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Isolation, culture and characterization of primary mouse RPE cells

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Abstract

Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1–2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

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Figure 1: Morphology of the primary mouse RPE cultured on Transwells.
Figure 2: Dissection of the anterior portion of the eye of a wild-type male C57BL/6J mouse aged 2 months old.
Figure 3: Sequential dissection of neural retina of a wild-type male C57BL/6J mouse aged 2 months old.
Figure 4: Collection of the RPE sheets of a wild-type female C57BL/6J mouse aged 3 months old.
Figure 5: Diagram illustrating the procedure of agarose embedding and vibratome sectioning.
Figure 6: Vibratome sections of primary mouse RPE cells taken from a 2.5-month-old wild-type male C57BL/6J mouse and grown on Transwells for 2 weeks.
Figure 7: Characterization of polarized RPE cultures (a–d).
Figure 8: Transepithelial electrical resistance (TER) of ten primary mouse RPE cultures measured with an EVOM epithelial tissue voltohmmeter every 3–4 d during 2 weeks in culture.

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Acknowledgements

This work was supported by the Ocular Genomics Institute. The TEM work was supported in part by the National Eye Institute Core (grant P30EY003790). We would like to thank Schepens Eye Research Institute Morphology Core Facility for performing the TEM, A. Tisdale for helpful technical assistance with SEM and A. Langsdorf for helpful comments that improved the manuscript.

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Authors and Affiliations

  1. Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA

    Rosario Fernandez-Godino, Donita L Garland & Eric A Pierce

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  1. Rosario Fernandez-Godino
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  2. Donita L Garland
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R.F.-G. conceived of, designed and performed experiments and wrote the manuscript. D.L.G. and E.A.P designed the experiments and edited the manuscript.

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Correspondence to Rosario Fernandez-Godino.

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Fernandez-Godino, R., Garland, D. & Pierce, E. Isolation, culture and characterization of primary mouse RPE cells. Nat Protoc 11, 1206–1218 (2016). https://doi.org/10.1038/nprot.2016.065

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