JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Published: February 10th, 2021

DOI:

10.3791/62228

1Ocular Genomics Institute of Massachusetts Eye and Ear, Harvard Medical School

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse retinal pigment epithelium (RPE) cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The RPE is a monolayer located in the eye between the neural retina and the Bruch's membrane. This single layer consists of highly polarized and pigmented epithelial cells joined by tight junctions, exhibiting a hexagonal shape that resembles a honeycomb2. Despite this apparent histological simplicity, the RPE performs a wide variety of func....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research were followed.

NOTE: This method has been proven successful with mice of different genetic backgrounds, including C57BL/6J, B10.D2-Hco H2d H2-T18c/oSnJ, and albino mice, at various ages. Preferably use 8 to 12-week-old mice to obtain RPE cells. RPE cells from older mice proliferate less in culture and younger mice have fewer and smaller cells, which requires pooling ey.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This protocol has been used to isolate and culture RPE cells from genetically modified mice1. No differences have been observed between mouse strains or gender. The results have helped to understand some important aspects of the mechanism underlying ocular diseases such as age-related macular degeneration, which is the most common cause of vision loss among the elderly9. RPE cells isolated following this protocol were completely attached to the membrane insert 24 hours afte.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

While several methods for mouse RPE cell isolation and culture had been developed before1,13,20,22,26,27, Fernandez-Godino's method first used membrane inserts allowing the efficient growth of the RPE cells in culture for weeks1,9. Another major change in their pr.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the Ocular Genomics Institute at Massachusetts Eye and Ear.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
10 ml BD Luer-Lok tip syringe, disposable BD Biosciences 309604
15 ml centrifuge tube VWR International 21008-103
50 ml centrifuge tube VWR International 21008-951
Alpha Minimum Essential Medium Sigma-Aldrich M4526-500ML
Angled micro forceps WPI 501727
Bench-top centrifuge any
CO2 incubator Thermo HERA VIOS 160I CO2 SST TC 120V
Dissecting microscope Any
Dulbecco’s Phospate Buffered Saline no Calcium, no Magnesium Gibco 14190144
Dumont #5 45° Medical Biology tweezers, 0.05 x 0.01 mm tip, 11 cm length WPI 14101
Ethanol Sigma-Aldrich E7023-500ML
Falcon Easy-Grip Clear Polystyrene Cell Culture Dish, 35mm BD Biosciences 353001
Fetal Bovine Serum Hyclone SH30071.03 Heat inactivated.
Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red Life Technologies 14175095
Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red B6 Life Technologies 14025092
HEPES 1M Gibco 15630106
Hyaluronidase Sigma-Aldrich H-3506 1G
Hydrocortisone Sigma-Aldrich H-0396
Laminar flow hood Thermo CLASS II A2 4 115V PACKAGECLA
Laminin 1mg/ml Sigma-Aldrich L2020-1 MG Dilute in PBS at 37C to 1mg/ml
McPherson-Vannas Micro Scissors 8 cm long WPI 503216
Non-essential amino acids 100X Gibco 11140050
N1 Supplement 100X Sigma-Aldrich N6530-5ML
Penicillin-Streptomycin Gibco 15140-148
Sterile Bard-Parker Carbon steel surgical blade size 11 Fisher-Scientific 08-914B
Taurine Sigma-Aldrich T-0625
Tissue culture treated 12-well plates Fisher-Scientific 08-772-29
Tissue culture treated 6-well plates Fisher-Scientific 14-832-11
Transwell supports 6.5 mm Sigma-Aldrich CLS3470-48EA
Triiodo-thyronin Sigma-Aldrich T-5516
Trypsin-EDTA (0.25%), phenol red Gibco 25200056
Tweezer, Dumont #5 Medical Biology 11 cm, curved, stainless steel 0.02 x 0.06 mm Mod tips WPI 500232
Vannas Scissors 8cm long, stainless steel WPI 501790
Whatman Puradisc 25mm Syringe Filters 0.45μm pore size Fisher-Scientific 6780-2504

  1. Fernandez-Godino, R., Garland, D. L., Pierce, E. A. Isolation, culture and characterization of primary mouse RPE cells. Nature Protocols. 11 (7), 1206-1218 (2016).
  2. Strauss, O. The retinal pigment epithelium in visual function. Physiological Reviews. 85 (3), 845-881 (2005).
  3. Konari, K., et al. Development of the blood-retinal barrier in vitro: Formation of tight junctions as revealed by occludin and ZO-1 correlates with the barrier function of chick retinal pigment epithelial cells. Experimental Eye Research. 61 (1), 99-108 (1995).
  4. Kay, P., Yang, Y. C., Paraoan, L. Directional protein secretion by the retinal pigment epithelium: Roles in retinal health and the development of age-related macular degeneration. Journal of Cellular and Molecular Medicine. 17 (7), 833-843 (2013).
  5. Johnson, L. V., Leitner, W. P., Staples, M. K., Anderson, D. H. Complement activation and inflammatory processes in drusen formation and age related macular degeneration. Experimental Eye Research. 73 (6), 887-896 (2001).
  6. Hageman, G. S., et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration. Progress in Retinal and Eye Research. 20 (6), 705-732 (2001).
  7. Lommatzsch, A., et al. Are low inflammatory reactions involved in exudative age-related macular degeneration. Graefe's Archive for Clinical and Experimental Ophthalmology. 246 (6), 803-810 (2008).
  8. Bandyopadhyay, M., Rohrer, B. Matrix metalloproteinase activity creates pro-angiogenic environment in primary human retinal pigment epithelial cells exposed to complement. Investigative Ophthalmology & Visual Science. 53 (4), 1953-1961 (2012).
  9. Fernandez-Godino, R., Garland, D. L., Pierce, E. A. A local complement response by RPE causes early-stage macular degeneration. Human Molecular Genetics. 24 (19), 5555-5569 (2015).
  10. Fernandez-Godino, R., Bujakowska, K. M., Pierce, E. A. Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway. Human Molecular Genetics. 27 (1), 147-159 (2018).
  11. Fernandez-Godino, R., Pierce, E. A. C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway. Scientific Reports. 8 (1), 1-14 (2018).
  12. Farkas, M. H., et al. Mutations in Pre-mRNA processing factors 3, 8, and 31 cause dysfunction of the retinal pigment epithelium. American Journal of Pathology. 184 (10), 2641-2652 (2014).
  13. Geisen, P., Mccolm, J. R., King, B. M., Hartnett, E. Characterization of Barrier Properties and Inducible VEGF Expression of Several Types of Retinal Pigment Epithelium in Medium-Term Culture. Current Eye Research. 31, 739 (2006).
  14. Schütze, C., et al. Retinal pigment epithelium findings in patients with albinism using wide-field polarization-sensitive optical coherence tomography. Retina. 34 (11), 2208-2217 (2014).
  15. Samuels, I. S., Bell, B. A., Pereira, A., Saxon, J., Peachey, N. S. Early retinal pigment epithelium dysfunction is concomitant with hyperglycemia in mouse models of type 1 and type 2 diabetes. Journal of Neurophysiology. 113 (4), 1085-1099 (2015).
  16. Garland, D. L., et al. Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration. Human Molecular Genetics. 23 (1), 52-68 (2014).
  17. Fu, L., et al. The R345W mutation in EFEMP1 is pathogenic and causes AMD-like deposits in mice. Human Molecular Genetics. 16 (20), 2411-2422 (2007).
  18. Greenwald, S. H., et al. Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease. American Journal of Pathology. 186 (7), 1925-1938 (2016).
  19. Gupta, P. R., et al. Ift172 conditional knock-out mice exhibit rapid retinal degeneration and protein trafficking defects. Human Molecular Genetics. 27 (11), 2012-2024 (2018).
  20. Gibbs, D., Williams, D. S. Isolation and culture of primary mouse retinal pigmented epithelial cells. Advances in Experimental Medicine and Biology. 533, 347-352 (2003).
  21. Bonilha, V. L., Finnemann, S. C., Rodriguez-Boulan, E. Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium. Journal of Cell Biology. 147 (7), 1533-1547 (1999).
  22. Nandrot, E. F., et al. Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking αvβ5 integrin. Journal of Experimental Medicine. 200 (12), 1539-1545 (2004).
  23. Maminishkis, A., et al. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Investigative Ophthalmology and Visual Science. 47 (8), 3612-3624 (2006).
  24. Maminishkis, A., Miller, S. S. Experimental models for study of retinal pigment epithelial physiology and pathophysiology. Journal of Visualized Experiments. (45), (2010).
  25. Brydon, E. M., et al. AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31+/− iPSC-Derived RPE Cells. Molecular Therapy - Methods and Clinical Development. 15, 392-402 (2019).
  26. Shang, P., Stepicheva, N. A., Hose, S., Zigler, J. S., Sinha, D. Primary cell cultures from the mouse retinal pigment epithelium. Journal of Visualized Experiments. 2018 (133), (2018).
  27. Bonilha, V. Age and disease-related structural changes in the retinal pigment epithelium. Clinical Ophthalmology. 2 (2), 413 (2008).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved