Basic Procedure for Cryopreservation and Reconstitution of Cultured Cells
NOTE: Not applicable to CloneticsTM and PoieticsTM Primary Human or Animal Cells.
Cryopreservation
1. Select a flask of cells at or near confluency.
2. Cells should be first removed by trypsinization.
3. Adjust the cell concentration to between 2 × 106 and 8 × 106 cells/mL with EMEM containing 20% Fetal Bovine Serum. Centrifugation may be necessary.
4. Add the above cell suspension to an equal volume of cold (+4°C) Cryoprotective Freezing Medium.
5. Mix continuously to ensure homogeneity.
6. Dispense either 4 mL of the cells suspended in Cryoprotective Freezing Medium into 5 mL glass ampoules, or 1 mL into plastic screw cap vials suitable for freezing in the liquid or vapor phase of liquid nitrogen.
7. Cells are now ready for the freezing cycle. Cells should not be allowed to remain in the Cryoprotective Freezing Medium for more than 1 hour before freezing.
8. The temperature of the contents in the ampoule must then be lowered at a rate of 0.5°C–2°C/minute throughout the range of +4°C to -30°C.
9. After the temperature has reached -30°C, the rate of the temperature drop to -70°C (which is the warmest temperature at which cells can be stored) can be done very quickly. An automatic programmable freezer system is the most reliable means of obtaining controlled rate freezing.
10. Storage of the ampoules or vials must be at -70°C or colder. A storage temperature of -196°C (liquid nitrogen) is best. It is essential that the temperature of the contents of the ampoule be -70°C or colder at all times until reconstitution. For prolonged or indefinite storage,the use of liquid nitrogen is strongly recommended. Storage in dry ice or a mechanical freezer (-70°C) should be limited to less than 3 months.
Ampoule Handling Recommendations – Receipt of Ampoule
Upon receipt, transfer the ampoule(s) from the shipping container to a -70°C freezer or a vapor phase nitrogen tank. For long-term storage (over 3 months), a vapor phase nitrogen tank is preferable to prevent significant loss of viability. Immersion of screw cap ampoules in liquid nitrogen is not recommended.
Reconstitution
Caution: Wear protective facemask and clothing as ampoule explosions can occur.
- Remove an ampoule from the freezer and place it into a 37°C waterbath. Do not submerge the ampoule or allow water to get under the cap.
- After thawing, disinfect the ampoule with 70% isopropanol, then open aseptically in a laminar flow safety cabinet. Dilute the contents 1:10 in the appropriate growth medium.
- Determine the viability and cell concentration of the thawed cells by using the Trypan Blue exclusion cell counting method.
- Adjust the cell concentration as desired for seeding culture vessels.
- Twenty-four hours after cells have been seeded, remove the medium and re-feed with the appropriate growth medium. This will remove the cryopreservative, if the alternative method described below is not used.
- As an alternative, the cryopreservative may be removed prior to cell viability determination by centrifugation. This is done by centrifuging the resuspended cells at low speed for 10 minutes (200 × g). The supernatant is removed and the cells are resuspended in the appropriate growth medium.
Reference:
- Freshney, R.I. (2000) Culture of Animal Cells: A Manual of Basic Technique, 4th edition, Wiley-Liss, Inc., New York, pp. 297–308.