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 dish or microplate, and gently wash the cells 1–2 times with DPBS (calcium- and magnesium-free).

2. Add 1 mL of UniZyme preheated to room temperature and incubate in a 37°C incubator for approximately 5 minutes. Alternatively, add 1 mL of UniZyme preheated to 37°C and incubate in a 37°C incubator for approximately 2 minutes.

3. Examine the cells under a microscope; if most of them are still adherent, extend the digestion time as needed.

4. Gently pipette up and down or tap the side of the container to dislodge the cells from the surface. Collect the digested cells into a pre-prepared 15 mL centrifuge tube containing 5 mL of iPSC maintenance medium (Cat. No.: R2-01-01) or DMEM/F-12. Rinse the surface of the container once with the medium from the centrifuge tube to collect as many cells as possible.

5. Centrifuge for 3 minutes, then discard the supernatant.

6. Resuspend in 1 mL of iPSC maintenance medium (containing CEPT) and count the cells.

7. 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.)

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

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

10. Proceed with medium changes and passaging according to standard culture protocols.