Tissue Culture - Storage of Cell Lines

1. Established hybridomas and other cell lines can be stored in liquid N₂ using this method.
   
2. Have everything ready before you start working with the cells (i.e. cool media, label vials, prepare cryovials, etc.)
   
3. Freeze only cultures that are healthy and in the mid-log phase of growth (ie., 2-5 x 10⁵ cells/ml).
   
4. Have your vials already labelled with a waterproof, cryogenic pen. Include cell line name, passage number, date frozen and number of cells per vial.
   
5. Centrifuge the cells and resuspend in 10% DMSO/90% FCS at about 1-5 x 10⁶ cells/ml. The freezing medium should be 5ºC, not warm.
   
6. Put 1ml of cell suspension into cryovials and tighten the lid finger tight. Do not over tighten or the vials could explode when thawed.
   
7. Use the Nunc cryovials with the O-ring and inner threads, not the Nalgene ones. Put the vials in one of the cryoracks specially designed for slow, even cooling.
   
   • The Nalgene cryocontainer holds 18 vials. The bottom is filled with isopropanol which must be changed after five uses. Follow the instructions on the container.
     
   • The blue cryorack holds 50 vials. It is filled with water, plugged and placed plug side down. Put the vials right side up into the spaces and place the rack in this position in the -70ºC freezer. Do not place them in a "pre-frozen rack". Do not fill 100% full or the plug will pop while freezing.
8. Record in the card file: cell line frozen; # cells; date frozen; number of vials; cane label/ #; which cane holder; your initials/name.