剂量水平下证明材料悬浮液的剂量控制雾化。这是朝着监管批准实施ALI肺细胞培养用于雾化材料的体外危害评估迈出的重要一步。
