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

Abstract

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 show a procedure to obtain ocular pigment epithelial cells from a rabbit model. The cells are helpful in developing therapeutic strategies for retinal degenerative diseases.

Protocol

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 wate....

Results

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

Materials

List of materials used in this article

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)