A refined protocol for the large-scale production of high-quality cerebral organoids
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Date
2026-07-03
Authors
Prokhorova, Tatyana
Hohmann, Sonja Simone
Fabozzi, Alessia
Saltarelli, Silvia
Akimov, Vyacheslav
Yde Ohki, Cristine Marie
McNeill, Rhiannon V.
Tvedesøe, Christian
Blagoev, Blagoy
Kittel-Schneider, Sarah
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Abstract
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.
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© 2026, the Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material.
Keywords
Cerebral organoids , iPSCs , Large-scale culture , Neuroepithelial formation , Organoid heterogeneity , Proteomics , [APCMicrobiome] , [Medicine] , Neurology , Neurology (clinical) , Psychiatry and Mental Health , Biological Psychiatry
Citation
Prokhorova, T, Hohmann, S S, Fabozzi, A, Saltarelli, S, Akimov, V, Yde Ohki, C M, McNeill, R V, Tvedesøe, C, Blagoev, B, Kittel-Schneider, S, Schneider-Kamp, P, Smigielski, L, Qin, J, Debrabant, B, Grünblatt, E & Michel, T M 2026, 'A refined protocol for the large-scale production of high-quality cerebral organoids', Journal of Neural Transmission, pp. 1--13. https://doi.org/10.1007/s00702-026-03216-x
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