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Routine culture and study of adult human brain cells from neurosurgical specimens

Nature Protocols volume 17, pages 190–221 (2022)Cite this article

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Abstract

When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens. The cell types that can be cultured include dissociated cultures of neurons, astrocytes, microglia, pericytes and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells. The initial setup of cultures takes ~2 h, and the cells are ready for further experiments within days to weeks. The resulting cells can be studied as purified or mixed population cultures, slice cultures and explant-derived cultures. This protocol therefore enables the investigation of human brain cells to facilitate translation of neuroscience research to the clinic.

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Fig. 1: Flow diagram of the step-by-step procedures in isolating neurons, brain endothelia, NPCs, microglia, mixed glia (astrocytes, microglia and pericytes) and brain tumor cells from post-operative brain specimens.
Fig. 2: Photomicrographs and electrophysiological recordings demonstrating the presence of mature primary adult human neurons in vitro.
Fig. 3: Photomicrographs showing the presence of GFAP-positive astrocytes and their response to inflammatory stimuli.
Fig. 4: Photomicrographs showing purified microglia expressing microglia markers and responding to pro-inflammatory cytokines.
Fig. 5: Photomicrographs showing isolated brain neurovascular cell types, brain endothelial cells (panel a) and brain pericytes (panel b).
Fig. 6: A timeline and representative figures showing the isolation, expansion and differentiation of primary adult human neural progenitor cells in vitro.
Fig. 7: Photomicrographs illustrating the growth and characterization of BTCs.
Fig. 8: Flow diagram of the step-by-step procedure for processing organotypic brain slice and explant cultures from post-operative human brain specimens and the leptomeninges.
Fig. 9: Photomicrographs and electrophysiological recordings demonstrating the presence of viable and functional adult human brain slice cultures.
Fig. 10: Photomicrographs of leptomeningeal explant cultures.

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The data in our protocol figures are composed entirely of photomicrographs and electrophysiological recordings displaying the results anticipated with the use of the protocols. Previous uses of the protocols are available in the literature and clearly cited, providing further examples of the types of data that can be obtained from using this protocol.

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Acknowledgements

We are tremendously grateful to the generous neurosurgical brain tissue donors and their families, without whom this research could not be performed; thank you so much. They are the real drivers of this work, and it is because of their special gifts of brain tissue that we can undertake this work for the benefit of future patients. We also thank our collaborators and research technicians: R. Faull, M. Curtis, J. Montgomery, H. Gibbons, M. Cheung, Y. Jung, S.-L. Kim, I. Semenyajenko and M. Eszes from Auckland University; and C. Turner, P. Bergin, E. Walker, L. Roberts and S. Tharakan from Auckland Hospital and LabPlus. This work was supported by the Hugh Green Foundation (funders of the Hugh Green Biobank), a Programme Grant from the Health Research Council of New Zealand, the Sir Thomas and Lady Duncan Trust, the Coker Charitable Trust, and Brain Research New Zealand to M.D. T.I.-H.P., the Centre for Brain Research Douglas Neurosurgical Postdoctoral Research Fellow, is funded by the Douglas Charitable Trust through a School of Medicine Foundation Grant to M.D. (director of the Hugh Green Biobank).

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Authors and Affiliations

  1. Hugh Green Biobank & Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

    Thomas I.-H. Park, Miranda Aalderink, Zoe R. Woolf, Amy Smith, Sheryl Feng & Mike Dragunow

  2. Neurosurgical Research Unit, Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

    Thomas I.-H. Park, Jason Correia, Peter Heppner, Patrick Schweder, Edward Mee & Mike Dragunow

  3. Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand

    Leon C. D. Smyth

  4. Center for Brain Immunology and Glia (BIG), Washington University, St. Louis, MO, USA

    Justin Rustenhoven

  5. Department of Physiology, Faculty of Medical Science and Health Sciences, The University of Auckland, Auckland, New Zealand

    Kevin Lee

  6. Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine VISN 20 Mental Illness Research, Education and Clinical Centre (MIRECC), VA Puget Sound Health Care System, Seattle, WA, USA

    Deidre Jansson

  7. Department of Neurosurgery, Auckland City Hospital, Auckland, New Zealand

    Jason Correia, Peter Heppner, Patrick Schweder & Edward Mee

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  1. Thomas I.-H. Park
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Contributions

T.I.-H.P. conceived, optimized and established the neuronal, neural progenitor, microglia, pericyte and endothelial cultures; optimized the tissue-collection process and wrote most of the manuscript. L.C.D.S. conceived, optimized and established the pericyte and endothelial cultures and wrote parts of the manuscript. M.A. conceived, optimized and established the pericyte and mixed glial cultures and wrote parts of the manuscript. Z.R.W. optimized the microglial cultures and wrote parts of the manuscript. J.R. conceived, optimized and established the pericyte and microglial cultures and wrote parts of the manuscript. K.L. conceived, optimized and established the neuronal and brain slice cultures and wrote parts of the manuscript. D.J. conceived, optimized and established the mixed glial and explant cultures. A.S. optimized the mixed glial cultures and inflammatory experiments. S.F. conceived, optimized and established the mixed glial and explant cultures. J.C., P.H. and P.S. are neurosurgeons and were part of the clinical research team; they aided in design and coordination of experiments, consented and provided tissue and optimized the biopsy tissue-collection process. E.M.is a neurosurgeon and was part of the clinical research team. He conceived these projects with M.D. and consented and provided biopsy tissue. M.D.was the study leader and Principal Investigator. He conceived, led and funded the entire program and supervised T.I.-H.P., L.C.D.S., M.A., Z.R.W., J.R., K.L., D.J., A.S. and S.F. in establishing the Hugh Green Biobank.

Corresponding author

Correspondence to Mike Dragunow.

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Competing interests

M.D. has developed a platform called ‘Neurovalida’ for CNS drug discovery and target validation.

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Nature Protocols thanks Henner Koch, Debomoy K. Lahiri, Jonas Ort and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Park, T. I.-H. et al. Brain Commun. 2, fcaa171 (2020): https://doi.org/10.1093/braincomms/fcaa171

Rustenhoven, J. et al. Sci. Rep. 6, 19371 (2016): https://doi.org/10.1038/srep19371

Jansson, D. et al. Commun. Biol. 4, 260 (2021): https://doi.org/10.1038/s42003-021-01787-x

Supplementary information

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Supplementary Figs. 1 and 2, Supplementary Tables 1–3 and Supplementary Methods.

Supplementary Data 1

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Park, T.IH., Smyth, L.C.D., Aalderink, M. et al. Routine culture and study of adult human brain cells from neurosurgical specimens. Nat Protoc 17, 190–221 (2022). https://doi.org/10.1038/s41596-021-00637-8

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