Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine Eye

Samenvatting

Source: Bascuas T. et al., Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. Exp. (2021).

In this video we describe a technique to isolate iris and retinal pigment epithelial cells from porcine eyes to obtain their primary cultures for in vivo and ex vivo studies. These pigment cells could be genetically modified to study regenerative approaches to treat ocular disorders.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Before preparation

1. Prepare complete medium (DMEM/Ham's F12 supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin / 80 µg/mL streptomycin, and 2.5 µg/mL amphotericin B). Heat the medium, 1x PBS, and 0.25% trypsin (if necessary) in a 37 °C water bath.
2. Put a sterile drape into the hood to prepare an aseptic working place. Introduce all needed sterile instruments and materials inside the hood.
   NOTE: Only the enucleation of the eyes and cleaning of remaining muscle tissue and skin are the procedures carried out outside the hood, the rest of the steps must be performed inside the hood.
3. Porcine eyes were obtained from a local slaughterhouse within 6 hours of sacrifice and were transported to the laboratory on ice

2. Isolation of pig PE cells

1. Use curved scissors and Colibri forceps to enucleate the eyes after euthanizing the animal. Clean the remaining muscle tissue and skin from the eyes using scissors and forceps (non-sterile).
   NOTE: The size of the scissors and forceps used for the enucleation and cleaning of the eyes depends on the species (e.g., for rat and mouse the instruments are going to be smaller than the ones used for pig and cattle) (see Figure 1).
   1. Collect the eyes in a 50 mL tube filled with non-sterile PBS and transfer the tube to the laminar flow hood. Disinfect the eyes by submerging for 2 min in iodine-based solution, then transfer them to a Petri dish filled with sterile PBS.
   2. After transferring the eyes to a sterile Petri dish, hold one firmly close to the optic nerve with Colibri or pointed forceps. Punch a hole near the limit of the iris (between pars plana and ora serrata) with an 18 G needle. Insert small scissors in the hole and cut around the iris. Remove the anterior segment (cornea, lens, and iris) and put it in a Petri dish. Leave the bulb with the vitreous until RPE cells are isolated.

3. Isolation of IPE cells

1. Remove the ciliary body from the iris by cutting with a scalpel #10. After the preparation of 2 iris, add 1 mL of complete medium and isolate the cells by carefully scratching with a flat fire-polished Pasteur pipette. Transfer the cell suspension into a 1.5 mL tube. Take 10 µL of the cell suspension and dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.
2. If not transfected immediately, seed 200,000 cells/well in a 24-well plate (100,000 cells/cm²) in 1 mL of complete medium (10% FBS) (for seeding see Table 1). Place the plate in an incubator and culture it at 37 °C, 5% CO₂.
   NOTE: It might be necessary to pool several eyes together to have enough cells for seeding.

4. Isolation of RPE cells

1. Perform step 1.1.1. Place the bulb in a Petri dish and wash with PBS. Fill the bulb with 1 mL complete medium.
2. Using a curved fire-polished Pasteur pipette, carefully remove RPE cells. Make sure to scrape from the bottom to the top to avoid slipping down of the choroid-Bruch's membrane complex. Collect the cell suspension within the bulb using a 1,000 µL pipette and transfer into a 1.5 mL tube for resuspension. Take 10 µL of the cell suspension and dilute 1:8 with trypan blue to count the cells in the Neubauer chamber.
3. Seed cells as described in step 3.2.

Resultaten

Species	Trypsin treatment	N° IPE cells	N° RPE cells	Plate for seeding (100,000 cells/cm2)
Pig	No	~1,000,000	~3,000,000	24-well plates

...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
12-well plates	 Corning	353043
24-well plates	Corning	353047
Betadine	Mundipharma
Bonn micro forceps flat
Colibri forceps (sterile)
DMEM/Ham`s F12	Sigma-Aldrich	D8062
FBS	Brunschwig	P40-37500
Forceps (different sizes) (sterile)
NaCl (0.9%)	 Laboratorium Dr. Bichsel AG	1000090
Needle (18G) 	Terumo	 TER-NN1838R
Neubauer chamber	Marienfeld-superior	640010
Pasteur pipette (fire-polish) 	Witeg	4100150
PBS 1X	Sigma-Aldrich	D8537
Penicillin/Streptomycin	Sigma-Aldrich	 P0781-100
Petri dish	ThermoFisher Scientific	150288
scarpel no. 10	 Swann-Morton	501
scarpel no. 11	Swann-Morton	503
Sharp-sharp tip curved Extra Fine Bonn Scissors (sterile) 
Sharp-sharp tip straight Extra Fine Bonn Scissors (sterile)
Tali Image-Based Cytometer	 ThermoFisher Scientific	T10796
Trypsin 0.25%	ThermoFisher Scientific	25050014
Trypsin 5%/EDTA 2% 	Sigma-Aldrich	T4174
Vannas spring scissors curved (sterile)