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Three-dimensional human neural culture on a chip recapitulating neuroinflammation and neurodegeneration

Nature Protocols volume 18, pages 2838–2867 (2023)Cite this article

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Abstract

Neuroinflammation has either beneficial or detrimental effects, depending on risk factors and neuron–glia interactions in neurological disorders. However, studying neuroinflammation has been challenging due to the complexity of cell–cell interactions and lack of physio-pathologically relevant neuroinflammatory models. Here, we describe our three-dimensional microfluidic multicellular human neural culture model, referred to as a ‘brain-on-a-chip’ (BoC). This elucidates neuron–glia interactions in a controlled manner and recapitulates pathological signatures of the major neurological disorders: dementia, brain tumor and brain edema. This platform includes a chemotaxis module offering a week-long, stable chemo-gradient compared with the few hours in other chemotaxis models. Additionally, compared with conventional brain models cultured with mixed phenotypes of microglia, our BoC can separate the disease-associated microglia out of heterogeneous population and allow selective neuro–glial engagement in three dimensions. This provides benefits of interpreting the neuro–glia interactions while revealing that the prominent activation of innate immune cells is the risk factor leading to synaptic impairment and neuronal loss, validated in our BoC models of disorders. This protocol describes how to fabricate and implement our human BoC, manipulate in real time and perform end-point analyses. It takes 2 d to set up the device and cell preparations, 1–9 weeks to develop brain models under disease conditions and 2–3 d to carry out analyses. This protocol requires at least 1 month training for researchers with basic molecular biology techniques. Taken together, our human BoCs serve as reliable and valuable platforms to investigate pathological mechanisms involving neuroinflammation and to assess therapeutic strategies modulating neuroinflammation in neurological disorders.

Key points

  • This protocol describes the fabrication of a microfluidic device and its implementation in three-dimensional human neural cultures, allowing the study of neuron–glia interactions in neuroinflammation and neurodegeneration.

  • Compared with conventional animal models, this system enables precise reconstitution of key physiological features of neuroinflammation found in patient tissues. In addition, compared with conventional well plates, this model achieves the separation of reactive microglia instead of heterogeneous populations.

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Fig. 1: Workflows to construct 3D human BoC modeling physio-pathological CNSs.
Fig. 2: The fabrication procedure of devices for 3D human BoC.
Fig. 3: Schematics of creating a physio-pathological 3D human BoC.
Fig. 4: Summary of on-chip analyses with either live or fixed cells, and off-chip analyses with conditioned media.
Fig. 5: The procedure of microglial chemotaxis.
Fig. 6: The procedure of on-chip immunostaining.

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Data availability

Datasets for this protocol are based on the published research articles3,8,12,14,19,21,22. Other data related to this paper can be requested from the authors.

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Acknowledgements

This work was supported by the National Research Foundation (NRF-2020R1A2C2010285, NRF-I21SS7606036), the Ministry of Health & Welfare and Ministry of Science and ICT (HU22C0115) through the Korea Health Industry Development Institute and Korea Dementia Research Center, Collabo R&D between Industry, Academy and Research Institutes funded by the Korea Ministry of SMEs and Startups in 2022 (no. S3247358) to H.C. and NRF-2022R1I1A1A01063094 to Y.J.K.

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Authors and Affiliations

  1. Institute of Quantum Biophysics, Sungkyunkwan University, Suwon, Republic of Korea

    You Jung Kang, Yen N. Diep, Minh Tran, Van Thi Ai Tran, Huyen Ngo & Hansang Cho

  2. Department of Biophysics, Sungkyunkwan University, Suwon, Republic of Korea

    You Jung Kang, Yen N. Diep, Minh Tran, Van Thi Ai Tran, Huyen Ngo & Hansang Cho

  3. Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University, Suwon, Republic of Korea

    Yen N. Diep, Minh Tran, Huyen Ngo & Hansang Cho

  4. Department of Psychiatry, School of Medicine, University of California San Diego, San Diego, CA, USA

    Ghuncha Ambrin

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Contributions

H.C. supervised the whole project. Y.J.K. mainly performed the experiments and wrote the manuscript. Y.N.D., M.T., V.T.A.T., G.A. and H.N. contributed to performing the experiments and writing the manuscript.

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Correspondence to Hansang Cho.

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Key references using this protocol

Cho, H. et al. Sci. Rep. 3, 1823 (2013): https://doi.org/10.1038/srep01823

Park, J. et al. Nat. Neurosci. 21, 941–951 (2018): https://doi.org/10.1038/s41593-018-0175-4

McQuade, A. et al. Nat. Commun. 11, 5370 (2020): https://doi.org/10.1038/s41467-020-19227-5

Chun, H. et al. Nat. Neurosci. 23, 1555–1566 (2020): https://doi.org/10.1038/s41593-020-00735-y

Kang, Y. J. et al. Adv. Sci. 8, 2101251 (2021): https://doi.org/10.1002/advs.202101251

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Kang, Y.J., Diep, Y.N., Tran, M. et al. Three-dimensional human neural culture on a chip recapitulating neuroinflammation and neurodegeneration. Nat Protoc 18, 2838–2867 (2023). https://doi.org/10.1038/s41596-023-00861-4

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