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Fungal brain infection modelled in a human-neurovascular-unit-on-a-chip with a functional blood–brain barrier

Nature Biomedical Engineering volume 5, pages 830–846 (2021)Cite this article

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Abstract

The neurovascular unit, which consists of vascular cells surrounded by astrocytic end-feet and neurons, controls cerebral blood flow and the permeability of the blood–brain barrier (BBB) to maintain homeostasis in the neuronal milieu. Studying how some pathogens and drugs can penetrate the human BBB and disrupt neuronal homeostasis requires in vitro microphysiological models of the neurovascular unit. Here we show that the neurotropism of Cryptococcus neoformans—the most common pathogen causing fungal meningitis—and its ability to penetrate the BBB can be modelled by the co-culture of human neural stem cells, brain microvascular endothelial cells and brain vascular pericytes in a human-neurovascular-unit-on-a-chip maintained by a stepwise gravity-driven unidirectional flow and recapitulating the structural and functional features of the BBB. We found that the pathogen forms clusters of cells that penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism. The neurovascular-unit-on-a-chip may facilitate the study of the mechanisms of brain infection by pathogens, and the development of drugs for a range of brain diseases.

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Fig. 1: Schematics of the 3D microfluidic hNVU chip study.
Fig. 2: Characterization of collagen I and BHEM hydrogels.
Fig. 3: Generation of a neurovascular unit with a BBB by in situ NSC differentiation in the hNVU chip.
Fig. 4: Characterization of BBB permeability in the hNVU chip.
Fig. 5: Barrier function and physiological responses of the BBB in the hNVU chip.
Fig. 6: Fungal brain-infection model using the hNVU chip.
Fig. 7: A multi-organ hNVU chip model for fungal neurotropism study.
Fig. 8: Screening for the pathogenicity of C. neoformans mutants in the multi-organ hNVU chip.

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Data availability

The main data supporting the findings of this study are available within the paper and its Supplementary Information. The datasets from the quantitative analyses generated during this study, including source data and the data used to make the figures as well as all images, are too large and numerous to be publicly shared, but they are available for research purposes from the corresponding authors on reasonable request. RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO), with accession number GSE171937.

Code availability

MATLAB was used to analyse results of fluorescence recovery after photobleaching experiments. The codes used for the analysis can be provided on request.

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Acknowledgements

This work was supported by grants (2017M3C7A1047659, 2016R1E1A1A01943365, 2021R1A2C3004262 and 2018R1A5A1025077) from the National Research Foundation of Korea funded by the Ministry of Science and ICT, Republic of Korea. This work was also supported by the Korea Evaluation Institute of Industrial Technology grant funded by the Korea government (MSIT) (no. 20009125) and the Institute for Basic Science (IBS-R026-D1), and the Strategic Initiative for Microbiomes in Agriculture and Food funded by Ministry of Agriculture, Food and Rural Affairs (916006-2). We thank J. Cheon (Yonsei University and IBS) for his support with facilities and equipment for analyses.

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  1. These authors contributed equally: Jin Kim, Kyung-Tae Lee.

Authors and Affiliations

  1. Department of Biotechnology, Yonsei University, Seoul, Republic of Korea

    Jin Kim, Kyung-Tae Lee, Jong Seung Lee, Jisoo Shin, Baofang Cui, Kisuk Yang, Yi Sun Choi, Yong-Sun Bahn & Seung-Woo Cho

  2. Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea

    Nakwon Choi & Soo Hyun Lee

  3. KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea

    Nakwon Choi

  4. Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology (UST), Seoul, Republic of Korea

    Nakwon Choi

  5. Institute for Basic Science (IBS), Center for Nanomedicine, Seoul, Republic of Korea

    Jae-Hyun Lee & Seung-Woo Cho

  6. Graduate Program of Nano Biomedical Engineering (NanoBME), Advanced Science Institute, Yonsei University, Seoul, Republic of Korea

    Jae-Hyun Lee & Seung-Woo Cho

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Contributions

J.K. and K.-T.L. contributed equally to this work. J.K. and K.-T.L. designed, led the experiments, performed data analysis and wrote the paper. J.K. led the overall work on human cell culture, analysis and microfluidic systems. K.-T.L. performed overall work on fungal experiments and transcriptome analysis. J.S.L. and K.Y. cultured NSCs, J.S. prepared and characterized hydrogels and B.C. fabricated microfluidic chips. Y.S.C. prepared and performed proteomic analysis of BEM. N.C. and S.H.L. provided materials and advised on the data analyses. J.-H.L. advised on the data analyses and wrote the manuscript. Y.-S.B. and S.-W.C. designed and supervised the experiments and wrote the paper.

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Correspondence to Yong-Sun Bahn or Seung-Woo Cho.

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J.K., K.-T.L., Y.-S.B. and S.-W.C. are co-inventors on patent applications (Korean Patent 10-2019-0115272, US patent 17/277,640 and EP patent 19 863 762.1) related to the hNVU chip developed in the manuscript.

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Supplementary methods, references, figures, notes,video captions, and tables.

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Supplementary Video 1

Live imaging of the fungal infection model. The video clip of C. neoformans wild-type cells stacking and penetrating the BBB in the hNVU chip.

Supplementary Video 2

3D confocal image of the C. neoformans penetration site. The 3D image was processed by IMARIS (Bitplane; http://www.bitplane.com).

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Kim, J., Lee, KT., Lee, J.S. et al. Fungal brain infection modelled in a human-neurovascular-unit-on-a-chip with a functional blood–brain barrier. Nat Biomed Eng 5, 830–846 (2021). https://doi.org/10.1038/s41551-021-00743-8

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