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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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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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.
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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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DOI: https://doi.org/10.1038/nprot.2013.033
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