This method can help answer key questions about the self therapy of the eye, such as what is the efficacy of a specifically designed bio-engineered tissue in vivo. With this technique we provide a method of producing and contouring the retinal tissue as well as a strategy for processing the tissue for eye implantation in an animal model. Begin by washing one to four 30 by 30 mm pieces of human amniotic membrane, or hAM.
Fix a nylon scaffolds two times in 80ml of PBS per wash in a 250ml bottle on a shaker, at high speed for five minutes. After the second wash, add 40ml of working Thermolysin solution to each bottle, and shake the bottles two times for five minutes at 450 rpm, followed by 30 seconds of vortexing. After the second round of Thermolysin treatment, wash the pieces two more times with 80ml of PBS per wash, as demonstrated.
Then, use long sterile forceps to transfer the hAMs individually, into a 10cm culture dish, containing 10ml of PBS. Next place one membrane with the nylon facing down in a second 10cm culture dish of PBS, and detach two of the four clips holding the membrane to nylon. Insert the smaller ring of the culture insert between the nylon and the membrane, making sure that the membrane covers the smaller ring completely.
Clip the second part of the culture insert to the top of the membrane so that the basement membrane of the hAM is facing right side up in the culture insert and detach the last two clips. Then use sterile scissors to trim any membrane outside of the culture insert as necessary. And use forceps to transfer the membrane into one well of a 12 well plate, containing PBS for storage at 37 degrees Celsius and 5%CO2.
Before seeding, warm cryo vials containing one times 10 to the sixth RPE cells in 3ml of RPE medium in one 15ml conical tube per vial. When the cells have thawed, pipette to obtain single cell suspensions in each of the tubes, and count the number of viable cells by tri pan blue exclusion. Collect the cells by centrifugation and re suspend the pellets at seven times 10 to the fifth cells per milliliter concentration in fresh RPE medium.
Next, aspirate the PBS from each well of the prepared 12 well plate, and add 500 micro liters of cells to the center of each culture insert, and 1ml of RPE medium to the bottom of each well. Then return the plate to the cell culture incubator for 30 days, changing the medium two times per week until the end of the culture. On culture day 30, warm an 8%gelatin solution in a 37 degrees Celsius water bath, for 30 minutes.
And fill the internal chamber of a vibratome with four degrees Celsius CO2 independent medium. Use a scalpel to cut a 5cm by 5cm block from a 20%gelatin block stored at four degrees Celsius. Secure the top side of the block to the support with an appropriate adhesive.
And place a new blade into the vibratome. Adjust the block until it is at the same level as the razor blade, and fill the bath around the support with fresh four degrees Celsius CO2 independent medium. Cut the block at a medium velocity until the block is cut uniformly with a smooth leading surface and set the position to zero.
Then aspirate the medium around the gelatin until the gelatin is completely dry. And use forceps to carefully open an RPE seeded culture insert on top of the block. Use scissors to remove all of the membrane outside of the insert ring, and aspirate the entire volume of residual medium.
Add up to 1ml of 8%liquid gelatin solution to the membrane until it is covered. And carefully remove any excess gelatin. After five to eight minutes, add fresh four degrees Celsius CO2 independent medium to the bath until the solidified membrane is covered.
And with the vibratome at the 100 micron position, cut a section of the membrane at a medium velocity, taking care to maintain the orientation of the tissue. Cut a corner of the section with a scalpel to allow identification of the orientation of the section. And use a spatula to transfer the gelatin-embedded membrane to one of a six well plate, filled with four degrees Celsius conservation medium.
The orientation of the slice could be inverted due to freed movement before the corner being cut. So take care to secure the position of the slice just after the section. The size of the implant can then be adjusted to the size of the recipient eye under a surgical microscope, prior transplantation.
Typically a few dead cells remain on the surface of the membrane, after fixation on the culture insert, but those cells are eliminated when the culture medium is changed. The fibers of the basement membrane are visible at a higher magnification when no cells remain, indicating that the Thermolysin treatment period has been optimized. The retinal epithelium becomes easier to see under a microscope, as it matures, forming a complete cobblestone-like monolayer at three weeks.
At four weeks, the epithelium is mature enough to be prepared for implantation. While attempting this procedure, it's important to remember that the state of the gelatin is dependent on the temperature. So take care to strictly follow recommended temperatures.
After its development, this technique paves the way for researchers in the field of stem cell therapy, to explore the therapeutic potential of a bio-engineered tissue in an animal model of retinitis pigmentosa. Though this method can be used to prepare an tissue on top of a scaffold, for its subsequent transplantation, it can also be applied to more complex systems containing photo receptor layers. Generally, individuals new to this method will struggle because of the difficulty in handling the human amniotic membranes, which is very thin and tends to fold easily.
Visual demonstration of this method is critical as human amniotic membrane fixation and gelatin embedding steps, are difficult to learn and require a particular handling approach.
