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 …

Disclosures

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)

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Cite This Article
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).

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