Characterization of tri-culture alveolar-like organoids to study fibrotic lung diseases

Guo, T. J. F.; Meng, Y.; Kim, S.; Machiri, T.; Miscampbell, A.; Lau, K. S. K.; Gibbons, C.; Penton, M. R.; Lee, C. P. S.; Li, I. T. S.; Goobie, G. C.; Halayko, A. J.; Ryerson, C. J.; Carlsten, C.; Ryu, M. H.; Osei, E. T.

doi:10.64898/2026.09.04.749305

bioRxiv · 10.64898/2026.09.04.749305

Characterization of tri-culture alveolar-like organoids to study fibrotic lung diseases

Guo, T. J. F.·Meng, Y.·Kim, S.·Machiri, T.·Miscampbell, A.·Lau, K. S. K.·Gibbons, C.·Penton, M. R.·Lee, C. P. S.·Li, I. T. S.·Goobie, G. C.·Halayko, A. J.·Ryerson, C. J.·Carlsten, C.·Ryu, M. H.·Osei, E. T.

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Abstract

Rationale: Fibrotic interstitial lung diseases are progressive disorders characterized by lung scarring and declining respiratory function. Repeated injury and dysregulated epithelial-mesenchymal crosstalk are implicated in disease pathogenesis but remain incompletely understood. Three-dimensional (3D) organoids incorporating epithelial-mesenchymal crosstalk provide a physiologically relevant platform for investigating these mechanisms. Here, we present a 3D tri-co-culture alveolar organoid model and evaluate its responses to two profibrotic stimuli, TGF-{beta} and bleomycin. Methods: CI-huArlo, NCI-H441, and MRC-5 cells were co-cultured for 14 days to generate alveolar-like organoids. Cell marker expression was assessed by immunofluorescence (IF). To test injury response, organoids were exposed to TGF-{beta} (50 ng/mL) or bleomycin (20 g/mL) for 48 hours and characterized. Supernatants were collected to assess interleukin 8 (IL-8) and procollagen I by ELISA. qPCR assessed expression of CDKN1A and COL1A1 following treatments. Bulk RNA sequencing (RNA-seq) evaluated the transcriptomic responses to fibrotic stimulus. Results: Cellular marker expression was confirmed using IF for aquaporin-5 (alveolar type I cell marker) and TE-7 (fibroblast marker). Bleomycin exposure reduced viability and significantly increased IL-8 (126.3 {+/-} 16.86 vs. 48.88 {+/-} 4.470 pg/mL; p = 0.0002; N=6), with no change in secreted procollagen I compared to control. TGF-{beta} stimulation significantly increased secreted procollagen I (149.6 {+/-} 27.07 vs. 53.38 {+/-} 5.672 pg/mL; p <0.0001; N=6) without affecting viability or IL-8 release. qPCR resulted in no change in CDKN1A expression, while COL1A1 expression was increased with TGF-{beta} treatment compared with control and bleomycin. RNA sequencing demonstrated distinct and reproducible transcriptional responses to TGF-{beta} and bleomycin. TGF-{beta} induced 750 significantly upregulated and 641 downregulated genes, including increased COL1A1, COL4A1, FN1, and TGFB1, with enrichment of epithelial-mesenchymal transition and TGF-{beta} signalling programs. In contrast, bleomycin induced 953 significantly upregulated and 552 downregulated genes relative to untreated controls and was characterized by p53 signalling, DNA-damage responses, and reduced cell-cycle progression. Reference-state analysis further indicated reduced normal alveolar epithelial signatures following both treatments, with TGF-{beta} producing the strongest aberrant basaloid and myofibroblast-associated signatures. Conclusions: Tri-culture alveolar-like organoids exhibited stimulus-specific responses in a 3D multicellular system, supporting their use for mechanistic studies of epithelial-mesenchymal crosstalk, environmental exposures, and therapeutic responses.

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Guo, T. J. F., Meng, Y., Kim, S., Machiri, T., Miscampbell, A., Lau, K. S. K., Gibbons, C., Penton, M. R., Lee, C. P. S., Li, I. T. S., Goobie, G. C., Halayko, A. J., Ryerson, C. J., Carlsten, C., Ryu, M. H., Osei, E. T.. 2026-09-09. Characterization of tri-culture alveolar-like organoids to study fibrotic lung diseases. https://doi.org/10.64898/2026.09.04.749305

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