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).