Procedure for Passaging iPSC Single Cells (Using One Well of a Six-Well Plate as an Example)

1. Remove the old culture medium from the culture dishes/plates and gently wash the cells 1–2 times with DPBS (calcium- and magnesium-free).

2. Add 1 mL of Gentlease that has been warmed to room temperature (never warm Gentlease in a 37°C water bath), and incubate in a 37°C incubator for approximately 5 minutes. Observe the cells under a microscope; if most cells are still adherent, extend the digestion time as needed.

Note: While Gentlease can terminate digestion by direct dilution, we still recommend the more reliable centrifugation and medium exchange method for critical experiments or extremely valuable cell samples to completely eliminate any potential minor effects. We recommend two methods for cell passaging:

3. Direct dilution method:

3.1 Add twice the volume of iPSC maintenance medium (containing CEPT).

3.2 Collect the cells into a 15-mL centrifuge tube, gently pipette to disperse them into a single-cell suspension, and count them.

3.3 Seed the cells at the appropriate density into the pre-coated microplate. (See the instructions for use for Soma Stem Cell Seeding Medium or fibronectin.)

3.4 Incubate in a 37°C, 5% CO₂ incubator.

3.5 After 24 hours, replace the medium with fresh iPSC maintenance medium without CEPT (Catalog No.: R2-01-01).

3.6 Proceed with medium changes and cell passaging according to standard culture procedures.

4. Centrifugal liquid exchange method:

4.1 Add twice the volume of DPBS (calcium- and magnesium-free).

4.2 Collect the cells into a 15-mL centrifuge tube, gently pipette to disperse them into a single-cell suspension, and count them.

4.3 Centrifuge at 300 g for 3 minutes, then discard the supernatant.

4.4 Resuspend the cells in an appropriate volume of iPSC maintenance medium (containing CEPT) and seed them into coated microplates.

4.5 Incubate in a 37°C, 5% CO₂ incubator.

4.6 After 24 hours, replace the medium with fresh iPSC maintenance medium without CEPT (Catalog No.: R2-01-01).

4.7 Proceed with medium changes and cell passaging according to standard culture procedures.