Precision-cut lung slices as an alternative model for repeated-dose inhalation toxicity

There is a growing need for appropriate alternatives for animal inhalation studies in order to test respiratory adverse effects of inhalable substances. These alternatives should comply with the three R principles. In this regard Precision-cut lung slices (PCLS) are a prevalent used ex vivo alternative reflecting the respiratory tract. However, most studies using PCLS have been conducted within a 72 h time window to investigate acute respiratory toxicity. In order to evaluate the feasibility of long-term PCLS cultivation to test e.g. toxicity of slowly metabolized substances, rat PCLS were cultivated for more than 14 days. Additionally, triple Triton X-100 treated rat PCLS were compared to single and double exposed PCLS in order to investigate their suitability for repetitive exposure studies. Markers for slice vitality were LDH and WST-1 assay. Moreover, physiological alterations were studied using methacholine-induced bronchoconstriction. Constant vitality and bronchoconstriction were observed over the 14 day period with slight decreases towards the end of cultivation. Repeated Triton X-100 exposure had no influence on the sensitivity of PCLS (DeltaEC50_1d/3d = 4 mikroM). Overall these results showed feasibility of long-term cultivation with a good preservation of vitality and physiology. Further it demonstrated that constant vitality remained despite repeated chemical exposure making PCLS a possible future model for slowly metabolized substances and repeated dose testing.