n/a n/a (In vitro cultured cells or hybridomas)

(General Cryopreservation Protocol for In Vitro Cultured Cells)


This general protocol for the cryopreservation of cell lines or hybridomas is adapted from an appendix in Current Protocols in Immunology and was last published in 2001. It is highly recommended that if you follow this protocol you optimize its parameters for your cell type.


• Cell line to be frozen
• Freezing medium (see recipe)
• 75-cm2 tissue culture flask, sterile
• Cryovials, sterile
• Indelible marker, resistant to cold, water, and ethanol
• 50-ml conical centrifuge tube, sterile
• Beckman TH-4 rotor (or equivalent)
• Freezer storage boxes
• 70% ethanol
• Medium used for growth of cell line
• Dry ice
• 12-ml tube with cap, sterile
• Tissue culture flasks


1. Grow cell line to be frozen to mid-log phase in a 75-cm2 tissue culture flask.
2. Label sterile cryovials using an indelible marker with name of cell line, medium, and date. Alternatively, a single log number corresponding to the listing of this information in a log book can be used.
3. Use a pipettor and a sterile 25-ml pipet to transfer the cells to a sterile 50-ml conical centrifuge tube. In cases where cells are adherent to the plastic surface, they should be dislodged (gently) with an appropriate scraper or removed by trypsinization.
4. Centrifuge the cells 5 min in TH-4 rotor at 1500 rpm (500 × g), room temperature. Discard the supernatant and save the pellet.
5. Add freezing medium to the cell pellet to achieve a final cell concentration of 2 × 106 cells/ml and resuspend cells gently. This concentration allows re-seeding of 75-cm2 flasks with a single cryovial. However, concentrations up to 107 cells/ml can be successfully frozen.
6. Transfer 1 ml of resuspended cells to each labeled cryovial. Cap the vial and immediately place in a freezer storage box in a −70°C freezer.
7. For short-term storage, leave cells at −70°C (see Critical Parameters). For long-term storage, transfer cells to a liquid nitrogen freezer.
8. Record the names and locations of all stored cell lines in a log book.

To thaw:
1. Retrieve cryovial containing cell line from storage area (−70°C or liquid nitrogen freezer) and place on dry ice to minimize thawing.
2. Thaw cells by placing bottom half of cryovial in a 37°C water bath (do not submerge completely). Swirl cryovial gently until cells are thawed.
3. Take cryovial to tissue culture hood and wipe vial with 70% ethanol.
4. Gently open cryovial and resuspend the cells gently using a sterile 1-ml pipet. Transfer the cells to a sterile 12-ml tube.
5. Add 9 ml medium, 1 to 2 drops at a time, with swirling. Cap the tube, centrifuge 5 min in TH-4 rotor at 1500 rpm (500 × g), room temperature, and discard supernatant.
6. Resuspend cells in medium to desired concentration and transfer to a tissue culture flask or other desired culture vessel.
7. Inspect cells using an inverted microscope for morphology. Examine an aliquot of cells for the ability to exclude trypan blue (APPENDIX 3B). If cells pass both inspections, they are ready for culture.


Yokoyama, W. M. 2001. Cryopreservation of Cells. Current Protocols in Immunology. 21:A.3G.1–A.3G.3.

Added by: Carlton Hoyt on July 25, 2012

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