Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Abstract

Source: Portela-Lomba, M., et al. Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration. J. Vis. Exp. (2020)

This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat's retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of ihOEG (Ts12 and Ts14) for the assay

NOTE: This step must be done 24 h before RGN dissection and coculture.

1. Treat 12 mm Ø coverslips with 10 µg/mL poly-L-lysine (PLL) for 1 h.
   NOTE: The coverslips can be left overnight in the PLL solution.
2. Wash the coverslips with 1x phosphate buffer saline (PBS) three times.
3. Detach Ts12 and Ts14 ihOEG cells from the p60 c....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B-27 Supplement	Gibco	17504044
D,L-2-amino-5-phosphonovaleric acid	Sigma	283967	NMDA receptor inhibitor
DMEM-F12	Gibco	11320033	Cell culture medium
FBS	Gibco	11573397	Fetal bovine serum
FBS-Hyclone	Fisher Scientific	16291082	Fetal bovine serum
L-Glutamine	Lonza	BE17-605F
Neurobasal Medium	Gibco	21103049	Neuronal cells culture medium
Papain Dissociation System	Worthington Biochemical Corporation	LK003150	For use in neural cell isolation
PBS	Home made
PBS-EDTA	Lonza	H3BE02-017F
Penicillin/Streptomycin/Amphotericin B	Lonza	17-745E	Bacteriostatic and bactericidal
Pituitary extract	Gibco	13028014	Bovine pituitary extract
Poly -L- lysine (PLL)	Sigma	A-003-M