Inhalation is an important route of exposure for chemical compounds as this is the route by which airborne compounds enter the human body.  These compounds come into contact with the pulmonary system through inhalation, potentially causing local effects in the lungs or systemic effects elsewhere in the body. To determine the effects of chemical compounds on the human respiratory tract, RIVM is using a model based on cells that can be exposed to airborne compounds, simulating conditions in human lungs.

Short video about RIVM's activities researching in vitro methods for inhaled substances


Lung toxicity testing in vitro
Speaker: Dr. Yvonne Staal, inhalation toxicologist

DR. YVONNE STAAL: Every day, we inhale airborne compounds. Such as nanoparticles, compounds from spraying cosmetics or compounds from smoking cigarettes. When these compounds are inhaled they may cause local effects in the airways or they may be absorbed into our bodies. To asses these effects, we use cell models of the airways cultured at the barrier between air and liquid. That allows the cells to be exposed to compounds via the air in a human relevant way. In addition, the cells have a human origin. Which means that they can be used for human mechanistic studies. In the future, finding a place in future risk assessment. Airway epithelial cells can be used to study the effect of inhaled compounds. There are two important aspects to consider. The first one is the cell model. Cells should be cultured at the air-liquid interface or ALI. This allows the cells to be exposed to compounds via the air. Which is a human relevant way of exposure. We're using two types of cells. Which are the epithelial lung cell layer and macrophages, which is an important immune cell in the airways for particle exposure. The second aspect is the exposure method. To determine the effects of airborne compounds, compounds need to be brought into the air. The dose to which the cells are exposed is dependent on the concentration of the exposure the duration of the exposure and the frequency. For particles, they may not always reach the cells. It's also dependent on the deposition of the particles on the cells. As a first site of contact macrophages and epithelial cells are the first to respond to a exposure with an inflammatory response or effects on cell barrier or cell viability. Inflammation is a key parameter in many different diseases, such as lung fibrosis. ALI-cultured cell models can therefore find a place in mechanism-based toxicity while avoiding animal studies as much as possible. We aim to further improve the relevance and standardization of our airway models and their exposure to contribute to animal replacement with realistic, reliable and human relevant in vitro data.

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Cell-based test systems

Every day we inhale particles, such as nanoparticles, air pollution and sensitising compounds from, for example, cosmetic products or cigarette smoke. These compounds can cause local effects in the lungs, but can also be absorbed into the blood and cause harmful effects elsewhere in the body (systemic effects).
The harmfulness of chemical compounds can be investigated by assessing the effects of these compounds on human cells. Immune system cells can also be added to the cell culture; immune cells play an important role in the immune response to airborne particles.

Air-liquid interface

These (combinations of) cells are cultured with air at the top and liquid with nutrients at the bottom. In a so-called 'air-liquid interface' (ALI) culture, the cells can be exposed to airborne particles via an airflow over the cells. As such, the ALI-cultured cells simulate human lungs. The harmfulness of chemical compounds is determined by the amount of compound that reaches the cell, the duration of the exposure and how often the cells are exposed to the airborne compounds.

Realistic exposure methods

This combination of human cells with realistic exposure methods can provide insight into the mechanism through which a compound can be harmful to human lungs. In the future, we may be able to use this information to predict whether a compound can cause lung diseases.


Key publications

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function Tulen, C. B. M., Duistermaat, E., Cremers, J. W. J. M., Klerx, W. N. M., Fokkens, P. H. B., et al. Cells 2022; 11(21):3481
Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method Staal, Y. C. M., Li, Y., Gerber, L.-S., Fokkens, P., Cremers, H., Cassee, F. R., et al. Inhalation Toxicology 2022:1-10
Optimization of an air-liquid interface in vitro cell co-culture model to estimate the hazard of aerosol exposures He, R. W., Braakhuis, H. M., Vandebriel, R. J., Staal, Y. C. M., Gremmer, E. R., Fokkens, et al. Journal of aerosol science 2021; 153:105703
An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing Braakhuis, H. M., He, R., Vandebriel, R. J., Gremmer, E. R., Zwart, E., Vermeulen, J. P., et al.  Journal of visualized experiments : JoVE 2020; 

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