A refined protocol for the large-scale production of high-quality cerebral organoids.

Human pluripotent stem cell-derived cerebral organoids have emerged as a valuable tool for investigating early brain development and modeling neurodevelopmental disorders. However, considerable variation in both organoid size and cellular composition has been widely reported. Moreover, the large-scale production of organoids using conventional methods is a significant challenge, particularly considering that some stem cell lines exhibit variable efficiency in neuroepithelial formation. This heterogeneity of cerebral organoids can complicate experimental reproducibility and comparability in studies of brain development and disease mechanisms. Therefore, we developed a protocol using CERO 3D bioreactors, which enables scalable organoid production and improves size homogeneity compared to conventional approaches. In addition, we conducted a pilot study, utilizing MS-based proteomic analysis of single organoids from two different lines, aiming to explore whether size variability is associated with differences in protein expression within cerebral organoids. The organoids produced in the CERO 3D bioreactors reproducibly form neuroepithelial structures and express key neurodevelopmental markers. The presented protocol provides an efficient way of producing cerebral organoids, supporting reproducibility and high-throughput applications in neurodevelopmental research.