Published: February 10th, 2021
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....
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.......
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.......
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.......
|10 ml BD Luer-Lok tip syringe, disposable
|15 ml centrifuge tube
|50 ml centrifuge tube
|Alpha Minimum Essential Medium
|Angled micro forceps
|HERA VIOS 160I CO2 SST TC 120V
|Dulbecco’s Phospate Buffered Saline no Calcium, no Magnesium
|Dumont #5 45° Medical Biology tweezers, 0.05 x 0.01 mm tip, 11 cm length
|Falcon Easy-Grip Clear Polystyrene Cell Culture Dish, 35mm
|Fetal Bovine Serum
|Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red
|Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red B6
|Laminar flow hood
|CLASS II A2 4 115V PACKAGECLA
|Dilute in PBS at 37C to 1mg/ml
|McPherson-Vannas Micro Scissors 8 cm long
|Non-essential amino acids 100X
|N1 Supplement 100X
|Sterile Bard-Parker Carbon steel surgical blade size 11
|Tissue culture treated 12-well plates
|Tissue culture treated 6-well plates
|Transwell supports 6.5 mm
|Trypsin-EDTA (0.25%), phenol red
|Tweezer, Dumont #5 Medical Biology 11 cm, curved, stainless steel 0.02 x 0.06 mm Mod tips
|Vannas Scissors 8cm long, stainless steel
|Whatman Puradisc 25mm Syringe Filters 0.45μm pore size
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