Differentiation of Human Lung Organoids from Induced Pluripotent Stem Cells Using a Stirred-Tank Bioreactor

As self-organizing, complex 3D cell culture structures that exhibit various organ-specific cell types and organ-like properties, organoids generated from induced pluripotent stem cells (iPSCs) offer an innovative and promising opportunity to serve as a patient- and disease-specific 3D in vitro model. Particularly human lung organoids herein have developed into patient-like in vitro model systems for lung diseases, which can largely be considered a useful alternative to preclinical animal models. We previously established a very practical and robust protocol for producing iPSC-derived lung organoids (iPSC-LuOrgs) in ultra-low attachment plates without relying on extracellular matrices. We have adapted this protocol for upscaling and process automation to allow for the use of a bioreactor to yield lung organoids from differentiated embryonic bodies in large numbers in a stirred-tank bioreactor. The protocol for this methodology is described in detail here. Using bioreactors is becoming increasingly important in lung bioengineering as it provides the basis for high-throughput assays for screening purposes in drug development, gene therapy, and/or organoid transplantation.