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.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Before preparation
2. Isolation of pig PE cells
3. Isolation of IPE cells
4. Isolation of RPE cells
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 |
Table 1: Number of primary PE cells isolated from eyes from different species.
Figure 1: Instruments used for PE isolation depending on the species. (A) Non-sterile instruments used for the enucleation of the eyes and cleaning of remaining muscle tissue and skin. Set 1 is used for rat, mouse, and rabbit, and set 2 is used for pig and cattle eyes. (B) Sterile instruments used for PE isolation. Note the different size of scissors and forceps used depending on the eye size. Round and flat fire-polished Pasteur pipettes are used for the scrape of RPE and IPE, respectively.
The authors have nothing to disclose.
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) |