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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    DiLuca, M. & Olesen, J. The cost of brain diseases: a burden or a challenge? Neuron 82, 1205–1208 (2014).

    CAS  Article  Google Scholar 

  2. 2.

    Gooch, C. L., Pracht, E. & Borenstein, A. R. The burden of neurological disease in the United States: a summary report and call to action. Ann. Neurol. 81, 479–484 (2017).

    Article  Google Scholar 

  3. 3.

    Abbott, N. J., Rönnbäck, L. & Hansson, E. Astrocyte-endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006).

    CAS  Article  Google Scholar 

  4. 4.

    Banks, W. A. From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery. Nat. Rev. Drug Discov. 15, 275–292 (2016).

    CAS  Article  Google Scholar 

  5. 5.

    Cho, C.-F. The blood–brain barrier: brain cancer therapy hits a wall. Oncol. Times 40, 1 (2018).

    Article  Google Scholar 

  6. 6.

    Cho, C.-F. et al. Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents. Nat. Commun. 8, 15623 (2017).

    CAS  Article  Google Scholar 

  7. 7.

    Urich, E. et al. Multicellular self-assembled spheroidal model of the blood brain barrier. Sci. Rep. 3, 1500 (2013).

    Article  Google Scholar 

  8. 8.

    Wolburg, H. & Lippoldt, A. Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul. Pharmacol. 38, 323–337 (2002).

    CAS  Article  Google Scholar 

  9. 9.

    Begley, D. J. & Brightman, M. W. Structural and functional aspects of the blood–brain barrier. Prog. Drug Res. 61, 39–78 (2003).

    CAS  PubMed  Google Scholar 

  10. 10.

    Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

    CAS  Article  Google Scholar 

  11. 11.

    Hervé, F., Ghinea, N. & Scherrmann, J.-M. CNS delivery via adsorptive transcytosis. AAPS J. 10, 455–472 (2008).

    Article  Google Scholar 

  12. 12.

    Schinkel, A. H. P-Glycoprotein, a gatekeeper in the blood–brain barrier. Adv. Drug Deliv. Rev. 36, 179–194 (1999).

    CAS  Article  Google Scholar 

  13. 13.

    Rodriguez, A., Tatter, S. B. & Debinski, W. Neurosurgical techniques for disruption of the blood–brain barrier for glioblastoma treatment. Pharmaceutics 7, 175–187 (2015).

    CAS  Article  Google Scholar 

  14. 14.

    Aryal, M., Arvanitis, C. D., Alexander, P. M. & McDannold, N. Ultrasound-mediated blood-brain barrier disruption for targeted drug delivery in the central nervous system. Adv. Drug Deliv. Rev. 72, 94–109 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Demeule, M. et al. Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2. J. Neurochem. 106, 1534–1544 (2008).

    CAS  Article  Google Scholar 

  16. 16.

    Stalmans, S. et al. Cell-penetrating peptides selectively cross the blood–brain barrier in vivo. PLoS ONE 10, e0139652 (2015).

    Article  Google Scholar 

  17. 17.

    Zhang, Y.-Y., Liu, H., Summerfield, S. G., Luscombe, C. N. & Sahi, J. Integrating in silico and in vitro approaches to predict drug accessibility to the central nervous system. Mol. Pharm. 13, 1540–1550 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Bowman, P. D., Ennis, S. R., Rarey, K. E., Betz, A. L. & Goldstein, G. W. Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability. Ann. Neurol. 14, 396–402 (1983).

    CAS  Article  Google Scholar 

  19. 19.

    Naik, P. & Cucullo, L. In vitro blood–brain barrier models: current and perspective technologies. J. Pharm. Sci. 101, 1337–1354 (2012).

    CAS  Article  Google Scholar 

  20. 20.

    Hatherell, K., Couraud, P.-O., Romero, I. A., Weksler, B. & Pilkington, G. J. Development of a three-dimensional, all-human in vitro model of the blood-brain barrier using mono-, co-, and tri-cultivation Transwell models. J. Neurosci. Methods 199, 223–229 (2011).

    Article  Google Scholar 

  21. 21.

    Cecchelli, R. et al. A stable and reproducible human blood–brain barrier model derived from hematopoietic stem cells. PLoS ONE 9, e99733 (2014).

    Article  Google Scholar 

  22. 22.

    Ribecco-Lutkiewicz, M. et al. A novel human induced pluripotent stem cell blood-brain barrier model: applicability to study antibody-triggered receptor-mediated transcytosis. Sci. Rep. 8, 1873 (2018).

    Article  Google Scholar 

  23. 23.

    Holman, D. W., Klein, R. S. & Ransohoff, R. M. The blood–brain barrier, chemokines and multiple sclerosis. Biochim. Biophys. Acta 1812, 220–230 (2011).

    CAS  Article  Google Scholar 

  24. 24.

    Hudecz, D., Rocks, L., Fitzpatrick, L. W., Herda, L.-M. & Dawson, K. A. Reproducibility in biological models of the blood-brain barrier. Eur. J. Nanomed. 6, 185 (2014).

    Article  Google Scholar 

  25. 25.

    Griep, L. M. et al. BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function. Biomed. Microdevices 15, 145–150 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Booth, R. & Kim, H. Characterization of a microfluidic in vitro model of the blood-brain barrier (μBBB). Lab Chip 12, 1784–1792 (2012).

    CAS  Article  Google Scholar 

  27. 27.

    Cecchelli, R. et al. Modelling of the blood-brain barrier in drug discovery and development. Nat. Rev. Drug Discov. 6, 650–661 (2007).

    CAS  Article  Google Scholar 

  28. 28.

    Fadzen, C. M. et al. Perfluoroarene-based peptide macrocycles to enhance penetration across the blood–brain barrier. J. Am. Chem. Soc. 139, 15628–15631 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Demeule, M. et al. Identification and design of peptides as a new drug delivery system for the brain. J. Pharmacol. Exp. Ther. 324, 1064–1072 (2008).

    CAS  Article  Google Scholar 

  30. 30.

    Maira, S.-M. et al. Identification and characterization of NVP-BKM120, an orally available pan-class I PI3-kinase inhibitor. Mol. Cancer Ther. 11, 317–328 (2012).

    CAS  Article  Google Scholar 

  31. 31.

    Liu, X. et al. Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging. Sci. Rep. 3, 2859 (2013).

    Article  Google Scholar 

  32. 32.

    Mittapalli, R. K., Vaidhyanathan, S., Dudek, A. Z. & Elmquist, W. F. Mechanisms limiting distribution of the threonine-protein kinase B-RaF(V600E) inhibitor dabrafenib to the brain: implications for the treatment of melanoma brain metastases. J. Pharmacol. Exp. Ther. 344, 655–664 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Zou, L.-L., Ma, J.-L., Wang, T., Yang, T.-B. & Liu, C.-B. Cell-penetrating peptide-mediated therapeutic molecule delivery into the central nervous system. Curr. Neuropharmacol. 11, 197–208 (2013).

    CAS  Article  Google Scholar 

  34. 34.

    Mijalis, A. J. et al. A fully automated flow-based approach for accelerated peptide synthesis. Nat. Chem. Biol. 13, 464–466 (2017).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Choi-Fong Cho.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing