Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output
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Abstract
Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.
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ALI culture has been widely employed for developing hiPSC-derived organoids such as cerebral organoids, kidney organoids, and intestinal organoids.
“ALI culture has been widely employed for developing hiPSC-derived organoids, such as cerebral organoids, kidney organoids, and intestinal organoids [15, 26, 27].”
From The empowering influence of air-liquid interface culture on skin organoid hair follicle development (2025) · cites this paper as [15]
The Methods sections on ALI-CO culture and generation of cerebral organoids describe using an air-liquid interface to culture cerebral organoids, supporting that example. However, the human pluripotent stem cell Methods section says the organoids shown were generally generated from H9 human embryonic stem cells, not hiPSCs. This source does not establish widespread use; the other organoid examples have separate citations in the citing sentence. (LLM, conf=0.88)
Experimental — accuracy not yet measuredhiPSC-derived astrocytes can be generated with a fast protocol in 2D layers or integrated in 3D systems such as spheroids and organoids.
“hiPSC-derived astrocytes, which can be generated with a fast protocol in 2D layers104 or integrated in 3D systems such as spheroids and organoids105–108”
From Reactive astrocyte nomenclature, definitions, and future directions (2021) · cites this paper as [108]
The Results section (‘Air-liquid interface culture of cerebral organoids leads to improved survival and maturation’; page unavailable) reports astrocytes in cerebral organoids, supporting the 3D-organoid part of the cited claim. The Methods section (‘Human pluripotent stem cell culture and genome editing’; page unavailable) specifies that the organoids were generated from human embryonic stem cells, not hiPSCs. The fast 2D protocol is attached to a different citation and is not assessed here. (LLM, conf=0.88)
Experimental — accuracy not yet measuredALI has previously been used for culturing other human organoids, such as cerebral organoids, where it improves organoid survival and functional development.
“ALI has previously been used for culturing other human organoids, such as cerebral organoids, where it is demonstrated to not only improve organoid survival but also organoid functional development [15].”
From The empowering influence of air-liquid interface culture on skin organoid hair follicle development (2025) · cites this paper as [15]
Long-range neural projections have been observed in cortico-thalamic assembloids, organotypic mouse co-cultures, and sliced cortical organoids with rodent spinal cord explants.
“Long-range neural projections have been observed in a cortico-thalamic assembloid (Xiang et al., 2019), in organotypic mouse co-cultures (Takuma et al., 2002) and in a preparation that used sliced cortical organoids and rodent spinal cord explants (Giandomenico et al., 2019).”
From Generation of Functional Human 3D Cortico-Motor Assembloids (2020) · cites this paper as Giandomenico et al., 2019
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- Authors
- Giandomenico, S. L., Mierau, S. B., Gibbons, G. M., Wenger, L. M. D., Masullo, L., Sit, T., Sutcliffe, M., Boulanger, J., Tripodi, M., Derivery, E., Paulsen, O., Lakatos, A., & Lancaster, M. A.
- Affiliations
- MRC Laboratory of Molecular Biology (GB)
- University of Cambridge (GB)
- Wellcome/MRC Cambridge Stem Cell Institute (GB)
- Corresponding author
- Lancaster, Madeline A.
- ORCID
- Venue
- Nature Neuroscience
- Published
- 2019-03-18
- Volume
- 22
- Issue
- 4
- Pages
- 669-679
- ISSN
- 1097-6256 (ISSN-L)
- 1546-1726
- PMC
- PMC6436729
- OpenAlex
- W2949410666
- Cited by
- 651
- References
- 35
- Citations by year
- 2026: 76 · 2025: 77 · 2024: 124 · 2023: 82 · 2022: 86 · 2021: 88 · 2020: 85 · 2019: 30 · 2018: 1 · 2017: 1
- FWCI
- 27.11
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc
- www.springer.com/tdm (tdm)
- Locations
- Nature Neuroscience (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Apollo (University of Cambridge) (submittedVersion)
- Apollo
- Funders
- Wellcome Trust
- Cancer Research UK
- Medical Research Council (MC_PC_12009, MC_PC_16036, MC_UP_1201/9, MR/P008658/1)
- Topics
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neural Engineering
- Keywords
- brain organoids, air-liquid interface culture, axon growth, corpus callosum, growth cone turning, retrograde tracing, muscle contraction, brain development, circuit formation
- MeSH terms
- Axons, Cell Survival, Cerebral Cortex, Female, Humans, Male, Neural Pathways, Neurons, Organoids, Pluripotent Stem Cells, Tissue Culture Techniques
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