FZStem™ Stem Cell Freezing Medium
Used for the cryopreservation of human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells.
2. Culture the cells in a 6-well plate until 60% to 80% confluent.
3. Aspirate the medium from the hES/hiPS cell culture and rinse with DPBS (2 mL/well).
4. Add 0.5 mL FZStem enzyme-free stem cell dissociation solution to each well. Let it stand at room temperature for 1-2 minutes.
5. Aspirate the dissociation solution, and then gently rinse each well with 2 mL DMEM/F-12 2-3 times.
6. Add 2 mL/well fresh medium, and scrape the colonies with a cell scraper.
7. Transfer the separated cell suspension to a 15 mL conical tube.
8. Centrifuge at 200 x g for 5 minutes at room temperature.
9. Aspirate the supernatant gently and loosen the cell pellet by tapping on the bottom of the test tube.
10. Gently resuspend the pellet in cold FZStem freezing medium, taking care to make the clumps larger than the usual passaging process.
11. Transfer 1 mL of cell suspension to each labeled cryogenic vial.
12. Place the vial in an isopropanol freezing container, and place the container at -80°C overnight.
13. Transfer to the liquid nitrogen tank the next day.