Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants

To generate iPSC-derived thymic emigrants, iTEs from immature T cells in a 3D thymic organ culture system, take deoxyguanosine, dGUO-treated mouse fetal thymic lobes in suitable media.

dGUO enters the thymic environment and destroys endogenous thymic lymphocytes, preventing their interference with iPSC-derived immature T cells during co-culture.

Make a deep incision at the center of each thymic lobe. Transfer the lobes to a fresh culture dish containing medium supplemented with the growth factors stem cell factor, FLT3 ligand, and interleukin-7.

Pipette the thymic lobe-containing media into the wells of a 3D culture plate. Add iPSC-derived immature T cells. Incubate. The T cells migrate into the thymic lobes through the incision.

Within the thymic environment, the growth factors FLT3 ligand and IL-7 bind to their respective receptors on immature T cells leading to the activation of transcription factors and the expression of specific genes.

Consequently, the immature T cells undergo a series of changes and acquire specific characteristics, ultimately maturing into CD8αβ+ T cells which express CD8 molecules, T cell receptors, and MHC class I molecules on their surface.

Remove the immature T cells that have failed to migrate into the lobes.

The mature iTEs migrate out of the thymic lobes which can be visualized as a halo-like pattern surrounding the lobes under a light microscope.

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 in vivo transplantation experiments.