Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts

Abstract

Organotypic 3D cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model.

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product

“…Thinning of the nasal epithelium was observed in H&E-stained sections of cultures exposed to 3R4F smoke (at a nicotine concentration of 0.25 mg/l (equivalent to 14% 3R4F smoke dilution). This was consistent with our previous findings, where we observed that 15% 3R4F smoke dilution resulted in thinning of the human 3-D bronchial epithelium (Iskandar et al, 2015). The current study further showed an increase of cytotoxicity by 30-40% in 3R4F (0.25)-exposed cultures rela-Although the impact of 3R4F (0.15) did not cause overt morphological changes in the cultures (as assessed by the histology analysis and AK assay), the systems level (omics) analyses demonstrate global mRNA (and miRNA) alterations relative to the air controls.…”

“…While, in 3R4F (0.15)-exposed cultures, where the epithelium remained intact (no thinning), a 12% increase in CYP1A1/CYP1B1 activity was detected in the 3R4F (0.15) group at 48 and 72 h post-exposure, the activity of CYP1A1/ CYP1B1 in the 3R4F (0.25)-exposed cultures was comparable to the air controls at these time points. This finding is consistent with our previous observations in human 3-D bronchial culture models (Iskandar et al, 2015). In contrast, the levels of CYP1A1/CYP1B1 activity in THS2.2-exposed cultures (at all concentrations tested) were nearly comparable to the air control.…”

“…These observations suggested that the cultures could eventually cope with the exposure-induced impact over time (or were capable of initiating a repair mechanism). This fading pattern of the biological impact over post-exposure time is in agreement with our previous observation where an exposure of 8% 3R4F smoke (containing a nicotine concentration of approximately 0.15 mg/l) was linked to a declining biological impact on in vitro human 3-D bronchial epithelial cultures (Iskandar et al, 2015). Furthermore, the current study shows that THS2.2 aerosol exposure (all concentrations tested) was also associated with a declining impact on the network perturbation/biological pathways.…”

“…systems toxicological assessment approach using a human in vitro 3-D nasal model will further support studies that enhance insights into the molecular biology mechanisms associated with the impact of exposure to CS and candidate modified risk tobacco products on the nasopharyngeal cavity of the human respiratory system. imately 0.15 mg/l) (Iskandar et al, 2015). Differently, upon a higher concentration of 3R4F (at a nicotine concentration 0.25 mg/l), which was associated with pronounced epithelium thinning and cytotoxicity, the secretion TIMP1 and VEGFA did not increase over the post-exposure time.…”

Optimization of a NovelIn SituHybridization Technology on 3D Organotypic Cell Cultures

“…[17][18][19][20][21] Herein, we illustrate an optimized protocol for applying the RNAscope ISH technology applied to human organotypic cultures (nasal, small airway, and gingival cultures), previously used for toxicological studies. [22][23][24][25][26][27][28][29][30][31][32] In these studies, gene expression alterations specific for each cell type were not assessed. In this regard, RNAscope technology would allow visualization of specific target genes in a single cell within the 3D context.…”