Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Podsumowanie

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Streszczenie

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

Wprowadzenie

Retinal pigmented epithelium (RPE) cells are located between the choroid and the neural retina, forming a simple monolayer of cuboidal cells that lies behind the photoreceptor (PR) cells¹. The RPE plays a critical role in maintaining a healthy environment for PR cells, mainly by reducing the excessive accumulation of reactive oxygen species (ROS) and consequent oxidative damage¹. RPE cells oversee many functions, such as the conversion and storage of retinoids, the absorption of scattered light, fluid and ion transport, and phagocytosis of the shed PR outer segment membrane^2,

Protokół

Animals were used as per the guidelines of Oakland university IACUC animal protocol number 21063 and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Solution preparation

1. Prepare the complete RPE cell culture media by supplementing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with 25% Fetal Bovine Serum (FBS), 1.5% penicillin/streptomycin, and 0.02% gentamicin.
2. Prepare Enzyme A by supplementing 741 µL of Hank's Balanced Salt Solution (HBSS) with 127 µL of Collagenase stock solution and 132 µL of Hyaluronidase s....

Wyniki

Validation of the specificity, purity, and barrier function/formation of isolated RPE cells
The isolated cells were examined under a light microscope to verify their viability, morphology, and pigmentation. Images from P0 and P1 (Figure 1A,B) and images from P0 and P4 were captured (Figure 1C,D) to show the changes in the cells; shape, size, and pigmentation as the passages proceeded to the fourth passage (black arrows.......

Dyskusje

The current protocol is a reported, modified, and simplified detailed procedure for RPE isolation from mouse eyes. The protocol includes enucleation, dissection, collection, seeding, culture, and characterization of RPE cells isolated from mouse eyes.

There are some limitations and critical steps that must be fulfilled for successful RPE isolation, such as mice age, the number of eyes dissected, the size of the tissue culture plate or dish, and cautions after seeding, storage, and passaging. T.......

Materiały

Name	Company	Catalog Number	Comments
Beaker : 100mL	KIMAX	14000
Collagenase from Clostridium histolyticum	Sigma-Aldrich	C7657-25MG	For working enzyme, A
Disposable Graduated Transfer Pipettes :3.2mL Sterile		13-711-20
DMEM/F12	gibco	11330	Media to grow RPE cells
Fetal Bovine Serum (FBS)	gibco	26140079	For complete RPE cell culture media
Gentamicin Reagent Solution	gibco	15750-060	For complete RPE cell culture media
Hanks' Balanced Salt Solution (HBSS)	Thermo Scientific	88284	For working enzymes (A&B)
Heracell VISO 160i CO2 Incubator	Thermo Scientific	50144906
Hyaluronidase from bovine testes	Sigma-Aldrich	H3506-500MG	For working enzyme A
Kimwipes	Kimberly-Clark	34155
Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL	B-D	309577
Micro Centrifuge Tube: 2 mL	Grainger	11L819
Mouse monoclonal anti-RPE65 antibody	Abcam, Cambridge, MA, USA	ab78036	For IF staining
Pen Strep	gibco	15140-122	For complete RPE cell culture media
Positive Action Tweezers, Style 5/45	Dumont	72703-DZ
Scissors Iris Standard Straight 11.5cm	GARANA INDUSTRIES	2595
Sorvall St8 Centrifuge	ThermoScientific	75007200
Stemi 305 Microscope	Zeiss	n/a
Surgical Blade, #11, Stainless Steel	Bard-Parker	371211
Suspension Culture Dish 60mm x 15mm Style	Corning	430589
Tissue Culture Dish : 100x20mm style	Corning	353003
Tornado Tubes: 15mL	Midsci	C15B
Tornado Tubes: 50mL	Midsci	C50R
Trypsin EDTA (1x) 0.25%	gibco	2186962	For working enzyme B
Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips	Dumont	501764
Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL	Electron Microscopy Sciences Dumont	50-241-57
Underpads, Moderate : 23" X 36"	McKesson	4033
Vannas Spring Scissors - 2.5mm Cutting Edge	FST	15000-08
Zeiss AxioImager Z2	Zeiss	n/a
Zeiss Zen Blue 2.6	Zeiss	n/a