This work investigates a completely novel and experimental concept of exposing L5178Y cells at the air-agar-interface to mainstream cigarette smoke aerosol (Kentucky reference 3R4F). This study highlights the associated challenges of combining a suspension cell line alongside an in vitro aerosol exposure system. To achieve a monolayer, cells were ‘seeded’ in a concentrated cell super-mix suspension onto an RPMI/agar-matrix -base. The resulting cell suspension media was adsorbed into the agar base leaving the L5178Y cells lightly suspended on the agar surface, approximating a monolayer. Cells were deemed supportable on the agar-matrix, viable and recoverable. Using Vitrocell VC 10 exposure system and the Ames 4 exposure module, L5178Y cells were successfully exposed to a dynamic cigarette smoke aerosol, recovered and assessed for mutant frequencies, using standard assay procedures. Method development included assessment of flowing air conditions, plating efficiency and recovery of L5178Y cells from the agar-matrix surface. Positive controls MMS and B[a]P were successfully incorporated into the agar-matrix and metabolic activation was achieved by S-9 incorporation into the same agar-base-matrix. B[a]P demonstrated metabolic activation and positive response, suggesting a clear cellular interaction with the agar-matrix. Whole smoke exposed cells in the presence of metabolic activation showed a clear dose response and increasing mutant frequencies, well in excess of the controls (air and incubator) and the global evaluation factor following a 2 or 3 day expression period. This experimental concept demonstrates that L5178Y cells can be exposed to cigarette smoke aerosol, using a completely novel and a previously untested approach. Although this work successfully demonstrates the approach is viable and cells can be plated and maintained on an agar-matrix, more optimisation and robustness assessment is required before it can be considered fully adapted and used alongside other whole aerosol methodologies for the assessment of cigarette smoke and other inhaled aerosols.

这项工作研究了一种全新的实验概念，将空气琼脂界面处的 L5178Y 细胞暴露于主流香烟烟雾气溶胶（肯塔基州参考文献 3R4F）。这项研究强调了将悬浮细胞系与体外细胞系相结合的相关挑战。气溶胶暴露系统。为了实现单层，将细胞在浓缩的细胞超级混合悬浮液中“接种”到 RPMI/琼脂基质基质上。将所得细胞悬浮培养基吸附到琼脂基质中，使 L5178Y 细胞轻轻悬浮在琼脂表面，接近单层。细胞被认为在琼脂基质上是可支持的、有活力的和可回收的。使用 Vitrocell VC 10 暴露系统和 Ames 4 暴露模块，L5178Y 细胞成功暴露于动态香烟烟雾气溶胶，使用标准检测程序恢复和评估突变频率。方法开发包括评估流动空气条件、接种效率和从琼脂基质表面回收 L5178Y 细胞。阳性对照 MMS 和 B[a]P 成功掺入琼脂基质中，代谢活化通过将 S-9 掺入相同的琼脂基质中实现。B[a]P 表现出代谢激活和阳性反应，表明与琼脂基质有明显的细胞相互作用。在代谢激活的情况下，暴露于全烟的细胞表现出明显的剂量反应和增加的突变频率，远远超过对照（空气和培养箱）和 2 或 3 天表达期后的整体评估因素。该实验概念表明，L5178Y 细胞可以使用一种全新且以前未经测试的方法暴露于香烟烟雾气溶胶中。尽管这项工作成功地证明了该方法是可行的，并且细胞可以在琼脂基质上接种和维持，