What are the procedures of cryopreserving cultured cells?

The steps for cryopreserving cultured cells are described below. 

1. Prepare the freezing medium and store at 2-8°C until necessary
2. If using adherent cells, gently detach them from the culture vessel using standard subculture procedure. Resuspend the cells in the complete medium appropriate for that cell type.
3. Calculate the total cell number and percent viability using a hemocytometer, cell counter, or Trypan Blue exclusion dye; then calculate the necessary volume of the freezing medium
4. Centrifuge the cell suspension at the recommended speed (100-200 x g) for 5-10 minutes. Carefully pour off the supernatant without disrupting the cell pellet.
5. Resuspend the cell pellet in the freezing medium at the target viable cell density for that specific type
6. Divide the cell suspension into cryogenic storage vials. While aliquoting, gently mix the cells constantly to ensure cell distribution within each vial.
7. Freeze the cells with a controlled freezing tool, decreasing the temperature to 1°C  per minute. Another option is to place the vials in an isopropanol chamber and leave them overnight at -80°C .
8. Transport the frozen cells to liquid nitrogen and store them (in gas phase) above the liquid nitrogen.