Background <p>Organoids are three-dimensional (3D) in vitro cell culture systems that grow and self-organize, with cells differentiating into functional types and forming miniature structures that replicate the architecture and function of in vivo organs. While the need for standardized organoid culture systems has been widely emphasized, the production of functionally complex and scalable lung organoids remains a significant challenge.</p> Methods <p>Here, we present a scalable lung organoid production platform using a 96-deep-well format with organoids derived from induced pluripotent stem cells (iPSCs). iPSCs were first differentiated into definitive endoderm (DE), followed by anterior foregut endoderm (AFE) in a two-dimensional (2D) culture system. The AFE cells were then transferred into deep wells to generate alveolar epithelial cells (AECs), and subsequently differentiated into SFTPA-positive lung cells within 3D organoids over a 15-day period.</p> Results <p>This new culture platform enables single cells to self-organize into organoids in a serum-free, antioxidant-enriched medium using a 96-deep-well plate. Compared to conventional organoid culture methods, this system significantly improves the consistency, uniformity, and reproducibility of organoid formation.</p> Conclusion <p>Notably, organoids generated in deep wells are functionally comparable to those cultured in Matrigel, but offer enhanced uniformity—an essential improvement for reliable drug screening applications.</p>