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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. 3D Thymic Organ Culture to Generate iTE
- Harvest mouse fetal thymic lobes and deploy endogenous lymphocytes by deoxyguanosine (dGUO) treatment.
- On day 7 of dGUO treatment, take four new 10 cm dishes and fill each with 20 mL of complete media (Roswell Park Memorial Institute Media 1640 [RPMI 1640] + 10% FBS + 1x L-alanyl-L-glutamine + 1x sodium pyruvate + 1x minimum essential medium with non-essential amino acids (MEM-NEAA) + 1x penicillin-streptomycin + [1:1000] 2-mercapto ethanol).
- Transfer all nitrocellulose membranes with thymic lobes into one 10 cm dish. Detach the individual lobes from the membrane with forceps, allowing them to be submerged in media. Discard the membranes. Incubate for 1 h at RT.
- Transfer the thymic lobes to a new 10 cm dish with complete media and incubate for 1 h at RT. Repeat this step 2 more times.
- Using forceps, fix the thymic lobes to the dish (one at a time), and with the other hand make a 100–200 µm deep incision in the center and extend half the diameter of the lobe to facilitate T cell progenitor migration into the lobe.
- Transfer the thymic lobes to a new 10 cm dish filled with complete differentiation media (complete media + 5 ng/mL mouse interleukin-7 (IL-7) + 5 ng/mL mouse Flt3 ligand (FLT3L) + 5 ng/mL stem cell factor (SCF)).
- Optionally, 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.
- Transfer 30 µL of complete media containing one dGuo-treated thymic lobe from step 1.6 into each well of the 3D culture plate.
- Collect non-adherent T lineage cells (iPSC-derived immature T cells) from OP9/DLL1 co-culture (days 16-21) and resuspend at 2–5 x 103 T lineage cells per 20 µL media.
- Add 20 µL of T lineage cell suspension to each thymic lobe in the 3D culture plate. Incubate overnight at 37 °C with 5% CO2.
- Set the P200 pipet to 30 µL and aspirate the media after pipetting several times from each well to remove all the cells surrounding the thymic lobes. Discard media and add 30 µL of complete media. Repeat this procedure 5–7 times to remove any extra immature T cells which do not migrate into the lobes. Change 25–30 µL of media daily thereafter.
- Confirm the formation of a halo of iPSC-derived thymic emigrants (iTE) around the lobes beginning on days 4–5 by light microscopy.
- Collect iTE daily by pipetting media without lobe disruption. Change media every day and continue collection for up to approximately 12 days.
- Harvested iTE are ready to use for molecular analyses (Figure 1, Figure 2, Figure 3, and Figure 4) or in vivo transplantation experiments.
