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