Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine Eye

Samenvatting

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

1. 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.
2. Put a sterile drape into the hood to prepare an aseptic working place. Introduce all needed sterile instruments and materials inside the hood.
   NOTE: Only the enucleation of the....

Resultaten

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

.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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)