Human PSC Maintenance Medium

Medium Compatibility

Human PSC Maintenance Medium is compatible with major feeder-free systems such as mTeSR, mTeSR Plus, and E8. Cells in an existing system should remain in the original medium until approximately 80% density before passaging. On day 1 after passage to P2, use a 1 : 1 mixture of the original medium and PSC Medium supplemented with CEPT Cocktail Plus (1000×). From day 2, use PSC Medium alone. By P3, cells are generally adapted to the system.

Reagents and Consumables Required

Basement Membrane Matrix IPSC-qualified (HY-K6006), Stem Cell Cryopreservation Medium (HY-K6019), iPSC/ESC Dissociation Solution (Enzyme-Free) (HY-K6020), CEPT Cocktail Plus (1000×) (HY-K6021), DMEM/F12 (HY-K3002), sterile 1.5/15/50 mL tubes, sterile 1 mL pipette tips, racks, and related consumables.

Aliquoting Basement Membrane Matrix

1. Thaw 5 mL Basement Membrane Matrix IPSC-qualified overnight at 4°C on ice.

2. Prepare twelve sterile 1.5 mL tubes, sterile 1 mL pipette tips, and a tube rack; pre-cool them at -20°C for at least 1 h.

3. Work on crushed ice in a biosafety cabinet. Gently mix the thawed matrix and aliquot 400 μL into each tube while keeping all materials cold.

4. Immediately transfer aliquots to -20°C for storage.

Coating a 6-Well Plate

1. Thaw one 400 μL matrix aliquot at 4°C on ice and pre-cool 1 mL tips at -20°C for at least 1 h.

2. Add 36 mL pre-chilled DMEM/F12 to a 50 mL tube.

3. On ice, add the 400 μL matrix aliquot to the cold DMEM/F12 and mix by gentle repeated pipetting.

4. Dispense 1.5 mL diluted matrix per well of a 6-well plate, gently tap for even coating, and incubate at 37°C for 1 h or at 4°C overnight. Use coated plates within about 2 weeks.

Thawing hESCs/iPSCs

1. Pre-warm a water bath to 37°C. Place the coated 6-well plate in the incubator for 1 h to equilibrate to approximately 15-30°C.

2. Prepare 4 mL Human PSC Maintenance Medium with CEPT Cocktail Plus (1000×) and allow it to equilibrate to 15-30°C. Do not warm this medium in a 37°C water bath.

3. Thaw the frozen cell vial in the 37°C water bath with gentle agitation and complete thawing within 2 min. Remove the vial when ice crystals are nearly gone, disinfect with 75% ethanol, and transfer to the biosafety cabinet.

4. Transfer the cell suspension to a 15 mL tube and slowly add 10 mL DMEM/F12 dropwise while gently mixing.

5. Centrifuge at 160 × g for 5 min and remove the supernatant. Gently resuspend in 4 mL PSC Maintenance Medium containing CEPT. Retaining approximately 200 μL supernatant and gently flicking the tube bottom can help disperse cells while avoiding large aggregates.

6. Remove the coating solution from two wells and seed 2 mL cell suspension per well.

7. Move the plate gently in a horizontal cross pattern three times after seeding and repeat once after placing the plate in the incubator.

8. Perform the first medium change 18-24 h after plating and then change medium daily. When hESC confluence exceeds 50%, use 3-4 mL medium per well.

9. Passage at approximately 85% confluence, generally every 4-8 d. Do not continuously culture a passage for more than 10 d even if colonies remain small.

10. At 4-8 d after passage, proceed to the next passage or cryopreservation according to cell condition.