Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants
Transcrição
On day 7 of dGUO treatment, take out four new 10-centimeter dishes. Fill each dish with milliliters of complete media. Transfer all of the nitrocellulose membranes with thymic lobes into one 10-centimeter dish. Using forceps, detach the individual lobes from the membrane, allowing them to be submerged in media.
Incubate the lobes for 1 hour at room temperature. Then, transfer the thymic lobes to a new 10-centimeter dish with complete media, and incubate for 1 hour at room temperature. Repeat this transfer and incubation an additional two times.
Using forceps, fix the thymic lobes to the dish, while using the other hand to make a 100 to 200-micrometer-deep incision in the center, and extending half the diameter of the lobe to facilitate T cell progenitor migration into the lobe.
Transfer the thymic lobes to a new 10-centimeter dish filled with complete differentiation media. If using 3D culture plates with lower and upper-level grids, fill both grids with sterile PBS to prevent the evaporation and drying of the hanging drops.
Next, transfer 30 microliters of complete media containing one dGUO-treated thymic lobe into each well of the 3D culture plate. Collect the non-adherent T lineage cells from the OP9/DLL1 co-culture, and re-suspend the cells at a density of 2,000 to 5,000 T cell lineage cells per 20 microliters of media. Add 20 microliters of the T lineage cell suspension to each thymic lobe in the 3D culture plate. Incubate overnight at 37 degrees Celsius with 5% carbon dioxide.
The next day, set the P200 pipette to 30 microliters. Pipette the media in each well up and down several times, to remove all the cells surrounding the thymic lobes. Then, aspirate the media from each well, and replace it with 30 microliters of complete media. Repeat this process — pipetting, removing, and replacing the media — 5 to 7 times to remove any extra immature T cells which does not migrate into the lobes.
Continue incubating the lobes making sure to change 25 to 30 microliters of media daily. On day 4 or 5, use light microscopy to confirm the formation of a halo of iPSC-derived thymic immigrants around the lobes. Collect the iTEs daily by pipetting media without lobe disruption. Change the media every day and continue the collection up to approximately 12 days.
Harvested iTEs are ready to use for molecular analysis or de vivo transplantation experiments.
