Air-Liquid Interface In Vitro Models for Respiratory Toxicology Research: Consensus Workshop and Recommendations

Abstract

In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

Keywords: air–liquid interface; in vitro; inhalation; lung cell models; toxicology; validation.

FIG. 1.

Main ALI exposure setups. (a) “Incubator/box-type” setup featuring a horizontal flow, a “smooth” cell exposure, and often using standard commercial culture plates. It results in a conjoined exposure of cultures with less defined fluid dynamics. (b) “Stagnation point flow” setup featuring individual exposure of single cultures, offering optimized fluid dynamics and an effective cell to gas contact although it usually calls for custom-made constructions and more sophisticated setups. ALI, air–liquid interface.

FIG. 2.

Main methods to improve particle deposition. (a) Electrostatic deposition on ALI cells to enhance particle deposition from aerosols. Aerosol particles are charged unipolar (left) or bipolar (right) and forced onto the cellular surface in an electric field with a constant (unipolar setup) or frequently changed (bipolar setup) polarity. (b) Droplet deposition on ALI cells. Particles are suspended in aerosol droplets to increase the particle size. Aerosol droplets are deposited on the cellular surface by gravitational forces. Depending on experimental conditions, this procedure may be enhanced by cloud movement effects of highly concentrated aerosols. (c) Enhancement of native particle deposition on ALI cells by thermophoresis. A thermal gradient of 15°C–20°C is applied between the cells and the aerosol, leading to a higher velocity of molecules in the aerosol gas at the warmer side. By this thermophoretic effect, aerosol particles are forced to the cellular surfaces of ALI cells.

FIG. 3.

Respiratory models available from simple to complex. Costs and also physiological relevance increase in line with the complexity of models.

FIG. 4.

Initiation of the validation process for the use of ALI systems in the field of respiratory toxicology. EURL ECVAM, European Union Reference Laboratory for alternatives to animal testing.