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

Published: April 30, 2023

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 Prepare complete medium (DMEM/Ham's F12 supplemented with 10% fetal bovine serum (FBS), 80 U/mL p…

Representative 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 <p class="jove_content…

Divulgaciones

The authors have nothing to disclose.

Materials

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)

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Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye. J. Vis. Exp. (Pending Publication), e20827, doi: (2023).

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