Protocol – Cryopreservation of adherent cell culture growing in serum-containing medium


  • Cryopreservation is an efficient way of preserving cell culture. Preserved cells can be revived whenever needed.
  • Cryopreservation not only stop the biological time and aging but also protect cell culture from accidental loss due to mishandling and contamination.
  • Cryopreservation involves freezing of cell at ultra low temperature below -135°C.
  • Cell suspension is prepared in specialized medium, the freezing medium, which contains DMSO and high concentration of serum.
  • DMSO, a cryoprotectant, protects cells from death by preventing ice crystal formation. High serum content also protects cells from death during cryopreservation and revival.
  • Cell suspension is subjected to slow cooling overnight at -80°C before storing them in liquid nitrogen. Slow cooling reduces the probability of of ice crystal formation, thus protects cells from lysis.
  • Serum containing cryopreservative medium is used to preserve cells lines which are maintained in serum containing growth medium.


  • Reagents
    • Freezing medium (Composition: 50% FBS + 10% DMSO + 40% growth medium)
    • Complete growth medium (e.g., DMEM supplemented with 10% FBS)
    • PBS without Ca2+/Mg2+
    • Trypsin EDTA (TE)
  • Equipment and disposables
    • Sterile cryogenic vials
    • Sterile conical tubes (15-mL or 50-mL)
    • Controlled rate freezing apparatus
    • Haemocytometer/Trypan Blue
    • -80°C freezer
    • Liquid nitrogen storage container/-150°C freezer
    • Benchtop centrifuge with 45° fixed-angle or swinging-bucket rotor (e.g., Eppendorf™ 5804 Series)
    • Personal protective equipment (sterile gloves, laboratory coat, Full-face protective mask/visor)
    • Laminar flow hood
    • Pipette tips and pipetman
    • Serological pipettes and Pipetboy
    • Inverted phase contrast microscope

Starting materials:

  • Late log-phase monolayer culture (80% confluent), growing in culture dish/flask
  • Visually inspect culture carefully under an inverted phase contrast microscope and make sure that cells are healthy and do not have any contamination.
  • Properly label Cryogenic vials. You must write cell line name, passage number and date.

Overview of procedure:

Harvested cells from monolayer culture is resuspended in ice-cold freezing medium at the recommended cell density (2 x 106 – 5 x 106). Cell suspension is aliquoted in ice-cold Cryogenic vials (1 ml/vial). All Cryogenic vials are subjected to slow cooling (1 – 3°C/min) overnight in -80°C freezer. Next day, all cryogenic vials are transferred to liquid nitrogen containers or -150°C incubator.

Here we describe a general procedure for cryopreserving adherent cell culture. For specific details, we recommend you to carefully read the manual provided with the cell line.


Step 1: Harvest cells from the culture using standard procedure (trypsinization)
  • Discard the culture medium and wash the monolayer with PBS. Add sufficient amount trypsin EDTA solution (1 -2 ml for T25 flask) and incubate at 37°C for 1 – 2 min. Inspect the dish for cell detachment. Incubate at 37°C for some more time if cells are not dislodged.
  • Once the cells are dislodged, tap the flask/dish 2-3 times to make sure all the cells have come out from the dish surface. Add serum containing complete medium (4 ml for T25 flask) and flush the entire surface of dish by pipetting to collect all cells from the dish.
  • Transfer cell suspension to a sterile centrifuge tube. Cells from two or more dishes from the same passage number (subculture) can be combined in one tube.
  • Serum in the complete growth medium has trypsin inactivating activity.
  • At this point, cells can be collected by centrifugation and cell pellet can be resuspended in sufficient amount of complete medium. This step is not necessary. In case of high cell death during trypsinization process, or if the cells are too diluted in suspension, centrifugation and resuspension of cells can be done.
  • Make sure that trypsinization of cells does not cause enormous cell death. A healthy culture is a prerequisite for cryopreservation.
Step 2: Determine viable cell density in cell suspension (optional)
  • Determine viable cell number using trypan blue exclusion assay and hemacytometer.
  • To count viable cell number, mix 20 µl cell suspension to 20 µl trypan blue solution. Place the solution onto haemocytometer chamber.
  • Count live and dead cells. Calculate viable cell count per ml and percentage of dead cells.
  • You can use other sophisticated methods like Countess® Automated Cell Counter or Moxi Flow Kit which can determine viability cell count quickly.
  • If cells are very diluted, collect cells by centrifugation and resuspend cell pellet in appropriate amount of complete medium.
  • In many cases, based on previous experience you can determine how many cryovials can be frozen from a culture dish. One can prepare 2- 4 ml cell suspension in freezing medium from a near confluent T25 flask which can be used to prepare 2 – 4 cryogenic vials (1 ml / vial).
  • Counting is required when cell number is limited and you want to freeze as many cryogenic vials as possible e.g., primary culture.
  • Make sure that cells are properly resuspended before counting viable cells.
Step 3: Resuspend the cells in ice-cold freezing medium at the recommended viable cell density  (2 x 106 – 5 x 106 cells/ml)
  • Harvest cells from cell suspension by centrifugation at 4°C for 5 – 10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge).
  • Carefully aspirate supernatant completely without disturbing the cell pellet.
  • Flick the tube with your finger several times to dislodge the pellet.
  • Add appropriate amount of ice cold freezing medium to obtain right viable cell density (between 2 x 106 – 5 x 106 cells/ml). Resuspend the cells thoroughly with gentle pipetting.
  • Cell density in freezing medium can vary with cell lines. In most cases, high cell density is good for cell recovery.
  • Since DMSO can be toxic to cells, it is advisable to use chilled freezing medium. Try your best to maintain the temperature of cell suspension 4°C.
  • Quickly resuspend pellet in freezing medium immediately after aspirating supernatant.
  • Centrifugation speed should be sufficient to get soft pellet. Pellet should not be too tight. Tight pellet will be difficult to resuspend and attempts to resuspend it by vigorous pipetting may cause cell death.
Step 4: Aliquot cell suspension to cryogenic vials
  • Place cryovials on ice.
  • Transfer 1 ml aliquots of cell suspension into cryovials.
  • Tighten caps on vials.
  • While aliquoting, frequently and gently mix the cells to maintain a homogeneous cell suspension.
Step 5: Subject cryovials to slow cooling (1 – 3°C/min) overnight in -80°C freezer
  • Place all cryogenic vials in controlled rate freezing apparatus (e.g., CoolCell® Cell Freezing Containers from Biocision) and immediately store in -80°C freezer overnight.
  • The most efficient way to freeze cryogenic vials is to use controlled-rate freezers (e.g., CryoMed™ Controlled-Rate Freezers from ThermoFisher Scientific). However, for most serum grown cell lines, one can use commercially cooling devices e.g., CoolCell® Cell Freezing Containers from Biocision, or  Mr. Frosty™ Freezing Container from ThermoFisher Scientific.
  • Homemade cooling devices – Thermocol box (small size) filled with cotton or tissue paper, can also used in place of commercial cooling devices.
Step 6: Store in liquid nitrogen
  • Transfer all frozen cryogenic vials to a liquid nitrogen container next day. Alternatively, frozen cryogenic vials can also be stored in -150°C freezer.
  • Cryogenic vials can be store in gas phase or liquid phase of liquid nitrogen.
  • Revive a vial after 2 weeks to ensure that frozen cells are viable and free of any contamination.
  • Biosafety level 2 cell lines should be stored in the gas phase of liquid nitrogen.
  • You must maintain the proper records of location of frozen cell lines.
  • Wear protective equipments when handling liquid nitrogen. Remember that cryogenic vials may explode if they are stored in liquid phase of liquid nitrogen.