By Pooja Toshniwal PahariaReviewed by Frances BriggsDec 10 2025Using a gently stirred bioreactor, scientists can now grow lung mini-organs by the thousands, producing lung organoids at scales previously unattainable.
Study: Upscaling: efficient generation of human lung organoids from induced pluripotent stem cells using a stirring bioreactor. Image Credit: ClareM/Shutterstock.com
Scientists developed this scalable method for growing human lung organoids from induced pluripotent stem cells (iPSCs) using a membrane-stirred bioreactor.
Reported in Frontiers in Bioengineering and Biotechnology, the approach increases output while maintaining the structural and molecular features needed to model respiratory disease and test new treatments.
Lung diseases continue to place a major burden on health systems worldwide. The COVID-19 pandemic further exposed the limits of animal models and traditional cell cultures, increasing the demand for human-relevant models that reflect the complexity of the lung’s branching airways and delicate alveoli.
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Generating Embryoid Bodies En Masse
The team generated embryoid bodies (EBs) from two human iPSC lines and directed early lung differentiation using branching lung organoid (BLO) medium.
To scale production, they transferred pre-differentiated EBs into a 2-litre stirred bioreactor fitted with a hollow-fiber membrane stirrer. This design enables bubble-free, low-shear aeration and controlled mixing at 80 rpm, improving oxygen and nutrient delivery without damaging fragile structures.
The bioreactor maintained tightly regulated conditions: dissolved oxygen typically 90 to 95 % (with a minimum 50 %), 5 % CO2, pH 7.2-7.4, and osmolality 310–350 mOsm/kg. The media was partially refreshed weekly.
A staged oxygen protocol, 5 % to mimic fetal development, then 10 % to reflect perinatal conditions, helped prevent hypoxic cores and supported epithelial maturation.
Bioreactor cultures ran for 28 days (35 days from iPSC seeding). In parallel, organoids were grown manually using ultralow-attachment plates to serve as a benchmark.
A Thousand Uniform Organoids
The system produced thousands of uniformly sized, viable lung organoids. Microscopy confirmed the presence of well-defined airways- and alveolar-like compartments, with minimal necrosis due to improved oxygenation.
Compared with manual cultures, bioreactor-grown organoids were structurally similar. However, they showed subtle differences in cell composition, with a higher proportion of basal and fibroblast populations in the bioreactor and slightly more alveolar cells in the manual samples.
Single-cell RNA sequencing identified a diverse set of epithelial and mesenchymal subtypes, including basal, secretory, goblet, multiciliated and early alveolar cells.
Classical markers such as EPCAM, ECAD, PDGFRB, and COL1A1 were expressed consistently across conditions, mirroring early human lung development.
The authors note that fresh, unfixed bioreactor organoids appeared to contain fewer alveolar vesicles, likely reflecting shear sensitivity that can be mitigated through further optimisation.
Despite this, overall reproducibility and yield were higher than in manual workflows, and the method avoids animal-derived components entirely.
Conclusions
The membrane-stirred bioreactor offers an efficient, scalable, and animal-material-free platform for producing human lung organoids with high structural and molecular fidelity. The organoids provide an improved way to study diseases such as COVID-19 and lung cancer, as well as for testing therapeutic compounds in patient-relevant models.
Moving forward, researchers plan to streamline iPSC differentiation, optimize oxygen and mechanical conditions, and integrate additional cell types.
These efforts aim to enhance physiological accuracy and support next-generation organoid systems for advanced therapeutic testing and regenerative medicine applications.
Journal reference
Budeus, B., Kroepel, C., Sevindik, Z. F., Buttler, L. F., & Klein, D. (2025). Upscaling: Efficient generation of human lung organoids from induced pluripotent stem cells using a stirring bioreactor. Frontiers in Bioengineering and Biotechnology, 13, 1684315. DOI: 10.3389/fbioe.2025.16