Abstract Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.

中文翻译：

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气液界面体外细胞共培养模型的优化以估计气溶胶暴露的危害

摘要 吸入暴露于环境和职业气溶胶污染物与许多呼吸系统健康问题有关。为了真实地模拟长期吸入暴露以进行毒性测试，肺上皮细胞需要在气液界面 (ALI) 条件下维持和暴露很长时间。此外，为了研究细胞对气溶胶颗粒的反应，肺上皮细胞必须与巨噬细胞共培养。为此，我们评估了人支气管上皮 Calu-3、16HBE14o- (16HBE)、H292 和 BEAS-2B 细胞系在长期 ALI 培养条件下的上皮形态、屏障功能和细胞活力。只有 Calu-3 细胞可以保留单层结构，并在至少长达 2 周的长期 ALI 培养下保持牢固的紧密连接。因此，Calu-3 细胞被用作结构屏障，以创建与人单核细胞衍生的巨噬细胞 (MDM) 和 THP-1 衍生的巨噬细胞 (TDM) 的共培养模型。允许巨噬细胞以 5 × 104 巨噬细胞/cm2 的密度粘附在上皮单层上 4 小时。与 Calu-3 单一培养模型相比，Calu-3 + TDM 和 Calu-3 + MDM 共培养模型在共培养的第 1 天对脂多糖 (LPS) 气溶胶的炎症反应敏感性增加， Calu-3 + MDM 模型提供比 Calu-3 + TDM 更强的响应。因此，上皮单层完整性和增加的敏感性使 Calu-3 + MDM 共培养模型成为 ALI 暴露于吸入气溶胶进行毒性测试的首选选项。