Attachment culture of cortical organoids at the microwell air-liquid interface

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Summary

The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.

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A method of air-liquid interface attachment culture for cortical organoids
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A stable fluid environment and effortless optical observation
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Same-region calcium imaging and immunohistochemical (IHC) cross-analysis

Abstract

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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Most cited references 8

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Cerebral organoids model human brain development and microcephaly

Madeline Lancaster, Magdalena Renner, Carol-Anne Martin … (2013)

The complexity of the human brain has made it difficult to study many brain disorders in model organisms, and highlights the need for an in vitro model of human brain development. We have developed a human pluripotent stem cell-derived 3D organoid culture system, termed cerebral organoid, which develops various discrete though interdependent brain regions. These include cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNAi and patient-specific iPS cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could explain the disease phenotype. Our data demonstrate that 3D organoids can recapitulate development and disease of even this most complex human tissue.

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Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output

Stefano Giandomenico, Susanna B. Mierau, George Gibbons … (2019)

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 extracortical projecting tracts can innervate mouse spinal cord 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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Generation of human brain region-specific organoids using a miniaturized spinning bioreactor.

Xuyu Qian, Fadi Jacob, Mingxi Song … (2018)

Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14-84 d to complete (depending on the type of brain region-specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

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Author and article information

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Shaohua Ma

Journal

Journal ID (nlm-ta): STAR Protoc

Journal ID (iso-abbrev): STAR Protoc

Title: STAR Protocols

Publisher: Elsevier

ISSN (Electronic): 2666-1667

Publication date PMC-release: 12 September 2023

Publication date Collection: 15 September 2023

Publication date (Electronic): 12 September 2023

Volume: 4

Issue: 3

Electronic Location Identifier: 102502

Affiliations

[1 ]Tsinghua Shenzhen International Graduate School (SIGS), Tsinghua University, Shenzhen 518055, China

[2 ]Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen 518055, China

Author notes

[∗ ]Corresponding author ma.shaohua@ 123456sz.tsinghua.edu.cn

[3]

These authors contributed equally

[4]

Technical contact: tjy21@mails.tsinghua.edu.cn, zhenghh20@mails.tsinghua.edu.cn

[5]

Lead contact

Article

Publisher Item ID: S2666-1667(23)00469-0 Publisher ID: 102502

DOI: 10.1016/j.xpro.2023.102502

PMC ID: 10502426

PubMed ID: 37715950

SO-VID: 5f41d049-bdca-4fc6-b551-3980971f2c2b

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Cortical brain organoid slices (cBOS) for the study of human neural cells in minimal networks
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Attachment culture of cortical organoids at the microwell air-liquid interface

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Karger: Neurology and Neuroscience

Most cited references 8

Cerebral organoids model human brain development and microcephaly

Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output

Generation of human brain region-specific organoids using a miniaturized spinning bioreactor.

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