Preparation of culture medium
Feeder cell culture medium: DMEM supplemented with 10% FBS and 1X penicillin-streptomycin.
2i/LIF mESC medium: Knock-out DMEM supplemented with 15% FBS, 1X L-glutaMAX, 1X penicillin-streptomycin, 1X non-essential amino acids (NEAA), 1X sodium pyruvate, 0.1 mM 2-mercaptoethanol, 1000 U/mL mouse leukemia inhibitory factor (mLIF), 3 µM CHIR-99021 and 1 µM PD0325901.
ciTotiSC medium: Knock-out DMEM supplemented with 5% KSR, CDL, 1% N2, 1X L-glutaMAX, 1X penicillin-streptomycin, 1X non-essential amino acids (NEAA), 1X sodium pyruvate, 0.1 mM 2-mercaptoethanol and 1000 U/mL mouse leukemia inhibitory factor (mLIF), 50 ng/ml Sodium L-ascorbyl-2-phosphate, 2.5 µM 1-Azakenpaullone, 0.5 µM WS6 and 0.2 µM TTNPB.
Note: 50 nM ~ 200 nM TTNPB was recommended to be tested in different cell lines.
Derivation of ciTotiSCs from mESCs:
Day 0:
1. Pre-coat 12-well plate with 1 ml 0.1% gelatin per well and incubate the plate at 37 °C for 30 min.
2. Remove the gelatin solution and seed inactivated MEF feeders at a density of 2X105 per well in gelatin pre-coated 12-well plate with feeder cell culture medium.
3. Incubate the feeder cells overnight in incubator at 37 °C with 5% CO2 and 95% air.
Day 1:
4. Aspirate out the feeder cell culture medium, change to 1 ml pre-warmed ciTotiSC medium, and equilibrate the culture medium in the incubator for 10 min.
5. mESCs are routinely cultured on inactivated MEF feeders using 2i/LIF mESC medium, and are passaged every 2-3 days using 0.05% trypsin-EDTA. For ciTotiSC derivation, upon mESCs reaching 80% confluency, aspirate out the medium, wash once with DPBS, and then incubate the cells with 500 µl 0.05% trypsin-EDTA at 37 °C for 3 min.
6. Add 2 ml 2i/LIF mESC medium, pipette cells up and down gently into single cells, transfer cell suspension to a 15 ml polystyrene conical tube and centrifuge cells at 1,000 rpm for 5 min.
7. Aspirate out the supernatant, resuspend the dissociated cell pellet with pre-warmed ciTotiSC medium and seed the cells with split ratios of 1:10~1:15 on inactivated MEF feeders.
8. Incubate the cells in incubator overnight at 37 °C with 5% CO2 and 95% air, and change the medium every day with freshly pre-warmed ciTotiSC medium.
Day 3/4:
9. After culturing with ciTotiSC medium for 2-3 days, ciTotiSC colonies will arise and reach 70-80% confluency.
10. Aspirate out ciTotiSC medium, wash once with DPBS, and then incubate the cells with 500 µl 0.05% trypsin-EDTA at 37 °C for 3 min.
11. Add 2 ml fresh ciTotiSC medium, pipette cells up and down gently into single cells, transfer cell suspension to a 15 ml polystyrene conical and centrifuge cells at 1,000 rpm for 5 min.
13. Aspirate out the supernatant, resuspend the dissociated cell pellet in pre-warmed ciTotiSC medium and seed the cells with split ratios of 1:3~1:5 on inactivated MEF feeders.
14. Incubate the cells in incubator overnight at 37 °C with 5% CO2 and 95% air, change the medium every day with freshly pre-warmed ciTotiSC medium.
15. ciTotiSCs are passaged every 2-4 days at high densities (1:3-1:5) when they reach 70-80% confluency.
Note: To ensure ciTotiSCs remain healthy, do not culture them on feeder cells pre-plated for more than one day in advance. To enhance the survival of passaged ciTotiSCs, Y27632 can be added on the first day after passaging.