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Blood–brain-barrier organoids for investigating the permeability of CNS therapeutics

Nature Protocols volume 13, pages 2827–2843 (2018)Cite this article

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Abstract

In vitro models of the blood–brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. ‘BBB organoids’ are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintain BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2–3 d. An additional day is required to analyze drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuropathologies.

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Fig. 1: Characterization of each cell type used for establishing BBB organoids.
Fig. 2: Culture, collection and assessment of BBB organoids.
Fig. 3: Setup and analysis of BKM120 and dabrafenib permeability by MALDI–MSI.
Fig. 4: Quantification of average fluorescence intensity in a BBB organoid.
Fig. 5: Analysis by confocal microscopy of angiopep-2 permeability using the BBB organoid platform.

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Acknowledgements

C.-F.C. was supported by the Canadian Institute of Health Research Post-Doctoral Fellowship and is currently supported by a Brigham and Women’s Hospital Women’s Brain Initiative/Connors Center Pilot Grant as well as the Sperling Family Fellowship. N.Y.R.A. is supported by National Institutes of Health/National Cancer Institute (NIH/NCI) grants R01CA201469 and U54CA210180. B.L.P. is supported by the Sontag Distinguished Scientist Award. S.E.L. was supported by a B*CURED Research Grant and a Brigham and Women’s Hospital Women’s Innovation Award. We thank H. Bridger and H. Schulze from the Brigham and Women’s Research Institute for providing assistance with photography.

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Authors and Affiliations

  1. Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Sonja Bergmann, Sean E. Lawler, Yuan Qu, Michael S. Regan, Nathalie Y. R. Agar & Choi-Fong Cho

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Colin M. Fadzen, Justin M. Wolfe, Bradley L. Pentelute & Choi-Fong Cho

  3. Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Nathalie Y. R. Agar

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  1. Sonja Bergmann
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Contributions

C.-F.C., N.Y.R.A. and S.E.L. designed the research. C.-F.C. performed the experiments. S.B. and C.-F.C. analyzed the data. S.B., S.E.L., M.S.R. and C.-F.C. wrote the manuscript. C.M.F., J.M.W. and C.-F.C. revised the manuscript. S.B., S.E.L., Y.Q., C.M.F., J.M.W., M.S.R., B.L.P., N.Y.R.A. and C.-F.C. read and approved the final manuscript.

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Correspondence to Choi-Fong Cho.

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Key references using this protocol

Cho, C. F. et al. Nat. Commun. 8, 15623 (2017): https://doi.org/10.1038/ncomms15623

Fadzen, C.M. J. Am. Chem. Soc. 139, 15628–15631 (2017): https://doi.org/10.1021/jacs.7b09790

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Bergmann, S., Lawler, S.E., Qu, Y. et al. Blood–brain-barrier organoids for investigating the permeability of CNS therapeutics. Nat Protoc 13, 2827–2843 (2018). https://doi.org/10.1038/s41596-018-0066-x

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