The overall goal of this procedure is to demonstrate the use of the EAK 16-II/EAKIIH6 peptide in promoting thymic epithelial cell aggregation in a three-dimensional structure for supporting thymic epithelial self-survival and function. This method may help answer key questions in the thymus bioengineering field. The main advantages of this technique is that it utilizes EAK-16-II/EAKIIH6 peptide, promotes thymic epithelial cell aggregation, is biodegradable, and has a potential to be used for injections.
To harvest the thymus, begin by placing a three to five week old C57 black 6J mouse in the supine position and wet the hair with 70%ethanol. Using sharp, straight dissecting scissors, make a small horizontal incision through the abdominal skin. Then pull the skin on both sides of the incision to expose the abdominal cavity.
Next, make a horizontal cut just under the lowest rib and cut sideways to the diaphragm. Using forceps, pull the xiphoid process and cut through the middle of the ribs along both sides of the chest. Pull up the ribs and sternum so that the thymus is clearly visible.
Then, using a curved forceps, gently scoop the lobes of the thymus off of the connective tissue and place them in washing solution. After the rinse, transfer the thymus into a 16 milliliter tissue culture dish containing five to six milliliters of RPMI 1640 and use a 28 gauge insulin needle to dissect the tissue into approximately one cubic millimeter square pieces. Next, use a glass pasteur pipette to rinse the tissue fragments with the medium in the dish.
Then, tilt the dish, letting the fragments settle to the bottom. Slowly remove the medium supernatant by aspiration. Then repeat the wash four to five more times with five to six milliliters of fresh medium each time until the solution becomes less opaque.
After the final wash, add three milliliters of digestion solution to the dish and transfer the thymus pieces and solution into a five milliliter round-bottom polystyrene tube. Incubate the tissues on a tube rotator at 37 degrees celsius for six minutes at 12 rpm. At the end of the rotation, let the fragments settle to the bottom of the tube and use a pasteur pipette to transfer the supernatant into a 15 milliliter conical tube containing 10 milliliters of washing solution.
Centrifuge the supernatant and resuspend the cells in 10 milliliters of fresh washing solution on ice. Then digest the thymic fragments two more times in the rotator as just demonstrated, pulling the supernatant in the same 50 milliliter tube. After the final digestion, use a plastic two milliliter serological pipette to triturate any remaining fragments and transfer the tissues'flurry to the 50 milliliter tube.
Spin down the tube to collect the cells and resuspend the pellet in 10 milliliters of washing buffer. Then, filter the cells through a 100 micron strainer while the tube is placed on ice. To enrich for the thymic epithelial cells, spin down the cells again and resuspend the pellet in 2.5 milliliters of washing buffer.
Next, add 20 milliliters of RPMI 1640 to the cells and use a 10 milliliter serological pipette to carefully layer 12 milliliters of freshly prepared 21%density gradient solution under the cells by gravity. Now, separate the cells by centrifugation in a swinging bucket rotor at the slowest deceleration rate. At the end of the centrifugation, transfer the top layer and the interface into a new 50 milliliter tube and add up to 40 milliliters of washing solution to the cells.
Remove the density gradient by three consecutive centrifugations using a pipette to aspirate the supernatant after each wash. Then resuspend the final pellet in two milliliters of washing solution for counting. To generate the thymic epithelial cell EAK aggregates, adjust the thymic epithelial cells to a five times 10 to the four cells per milliliter dilution in washing solution and seed 100 microliters of cells per well into a low evaporative 96 well, round-bottom, tissue culture plate.
Next, add 14.6 microliters of freshly prepared protein AG adapter complexes to each well and place the plate on a rocking platform at four degrees celsius. After 20 minutes, wash the cells with 200 microliters of washing solution per well, using a micropipette to remove the supernatant. Then, wash the cells again with 200 microliters of 10%sucrose solution.
Resuspend the pellets in 30 microliters of 10%sterile sucrose solution per well. Then, incubate the cells in 10 microliters of EAKIIH6 for five minutes at room temperature. At the end of the incubation, add 20 microliters of EAK 16-II, followed by 100 microliters of complete medium and place the plate on a platform at room temperature to initiate the gelation.
After 15 to 20 minutes, transfer the plate to a 37 degrees celsius and 5%carbon dioxide incubator for two to four hours. Then add another 100 microliters of complete medium and return the plate to the incubator. To change the medium, use a micropipette to aspirate 100 microliters of the medium from the surface of the cultures without disturbing the gel and place the tip against the walls of each well to seed the cultures with 100 microliters of pre-warmed medium.
Using the density gradient separation protocol as just demonstrated, CD45 negative stromal cells can routinely be enriched 15 to 20 times as illustrated in these representative dot plots. Here, thymic epithelial cell aggregates, prepared from four week old red fluorescent expressing transgenic mice as just demonstrated, were cultured for two days in vitro and examined by confocal microscopy. Once mastered, thymic epithelial cells can be enriched for flow sorting in three to four hours.
After its development, this technique paved the way for researchers in the field of thymus tissue engineering to develop injectable, functional mini thymus units to modulate adaptive immune system.
