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Isolation and expansion of human and mouse brain microvascular endothelial cells

Nature Protocols volume 8, pages 1680–1693 (2013)Cite this article

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Abstract

Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I–precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d.

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Figure 1: Brain isolation procedure.
Figure 2: Culture characteristics of human and mouse BMVECs.
Figure 3: Characterization and functional features of human and mouse BMVECs.
Figure 4: Vascular permeability assay and the expression of tight-junction markers.
Figure 5: Angiogenic gene expression profile and in vivo engraftment.

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Acknowledgements

We thank A. Smith for the English review of the paper and A. Canazza, G. Bedini for critical support. This work was supported by the IRCCS Foundation Neurological Institute Carlo Besta (LR8) and by the Italian Ministry of Health (RF2008.22).

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Authors and Affiliations

  1. Laboratory of Cellular Neurobiology, Istituto Di Ricovero e Cura a Carattere Scientifico (IRCCS) Foundation Neurological Institute Carlo Besta, Milan, Italy

    Stefania E Navone, Giovanni Marfia, Gloria Invernici, Silvia Cristini, Giulio Alessandri & Eugenio A Parati

  2. Dipartimento Fisiopatologia Medico-Chirurgica e dei Trapianti, Unità di Neurochirurgia, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy

    Giovanni Marfia

  3. Unit di Produttiva per Terapie Cellulari (UPTC), IRCCS Foundation Neurological Institute Carlo Besta, Milan, Italy

    Sara Nava

  4. Department of Biotechnologies and Life Sciences, University of Insubria, Varese, Italy

    Sergio Balbi & Simone Sangiorgi

  5. Laboratory of Clinical Pathology and Medical Genetics, IRCCS Foundation Neurological Institute Carlo Besta, Milan, Italy

    Emilio Ciusani

  6. School of Healthcare Science, Manchester Metropolitan University, Manchester, UK

    Alessandra Bosutti & Mark Slevin

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  1. Stefania E Navone
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Contributions

S.E.N. designed and performed experiments, analyzed data and wrote the paper; G.M. gave his contribution to write the paper, supervised and undertook revision of the paper; S.N. performed analysis on cellular cycles and blood-brain barrier; M.S. and A.B. contributed in the isolation of mouse BMVECs; G.A., G.I. and S.C. analyzed molecular data; S.B. and S.S. collected human samples; E.C. performed flow cytometric analysis; E.A.P. supervised and undertook revision of the paper.

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Correspondence to Stefania E Navone.

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Competing interests

The authors have applied for a patent on the EndoPM medium (MI2011000201; PCT and Italian application pending).

Supplementary information

Supplementary Table 1

RT-PCR primer sequences and expected product size. (PDF 67 kb)

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Navone, S., Marfia, G., Invernici, G. et al. Isolation and expansion of human and mouse brain microvascular endothelial cells. Nat Protoc 8, 1680–1693 (2013). https://doi.org/10.1038/nprot.2013.107

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