Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye

Protokoll

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

New Zealand white rabbits were euthanized by an overdose of Pentobarbital (150 mg/kg) diluted in 0.9% NaCl injected intraperitoneal and the eyes were enucleated immediately after sacrifice.

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.

2. Isolation of rabbit 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. Opening of the bulb
   1. Put one eye on a sterile gauze compress and hold it firmly close to the optic nerve. Open the eye with the scalpel #11 and scissors about 2 mm under the limbus. 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 lens and with fine forceps delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish, wash with sterile PBS and leave it in PBS until more iris are prepared. Remove the ciliary body from the iris by cutting with a scalpel #10.
   2. After the preparation of 2 iris, incubate with 1 mL of 0.25% trypsin at 37 °C for 10 min; during this time, the RPE cells can be isolated (see step 2.4). Remove the trypsin and add 1 mL complete medium to the iris and isolate the cells by carefully scratching with a flat fire-polished Pasteur pipette. Resuspend the cells carefully by pipetting and transfer the cell suspension into a 1.5 mL tube. Take 10 µL of the cell suspension and dilute 1:3 with trypan blue to count the cells in the Neubauer chamber.
   3. 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. Remove vitreous humor and retina from the posterior segment with thin forceps. Avoid damaging the retinal pigment epithelium.
   2. Put a sterile gauze into a 12 well plate and put the bulb over the gauze.
   3. Wash with PBS. Add 50 µL of 0.25% trypsin per eye and incubate for 10 min at 37 °C. Remove trypsin and add 150 µL of complete medium per globe and scrape the RPE cells delicately with a curved fire-polished Pasteur pipette; use fine forceps to immobilize the tissue. Collect the cell suspension and put it in a 1.5 mL tube. Take 10 µL of the cell suspension; dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.
   4. Centrifuge the cells 10 min at 120 x g.
   5. 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.

Ergebnisse

Species	Trypsin treatment	N° IPE cells	N° RPE cells	Plate for seeding (100,000 cells/cm2)
Rabbit	Yes	~350,000	~2,500,000	24-well plates

Materialien

Name	Company	Catalog Number	Comments
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
Drape (sterile)	Mölnlycke Health Care	800530
FBS	Brunschwig	P40-37500
Forceps (different size) (sterile)
Gauze compress	PROMEDICAL AG	25403
NaCl (0.9%)	Laboratorium Dr. Bichsel AG	1000090
Neubauer chamber	Marienfeld-superior	640010
Pasteur pipette (fire-polish)	Witeg	4100150
PBS 1X	Sigma-Aldrich	D8537
Penicillin/Streptomycin	Sigma-Aldrich	P0781-100
Pentobarbital (Thiopental Inresa)	Ospedalia AG	31408025
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)
Trypsin 0.25%	ThermoFisher Scientific	25050014
Vannas spring scissors curved (sterile)