Preprint Human Lung Tri-Culture for Acute Injury and Matrix-Response Comparisons

Research Applications

Compare acute pulmonary drug injury and profibrotic pathway responses in a controlled epithelial–fibroblast model.

Model & Design

Human NCI-H441 adenocarcinoma epithelial cells, immortalized CI-huArlo alveolar epithelial cells and MRC-5 fetal lung fibroblasts.

Experimental Format

Three-dimensional basement-membrane domes formed for fourteen days; matched forty-eight-hour TGF-β1 or bleomycin exposure.

Model Components

• Component

  Cell mixture

  Description

  NCI-H441:CI-huArlo:MRC-5 ratio 11:7:4, with shared terminal-airway organoid medium and basement-membrane matrix.

• Component

  Stimuli

  Description

  TGF-β1 at 50 ng/mL or bleomycin at 20 μg/mL for 48 hours after model formation.

• Component

  Molecular comparisons

  Description

  Bulk RNA profiles compared with lung-atlas reference states using the bulk-RNA deconvolution tool CIBERSORTx; ligand–receptor candidates prioritized computationally.

Measurements & Endpoints

• Resazurin metabolic activity
• Secreted IL-8 and procollagen I
• COL1A1/CDKN1A targeted expression
• Differential gene expression and pathway enrichment
• Reference-state resemblance and candidate ligand–receptor scores

Analysis

Assess injury, inflammation and matrix synthesis separately. Bulk RNA analysis adjusts batch and condition; differential genes require FDR < 0.05 and absolute log2 fold change > 1. Reference scores describe similarity rather than cell fractions or conversion.

Published Evidence

• Study Design

  Compare matched tri-cultures after TGF-β1 or bleomycin.

  Study Sample

  Six replicates for reported secreted-protein comparisons; six biological RNA-sequencing replicates per condition.

  Comparison

  Untreated/vehicle controls and two stimulus conditions.

  Finding

  Values below are mean ± standard error of the mean (SEM). Bleomycin lowered metabolic activity and increased IL-8 (126.3 ± 16.86 versus 48.88 ± 4.470 pg/mL, P = 0.0002), without procollagen increase. TGF-β1 increased procollagen (149.6 ± 27.07 versus 53.38 ± 5.672 pg/mL, P < 0.0001), without significant metabolic/IL-8 change.

  Interpretation & Limits

  • The forty-eight-hour single-dose design demonstrates stimulus-specific acute phenotypes, not chronic progressive fibrosis.
  • Senescence, deposited mature matrix and durable post-withdrawal fibrosis were not directly established.
  • Methods specifies 35 μL domes while Results describes 30 μL; no single reconciled dome volume is asserted.
  • Established malignant/immortalized/fetal cell sources lack patient aging/genetic context and immune/endothelial compartments.