Cell Freezing Procedure

Cell freezing is a crucial step in preserving cell lines for long-term storage and use in various research applications. Freezing cells helps maintain their viability, prevents contamination, and allows for easier transportation. Here is a general protocol for cell freezing:


  1. Cell culture flask or plate containing the cells
  2. Trypsin-EDTA or other suitable cell detachment reagent
  3. Cell culture media with serum
  4. Freezing medium (e.g., cell culture media with 10% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS))
  5. Sterile centrifuge tubes
  6. Cryovials (sterile and pre-chilled)
  7. Pipettes and tips
  8. Cell counter (e.g., hemocytometer or automated cell counter)
  9. Incubator (37°C, 5% CO2)
  10. Sterile biosafety cabinet
  11. Centrifuge
  12. Ice or ice-water bath
  13. Freezer (-80°C)
  14. Liquid nitrogen storage system


  1. Begin by working in a sterile biosafety cabinet to prevent contamination.
  2. Gently aspirate the media from the cell culture flask or plate.
  3. Wash the cell monolayer with sterile PBS or a suitable buffer to remove any residual media.
  4. Add an appropriate volume of trypsin-EDTA to detach the cells from the surface. Incubate the flask or plate for 2-5 minutes at 37°C.
  5. Periodically check for cell detachment under a microscope. Gently tap the flask or plate to facilitate cell detachment if necessary.
  6. Once the cells have detached, add an equal volume of cell culture media with serum to neutralize the trypsin.
  7. Transfer the cell suspension to a sterile centrifuge tube.
  8. Centrifuge the cell suspension at 200-300 x g for 5 minutes to pellet the cells.
  9. Carefully aspirate the supernatant without disturbing the cell pellet.
  10. Resuspend the cell pellet in an appropriate volume of pre-chilled freezing medium. Mix gently but thoroughly.
  11. Determine the cell concentration and viability using a cell counter.
  12. Adjust the cell concentration to the desired level (e.g., 1 x 10^6 cells/mL) with the freezing medium.
  13. Aliquot the cell suspension into pre-chilled cryovials (1-2 mL per vial).
  14. Place the cryovials in an ice-water bath for 10-15 minutes to begin the cooling process.
  15. Transfer the cryovials to a controlled-rate freezing container or a -80°C freezer for 24 hours. This step allows for a slow decrease in temperature, which is essential for optimal cell viability.
  16. After 24 hours, transfer the cryovials to a liquid nitrogen storage system for long-term storage.

Remember to always work under sterile conditions and follow specific guidelines for the cell line you are working with. This protocol is a general guideline, and specific conditions may vary depending on the cell type and the requirements of your laboratory.