Isolating RPE and IPE Cells from Bovine Eye: A Procedure to Harvest and Culture Bovine Primary Pigment Epithelial Cells

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).

This video demonstrates the procedure to isolate the retinal pigment epithelial, or RPE, cells and iris pigment epithelial, or IPE, cells from harvested bovine eyes. The isolated cells can be cultured to study their role in the treatment of ocular diseases.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Isolation of bovine PE cells Use curved scissors and Colibri forceps to enucleate the eyes after euthanizing the animal. Clean the remaining muscle tissue and skin from the eyes using scissors and forceps (non-sterile). NOTE: The size of the scissors and forceps used for the enucleation and cleaning of the eyes d…

Representative Results

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 …

Materials

6-well plate	Greiner	7657160
Betadine	Mundipharma
Colibri forceps (sterile)
Forceps (different size) (sterile)
Gauze compress	PROMEDICAL AG	25403
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
DMEM/Ham`s F12	Sigma-Aldrich	D8062
FBS	Brunschwig	P40-37500
Scalpel no. 10	Swann-Morton	501
Scalpel 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)