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Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear

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Abstract

This protocol describes a growth medium–based approach for obtaining cochlear endothelial cells (ECs), pericytes (PCs) and perivascular resident macrophage-like melanocytes (PVM/Ms) from the stria vascularis of mice aged between P10 and P15 (P, postnatal day). The procedure does not involve mechanical or enzymatic digestion of the sample tissue. Explants of stria vascularis, 'mini-chips', are selectively cultured in growth medium, and primary cell lines are obtained in 7–10 d. The method is simple and reliable, and it provides high-quality ECs, PVM/Ms and PCs with a purity >90% after two passages. This protocol is suitable for producing primary culture cells from organs and tissues of small volume and high anatomical complexity, such as the inner ear capillaries. The highly purified primary cell lines enable cell culture–based in vitro modeling of cell-cell interactions, barrier control function and drug action.

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Figure 1: Outline of the steps in the explant procedure.
Figure 2: Outline of the selective culturing procedure.
Figure 3: Validating the phenotype of PCs, PVM/Ms and ECs.
Figure 4: Morphological validation of cell lines.
Figure 5: FACS was used to assess cell purity and validate phenotype.
Figure 6: The schematic illustrates several variations of the in vitro blood-labyrinth barrier model.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) National Institute on Deafness and Other Communication Disorders (NIDCD) grant no. DC008888-02A1 (X.S.), NIH NIDCD grant no. DC008888-02S1 (X.S.), NIH NIDCD grant no. R01-DC010844 (X.S.), NIH NIDCD grant no. R21-DC12398-01 (X.S.), NIH grant no. P30-DC005983 and NIH National Institute of General Medical Services (NIGMS) grant no. P01-051487-15 (M.A.).

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Author notes

  1. Lingling Neng and Wenjing Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Otolaryngology/Head and Neck Surgery, Oregon Hearing Research Center, Oregon Health & Science University, Portland

    Lingling Neng, Wenjing Zhang, Allan Kachelmeier & Xiaorui Shi

  2. Oregon, USA

    Lingling Neng, Wenjing Zhang, Allan Kachelmeier & Xiaorui Shi

  3. Department of Otolaryngology/Head and Neck Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China

    Lingling Neng & Wenjing Zhang

  4. Lawrence Berkeley National Laboratory,

    Ahmed Hassan, Marcin Zemla & Manfred Auer

  5. Life Sciences Division, Berkeley, California, USA

    Ahmed Hassan, Marcin Zemla & Manfred Auer

  6. Department of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden

    Anders Fridberger

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  1. Lingling Neng
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  3. Ahmed Hassan
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Contributions

W.Z. and L.N. created the initial protocol. L.N. refined the protocol. A.H. and M.Z. performed the SEM imaging. A.K. supervised the flow cytometry. X.S., A.F. and M.A. supervised the project. X.S. wrote the manuscript. All the authors discussed the results, procedures and commented on the manuscript at different stages.

Corresponding author

Correspondence to Xiaorui Shi.

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The authors declare no competing financial interests.

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Neng, L., Zhang, W., Hassan, A. et al. Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear. Nat Protoc 8, 709–720 (2013). https://doi.org/10.1038/nprot.2013.033

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