This method is used to study cone receptors which are essential for daylight vision. The advantage of the methods is that it use fertilized chicken eggs, which are easy accessible, and one of the only source of primary cone cultures. The protocol was used to identify Rod-Derived Cone Viability Factor, a future promising treatment for inherited retinal degeneration.
Furthermore, the protocol was used to study cone photoreceptor metabolism. By carefully following the described protocol, an individual whose expertise is in primary cell culture, should be able to obtain cone enriched culture after only a few attempts. Begin by collecting weekly fertilized eggs from an industrial hatchery.
Maintain the fertilized eggs at 17 degrees Celsius in the laboratory. For each culture, incubate seven fertilized eggs for 24 hours at 20 degrees Celsius, and then 136 hours at 37 degrees Celsius in a humidified chamber with intermittent reversion of inclination. To recover the chicken embryos, wash the surface of the eggs with disinfectant, break the eggshell by making a hole at the top of the shell with large straight pliers, then cut the shell to remove the hat from the egg.
Gently extract each embryo from the eggshell with curved forceps and transfer it to a Petri dish containing sterile PBS previously heated to 37 degrees Celsius. Carefully remove the envelope that surrounds the embryos. Verify the stage of development of each embryo by visual comparison to Hamburger and Hamilton and select two embryos at the 29th stage of development Enucleate the eyes of these selected embryos and transfer them into carbon dioxide-independent medium.
Working in carbon dioxide-independent medium, position four eyes with the cornea facing down and the optic nerve facing the experimenter. Drill a hole in the optic nerve using two straight forceps. Insert a branch of each forceps between the retina and the pigment epithelium, then pull and rotate the eye to detach the epithelium from the retina.
Remove the cornea, followed by the lens and the vitreous. Transfer the four retinas into a Petri dish containing Ringer's medium at pH 7.2. Cut the four retinas into very small pieces using two straight pliers and wash the pieces twice with Ringer's medium.
After the second wash, let the pieces of retina fall to the bottom of the tube and remove the media. Treat the retinal pieces with a solution of Trypsin for 20 minutes at 37 degrees Celsius. After 20 minutes, stop the reaction by adding culture media supplemented with 10%inactivated fetal calf serum.
Incubate the cell suspension with 0.05 mg of DNase 1 Then immediately dissociate the cell clusters and the DNA by pipetting up and down with a pipette. Wash the retinal cell suspension twice with chemically defined culture medium or CDM. Treat two black 96-well culture plates with a transparent bottom with polylysine for two hours at 37 degrees Celsius.
When finished, rinse the plates twice with M199 culture medium. Add Trypan blue to an aliquot of 10 microliters of the cell suspension to stain the living cells. Then add the cell suspension specimen to a hemocytometer.
After counting the cells, bring the cell suspension to the appropriate concentrations using CDM. Add 50 microliters of the library of molecules to be screened using a predefined pattern. Seed the 50 microliters of the two cell suspensions into the two pretreated black 96-well culture plates.
Distribute the cells in the plates with a multi-channel pipette from the right of the plate to the left, homogenizing between each column. Then incubate the plates for seven days at 37 degrees Celsius, under 5%carbon dioxide with no change of media. To count the viable cells, add 2.7 micromolar calcein AM and 0.3 millimolar ethidium homodimer to each well of the plate.
Incubate the plates for one hour at room temperature in the absence of light. Read the fluorescence on an automated plate reader composed of an inverted microscope equipped with a mercury lamp with two excitation filters at 485 and 520 nanometers, two emission filters at 520 and 635 nanometers and a charge-coupled device camera. This protocol was used to screen a normalized cDNA library made of choroid and retinal pigmented epithelium from 400 eyes of an eight week old, Long-Evans Rats.
A total of 2, 112 sets of 100 clones corresponding to 211, 200 individual clones were evaluated. Among the 42 pools of clones with a ratio greater than two, pools 0080 and 0073 had a viability ratio 16 and 14 times higher than the negative control after seven days of culture. Each selected of 100 clones was subdivided into 16 sets of 10 clones from their glycerol stock.
The sub pool 0073-09 gave the strongest viability ratio and was subdivided to produce 16 individual clones that were tested in a third round of screening on cone-enriched cultures. The clone 0073-09-37 stood out with a viability ratio of 2.5. Further analysis confirmed that this clone has a robust and reproducible effect on cone survival.
The test was repeated independently and the insert of 1.8 kilobases was sequenced. A bioinformatic analysis revealed that the clone 0073-09-37, which was named Epithelium-Derived Cone Viability Factor, or EdCVF contains three open reading frames. When tested independently, only ORF1 exerted a protective effect on the cones.
ORF1 was produced as a Glutathione S-transferase protein. The epithelium-derived cone viability factor was purified and the GST tag was removed. Analysis of trophic activity demonstrated that EdCVF is able to prevent cone degeneration in the cone-enriched culture system.
When attempting this procedure, the stage of development of the embryos should be carefully checked in order to get the cone-enriched cultures. Following this protocol, the cells can be electroporated with plasmid DNA to study the molecular mechanisms of any survival factors such as RdCVF. This technique paves the of metabolic research including the development of mathematical models of cone survival.
