Investigations of the Biological Effects of Airborne and Inhalable Substances by Cell-Based In Vitro Methods: Fundamental Improvements to the ALI Concept
The state of the art for cell-based in vitro investigations of airborne and inhalable material is “air-liquid interface” (ALI) technology. Cell lines, primary cells, complex 3D models, or precision-cut lung slices (PCLS) are used to represent the lung or skin by way of an in vitro barrier model. These models have been applied in toxicity or pharmacological testing. However, contrasting with a clear demand for alternative methods, there is still no widely accepted procedure for cell-based in vitro testing of inhalable substances. In the light of this, an analysis was undertaken of common drawbacks of current approaches. Hence, the pivotal improvements aimed at were the cellular exposure environment, overall performance and applicability, operability of online investigations during exposure and routine setup. It resulted in an improved device (P.R.I.T. ExpoCube) based on an “all-in-one-plate” concept including all phases of the experiment (cell culture, exposure, and read-out) and all experimental groups (two test gas groups, controls) in one single commercial multiwell plate. Verification of the concept was demonstrated in a first experimental series using reference substances (formaldehyde, ozone, and clean air). The resulting ALI procedure enables the application of inhalable substances and mixtures under highly effective exposure conditions in routine utilization. 1. Introduction There are many different reasons for using cell-based in vitro methods in inhalation toxicology or pharmacology approaches. Some of them belong to the field of mechanistic research (e.g., ADME studies, drug administration via the lung, and “omics” technologies), some of them belong to legislation and regulation (REACH, European chemical legislation), some have economic reasons (time and money, screening approach, and fast results), and some have ethical reasons (animal welfare, “3R” principle). Although toxicological testing of chemicals and pharmacological investigations based on in vitro methods have developed very quickly in recent years and often ended up in standardised and accepted procedures [1, 2], this is not the case for the field of research related to airborne material or inhalable substances. For in vivo testing, too, inhalational toxicological and pharmacological approaches still represent a challenging field owing to additional tasks in comparison with oral administration, such as generation and application of gaseous or airborne test substances and specific animal and study design-related aspects [3], including animal handling, behaviour, dosimetry, and many
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