| The following is the recipe of preparing 50 mL TOM. | | |
| Ingredient | Volume(μl) | Final Concentration |
| 100 × N2 (Life Technologies, 17502-048( | 500 | 1× |
| 50 × B27 (Life Technologies, 17504-044) | 1000 | 1× |
| 500 × Primocin (InvivoGen, ant-pm-1) | 100 | 100 μg/ml |
| 80 × NAC (Sigma, A9165) | 625 | 1.25 mM |
| 100 × L-glutamine (Life Technologies, 35050-061) | 500 | 2 mM |
| 10000 × A83-01 (Tocris, 2939) | 5 | 500 nM |
| 10000 × CHIR99021 (Tocris, 4423) | 5 | 1.5 μM |
| 2000 × recombinant hEGF (Gibco, PHG0314) | 25 | 50 ng/ml |
| 2000 × recombinant R-spondin1 (R & D systems, 4645-RS-100) | 25 | 80 ng/ml |
| 2000 × recombinant hFGF2 (Peprotech, 100-18C) | 25 | 100 ng/ml |
| 2000 × recombinant hHGF (Peprotech, 100-39) | 25 | 50 ng/ml |
| 100 × Nicotinamide (NTM)(Sigma, N0636-100G | 500 | 10 mM |
| 500 × Y-27632 (Sigma, Y0503-1MG) | 250 | 5 μM ↑ |
| 2000 × PGE2 (R & D systems, 22-961-0) | 25 | 2.5 μM |
| FBS (heat inactivated) (Cytiva HyClone, SH30070.03( | 5 mL | 10% (vol/vol) |
| Advanced DMEM/F12 (Life Technologies, 12634-010) | Adjust to 50 mL | N/A |
| Annotation: First add about 35 mL Advanced DMEM/F12 to the 50 mL centrifuge tube, then add the above supplements, adjust the final volume to 50 mL with Advanced DMEM/F12. Use the full medium within 1 month. | | |