The overall goal of this procedure is to culture iris pigmented epithelial cells to study lens regeneration. In Newt. This is accomplished by first anesthetizing, a new in sleeping solution, and removing the eyes.
The second step of the procedure is to isolate iris pigmented epithelial cells from the eye tissue by first removing the retinal tissue, followed by cutting corneal iris fragments into dorsal and ventral halves. The iris cells are then separated from the stroma and are cultured in vitro for two weeks. After two weeks of Turing, the IPE cells are aggregated and are then implanted into a recipient new that has been lin optimiz.
After a month of healing histology reveals the lens regeneration potential of dorsal and ventral IPE cells. Visual demonstration of this method is critical at cell culture and implantation steps difficult to learn because it requires a good hundred skills for micro dissection and surgeries Begin by anesthetizing at least seven nudes with 0.1%Ethel three amino benzoate methane sul acid surgically remove their eyes by using forceps and surgical scissors by cutting off the eyelid along with the conjunctiva. Then cut the optic nerve.
Now quickly move the dissected eyes to a hood and sterilize them in lugo's ethanol for three seconds. Then wash the eyes twice in calcium and magnesium free Hank solution. Transfer them to fresh hanks and proceed with the dissection.
Using a scalpel, make a slit in the eye. Then use scissors to cut around the corneal margin. Remove the lens and cut off the remaining retinal tissue along with the sclera.
Transfer the iris corneal complexes to a dish, a fresh hanks using a number 15 scalpel. Separate the complexes into their dorsal and ventral halves. The dorsal and ventral irises are identified by the presence of black spots on the sclera of the dorsal side, and a V-shaped cut on the ventral iris.
Separate the ventral iris and the dorsal iris. Collect the iris fragments in a 35 millimeter dish containing one milliliter of L 15 solution. Keep the dorsal and ventral halves in separate dishes.
Now add 150 microliters of dispa solution to the dishes, and incubate them for two hours at 27 degrees Celsius. To collect IPE cells from stroma. With the help of forceps, gently peel off the IPE cells from all the collected tissue fragments.
Next, transfer the IPE cells into a 1.5 milliliter tube and centrifuge at 1000 RP M for two minutes at room temperature, discard the supernatant. Resuspend the pellet in one milliliter of Hanks solution, and repeat the centrifugation to separate the cells. Discard the hanks and resuspend the pellet.
In one milliliter of a one to 250 trypsin solution, incubate for two hours at 27 degrees Celsius to wash the cells centrifuge and remove the trypsin. Add one milliliter of L 15 solution and centrifuge again, repeat the wash once more and replace with 800 microliters of fresh L 15, and gently pipee the cells into solution. Now plate the cells into 24 well plates coated with collagen.
The usual yield from seven Ns is four wells of dorsal cells and four wells of ventral cells. Incubate the dish for two weeks at 27 degrees Celsius after two weeks. Shown here, the IPE can be transfected by genes to examine their function in lens regeneration.
This two week culture of ventral IPE cells exhibits the same morphology as the dorsal cells. Note that pigmentation persists to this stage in both dorsal and ventral iris cells. Prior to implanting the IPE cells, they must be aggregated by adding 20 microliters of dyse in 400 microliters of fresh culture, medium to the cells, swirl the plate gently and leave the cells overnight in an incubator at 27 degrees Celsius.
The next day, use gentle pipetting to dislodge the cells and then transfer them to 1.5 milliliter tubes now wash the cells by centrifuging the tubes and replacing the supernatant with one milliliter of L 15. Repeat this step twice. Use 10 microliters of the cell suspension to calculate the cell count using standard methods.
Once calculated, prepare 3000 to 5, 000 cells in 200 microliters of L 15 per aggregate. Now spin down the cells and incubate them for 48 hours. At 27 degrees Celsius cells can now be implanted into a recipient animal.
Begin by anesthetizing an adult new in the sleeping solution until it stops moving. Now use a scalpel blade to make a slit in the cornea of the eye. Then using forceps, remove the lens.
After the lens is removed, place the IPE cell aggregate just below the corneal tissue on the ventral side of the eye. Now return the newt to a separate container and allow it to heal. During this time, handle the newt carefully to avoid damaging the healing tissue.
The transplanted cells require a month to regenerate into a new lens. After a month has passed, remove the new eyes implanted with the aggregate cells and fix the tissue. Using a standard dehydration protocol, the fixed can be embedded in embedding molds suitable for cutting 15 micrometer sections sliced and stained appropriately.
After the implantation of aggregate into lento new to eye, the aggregate from the dorsal IPE cells induces new lens formation. Host lens induction from the dorsal iris is also observed in the eye with the ventral implant. No lens induction is observed from the aggregate prepared from the ventral IPE cells when the gene six three is over, expressed in the presence of retinoic acid lens induction is observed from the ventral aggregate.
Also, host lens induction from the dorsal iris of the eye is observed after its development. This technique paved the way for the researchers in the field of lens regeneration to explore role of different genes in new tile.
