Abstract
Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation. The method builds upon earlier cerebral organoid methodology, with modifications of embryoid body size and shape to increase surface area and slice culture to maintain nutrient and oxygen access to the interior regions of the organoid, enabling long-term culture. We also describe approaches for introducing exogenous plasmid constructs and for sparse cell labeling to image neuronal axon outgrowth and maturation over time. Together, these methods allow for modeling of later events in cortical development, which are important for neurodevelopmental disease modeling. The protocols described can be easily performed by an experimenter with stem cell culture experience and take 2–3 months to complete, with long-term maturation occurring over several months.
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All data generated or analyzed during the development of this protocol are included in the figures.
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Acknowledgements
The authors thank members of the Lancaster lab for helpful discussions, especially I. Kelava, as well as Z. Neuburger for photos of the ALI-CO procedure. We also thank the LMB light microscopy facility for assistance with imaging. Work in the Lancaster lab is supported by the Medical Research Council (MC_UP_1201/9) and the European Research Council (ERC STG 757710).
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M.A.L. is an inventor on two cerebral organoid patents with licensing agreements with third parties, including STEMCELL Technologies.
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Key references using this protocol
Lancaster, M. A. & Knoblich, J. A. Nat. Protoc. 9, 2329–2340 (2014): https://doi.org/10.1038/nprot.2014.158
Lancaster, M. A. et al. Nat. Biotechnol. 35, 659–666 (2017): https://doi.org/10.1038/nbt.3906
Giandomenico, S. L. et al. Nat. Neurosci. 22, 669–679 (2019): https://doi.org/10.1038/s41593-019-0350-2
This protocol is an extension to Nat. Protoc. 9, 2329–2340 (2014): https://doi.org/10.1038/nprot.2014.158
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Giandomenico, S.L., Sutcliffe, M. & Lancaster, M.A. Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development. Nat Protoc 16, 579–602 (2021). https://doi.org/10.1038/s41596-020-00433-w
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DOI: https://doi.org/10.1038/s41596-020-00433-w
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