Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine 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 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 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. Put a sterile drape into the …
Representative Results
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 …
Divulgazioni
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 | |
| 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) |
Tags
Citazione di questo articolo
Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine Eye. J. Vis. Exp. (Pending Publication), e20828, doi: (2023).