First, observe the cells; when they reach approximately 80% confluence and are in good condition, passaging can be performed. Generally, passage every 3–6 days. Even if the colonies are small and confluence is low, it is not recommended to culture continuously for more than 7 days. Alternatively, if confluence is low but the stem cell colonies are too large with poor growth in the center, passaging should also be carried out. The passaging ratio can be 1:5 to 1:10, depending on cell growth status and experimental needs. During digestion, when cracks appear in the colonies under the microscope, this indicates complete digestion, and the digestion should be terminated. If the cells after passaging and seeding are mostly single cells rather than small clumps, or if cell viability is low, it is recommended to add Y27632 within the first 24 hours to help cells attach and survive. After 18–24 hours, replace with new medium without Y27632, and then change the medium daily thereafter.

The iPSC cells shipped by our company are generally cryopreserved as clumps after passaging. The main reason why most cells appear as single cells upon seeding is excessive pipetting after thawing and centrifugation, which breaks apart the cell clumps. iPSCs have low viability in single‑cell form and are more difficult to attach. If, 24 hours after thawing, you observe low cell viability and poor attachment, you should still change the medium immediately. iPSCs have a very strong proliferation capacity; even if only a few cells attach, they may still grow out. However, if the medium is not changed promptly, they will definitely not recover.