Posters

      Organotypical epithelial 3D tissues (MatTek®) exposed in vitro to whole smoke as a potential alternative to rodent inhalation studies

      Weisensee, D.; Kurkowsky, B.; Hebestreit, M.; Wagner, S.; Schueller, J.
      Conference date
      Apr 26, 2011
      Conference name

      In Vitro Testing Industrial Platform
      Link to more information
      Topic
      Summary

      The use of reconstituted human airway epithelia exposed in vitro to cigarette smoke (CS) closely mimics the situation in human smokers, where lung epithelial cells are exposed to fresh whole smoke at the air-liquid interface (ALI). It is known that CS induces inflammatory processes that can lead to tissue remodeling (e.g., via increased expression of matrix metalloproteinases, MMP), which may eventually result in a diseased lung phenotype. Here we describe an in vitro test system to investigate the toxicological effects of single and repeated CS exposure on human organotypical 3D epithelial tissues (MatTek®). Tissues grown on culture inserts (12- or 24-well format) were supplied by MatTek® (Ashland, USA) and stored at 4-8°c until use. After one week, tissues received fresh culture medium and were transferred to an incubator. The next day, tissues were exposed at the air liquid interface to fresh air or to mainstream smoke from the reference cigarette 3R4F in the Vitrocell® system. CS concentration was adjusted to 15% by dilution with fresh air and tissues were exposed once for up to 28 min. In a second approach tissues were repeatedly exposed to 20% CS on 3 subsequent days (20 min/day). Cell viability, as determined by resazurin reduction 24h after CS exposure, was greater than 80% under all conditions. Morphological characterization of the reconstituted tissue by H&E staining, staining for proteoglycans (AB-PAS), and staining for epithelial stratification (P63), revealed an intact basal and pseudostratified epithelium with mucociliary phenotype similar to in vivo proximal airway epithelium. Expression of genes or proteins functionally involved in (oxidative) cell stress regulation, i.e., hemoxygenase-1 (HO-1), heat shock 70KDA protein A6 (HSPA6), xenobiotic drug detoxification (cytochrome P450 1A1 (CYP1A1)), and tissue remodeling (transforming growth factor ß1 (TGF ß1, MMP-1)) was investigated. Differential gene expression showed a strong activation of HO-1 (~120-fold), hspa6 (~80-fold), and CYP1A1 (~900-fold), and moderate up-regulation of TGFß1 (~3-fold) in response to CS exposure as determined by QRT-PCR. The release of MMP-1, an enzyme possibly involved in the pathogenesis of COPD, was dose-dependently increased (up to 14-fold). Results for HO-1 and CYP1A1 and MMP-1 are comparable to in vivo data from previous rodent inhalation studies; thus, this in vitro study design may serve as a potential alternative to in vivo inhalation studies for the investigation of the toxicology of aerosols, and supports the 3R strategy of refinement, reduction, and replacement of animal experimentation. 

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