01887nas a2200193   4500000000100000000000100001008004100002260001500043100001900058700001900077700002500096700002400121700001600145245012400161856011500285490000700400520127200407022001401679       2025                            d  c2025-12-011 aBettina Budeus1 aChiara Kroepel1 aZehra Fatma Sevindik1 aLuca Fabian Buttler1 aDiana Klein00aUpscaling: efficient generation of human lung organoids from induced pluripotent stem cells using a stirring bioreactor  uhttps://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2025.1684315/full0 v133 aIntroductionHuman induced pluripotent stem cells (iPSCs) can be successfully differentiated into complex (three-dimensional) lung spheroids or organoids and have thus proven to be promising in vitro tools that provide a robust system for simulating lung disease and modeling drug response. We previously described a very simple and practical protocol for producing iPSC-derived lung organoids (iPSC-LuOrgs) in ultra-low attachment plates without relying on a gel-like extracellular matrix.MethodsHere, we produced these organoids in a stirred-tank bioreactor equipped with a unique membrane stirrer and compared this type of up-scaled and more automated cultivation with the manual variant. Detailed morphological and molecular analyses, including single-cell RNA sequencing, of the differently generated LuOrgs were performed.ResultsJust like in the manual variant, using the bioreactor morphologically comparable lung organoids could be obtained that showed a very similar cellular composition. This type of generation can also be considered as animal-component-free production.DiscussionThese freely floating LuOrgs are now available in large numbers for the investigation of, for example, cancer therapy approaches as a new and patient-oriented in vitro platform.  a2296-4185