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Preservation (Cell Freezing)

Materials:

• Medium (with 15% FBS)
• DMSO (final concentration is 10% v/v)
• 1.8 ml sterile cryovial (Nunc #3-66656)
• Cryo 1 freezing container(cryogenic controlled rate freezing container), Nalgene (VWR Cat. No. 55710-200) or (Fisher Cat No. 15-350-50)

Protocol:

1. Renew (change medium) cells 24 hours prior to freezing. Cells should be healthy and at the edge of confluence at time of freezing. They should also have many mitoses evident by phase microscopy (~ day 8 of propagation).
2. Trypsinize cells as described in the subculture methods. Count cells and determine the amount per vial to be frozen (a good cell number per vial is between 1 and 5 x 10⁶).
3. After cells have been harvested, as described in the subculture methods and counted, the cells should then be centrifuged again, the supernatant aspirated and the cells resuspend in the predetermined volume of medium with 15% FBS (the volume of the medium in which the cells are resuspended is determined as follows: 0.45 ml medium x vials to be frozen). This should be performed in a stock Gently pipette cell suspension up and down a few times.
4. Slowly (dropwise) add predetermined volume of DMSO to the stock tube (50 m l DMSO x vials to be frozen = 10% v/v of DMSO), while gently mixing the suspension.
5. Place 0.5 ml aliquots into cryovials labeled with cell type, passage number, date, number of cells and initials.
6. Transfer vials into a cryo 1° freezing container and place into a -80° C freezer overnight. Freeze rate down to approximately 1° /min.
7. Transfer vial to liquid nitrogen and record location and all other information on vial.

Alternate Protocol:

1. Renew (change medium) cells 24 hours prior to freezing. Cells should be healthy and at the edge of confluence at time of freezing. They should also have many mitoses evident by phase microscopy.
2. Trypsinize cells as described in the subculture methods. Count cells and determine the amount per vial to be frozen (a good cell number per vial is between 1 and 5 x 10⁶).
3. Resuspend cells in the appropriate volume of Freezing Medium (90% Medium with 15% FBS; 10% DMSO). Please work quickly once cells are resuspended in Freezing Medium, DMSO is toxic to cells.
4. Place 0.5 à 1 ml/cryo vial (10⁶ cells) labeled with cell type, passage number, date, number of cells and initials.
5. Transfer vials into a Cryo 1° freezing container(cryogenic controlled rate freezing container) and immediately place into a -80° C freezer overnight (freeze rate = ~ 1° /min.).
6. Transfer vial to liquid nitrogen and record location and all other information on vial.