Optimization of an air-liquid interface in vitro cell co-culture model to estimate the hazard of aerosol exposures

October 31, 2020



Rui-Wen He a,b, Hedwig M. Braakhuis a, Rob J. Vandebriel a, Yvonne C.M. Staal a, Eric R. Gremmer a, Paul H.B. Fokkens a, Claudia Kemp a, Jolanda Vermeulen a, Remco H.S. Westerink b, Flemming R. Cassee a,b
a National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720, BA, Bilthoven, the Netherlands
b Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80178, 3508, TD, Utrecht, the Netherlands


The results demonstrate the substantial differences in cellular responses including cell morphology, TEER changes, and cytotoxicity for epithelial mono-culture of 16HBE, Calu-3, H292 and BEAS-2B cells as well as the macrophage/epithelial cell co-culture models under ALI conditions. With the optimization of the co-culture procedure at the ALI, the cell co-culture models showed epithelial monolayer integrity, and increased sensitivity in inflammatory responses to LPS exposure, with the Calu-3 + MDM model giving the strongest responses.


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.

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