3.7 Reproduction and verification of SI-ALI by human-derived lung organoid on chip
Acknowledging the distinct differences between cell lines and primary cells, we sought to validate our findings using human-derived lung organoids. Bright-field microscopy illustrated the development and characteristic self-organization of lung organoids (Figure 8A). Immunofluorescence staining confirmed their terminal differentiation into alveoli, identified by SFTPC and E-cadherin biomarkers (Figure 8B), without expression of the airway basal cell marker KRT5 (not shown). Subsequently, we incorporated lung-organoid-derived epithelial cells into the lung chip system. Immunofluorescence images revealed the morphological features of the epithelium on the chip (Figure 8C). These cells retained the expression of alveolar-specific markers (SFTPC and E-cadherin), resembling normal lung tissue to some extent.
We further validated the SI-ALI-related genes identified in the previous experiment. The mRNA expression levels of TST, NIT1, and RAP1GDS1 were reduced in both lung-organoid-derived epithelial cells and endothelial cells in the smoke-exposed group (Figures 8E-b, d, e). The mRNA expression of KRR1 increased in epithelial cells but decreased in endothelial cells in the smoke-exposed group (Figure 8E-b). The expression of COMT and DOCK2 tended to increase in both lung-organoid-derived epithelial cells and endothelial cells in the smoke-exposed group (Figures 8E-a, f), consistent with the proteomic findings from the chip model.
Upon administering the drug combination, qPCR analysis revealed varying degrees of reduction in the expression of inflammation-related genes (IL-1β, TNF-α, NF-κB, IL-6, and IL-10) across different cell components (Figures 8E-a, b, c, d, f). These results were consistent with findings from the cell line chip model. However, the expression of the apoptosis-related gene caspase-3 showed no significant change in the treated group of lung-organoid-derived epithelial cells (Figure 8E-e).