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Background <p>Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II-associated features, enable cryopreservation, and capture disease-associated metabolic alterations.</p> Methods <p>HT-II-280<sup>+</sup> AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. Metabolic profiling was performed using extracellular flux assays.</p> Results <p>AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (&gt; 90% in P1), especially in AT-II cells from fibrotic explants. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production.</p> Conclusion <p>We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.</p>

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Optimized culture of primary human alveolar type II cell–derived 3D organoids from fibrotic lung tissue with phenotypic and metabolic profiling

  • 期刊论文

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  • 发表日期：

    2026年3月7日

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  • Lara-Jasmin Schröder,
  • Julia Rückoldt,
  • Stephanie Schubert,
  • Lars Knudsen,
  • Sabina-Marija Janciauskiene,
  • Christopher Werlein,
  • Mareike Knoll,
  • Regina Engelhardt,
  • Christina Petzold-Mügge,
  • Jonas C. Schupp,
  • Marius M. Hoeper,
  • Jens Gottlieb,
  • Fabio Ius,
  • Patrick Zardo,
  • Marc Lindenberg,
  • Christian Riehle,
  • Lavinia Neubert,
  • Jan C. Kamp

摘要

Background

Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II-associated features, enable cryopreservation, and capture disease-associated metabolic alterations.

Methods

HT-II-280+ AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. Metabolic profiling was performed using extracellular flux assays.

Results

AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production.

Conclusion

We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.