updated 10/30/08 MDR



  1. Cell-appropriate freezing medium (no antibiotics)
  2. Cells to freeze
  3. PBS and cell-appropriate trypsin-EDTA solution (for adherent cells)
  4. Sterile dimethyl sulfoxide, DMSO
  5. 15-mL tube
  6. cryovials
  7. ice
  8. Isopropanol-filled freezing jar (Mr. Frosty)
  9. cryogloves


  • The following protocol prepares frozen stocks from 25-cm2 (T25) tissue culture flasks; modify volumes as necessary; ALWAYS FOLLOW SUPPLIER'S RECOMMENDATIONS
  • Feed the cells one day prior to preparing liquid nitrogen stocks to improve viability
  • For AAV-293 cells, it is important to maintain cells that are propagated to establish a liquid nitrogen stock at no greater than 50% confluency to ensure the integrity of the stock, maintaining the higher titer production phenotype
  • For other cell lines, collect cells from a healthy log-phase culture (approximately 80% confluent)
  • DMSO is a toxic cryoprotectant used to prevent intracellular ice crystal formation during freezing. As it it less toxic at cooler temperatures, be sure to only add chilled solutions to chilled cell suspensions and work quickly
  1. Place isopropanol-filled jar in -20C freezer
  2. Label cryovials with cell line, date, passage number, # of cells, and your initials; chill on ice
  3. Prepare 20%DMSO/freezing medium mixture; chill on ice
  4. Prewarm medium to 37C in water bath (for suspension cultures, skip to step 10)
  5. Prewarm Trypsin-EDTA solution to 37C in water bath
  6. Aspirate culture medium from flask
  7. Wash cells twice with 1mL PBS
  8. Trypsinize cells with 1mL of Trypsin-EDTA solution for 1 minute in the 37C incubator, check cells under the microscope. If more time is needed, check cells every 30 seconds to ensure that you only incubate for the minimum time required to release adherent cells; excess trypsinization may damage or kill the cells
  9. Dilute the cells with 2mL of warm medium to inactivate the trypsin. Transfer the cell suspension into a 15-mL tube. Wash inside of flask with an additional 4-6mL of medium to collect all of the cells; transfer to the 15-mL tube
  10. If applicable, use ~150ul cell suspension for counting
  11. Pellet the cells by centrifugation at 700 rpm for 5 minutes
  12. Aspirate off medium. Resuspend cells by gently pipetting up and down in half the volume of cold medium that will be used for freezing. (i.e. use 1mL if you'll be freezing 2 aliquotts). Place on ice.
  13. Add cold 20%DMSO/freezing medium to vial in a drop wise fashion, swirling gently between drops.
  14. Transfer 1mL aliquots to cold cryovials, cap tightly.
  15. IMMEDIATELY Place vials in isopropanol-filled freezing jar (for gradual freezing, -1C/min) and leave in -80C freezer overnight. Then, transfer vials to liquid nitrogen tank (in the cold room). Be sure to use cryogloves and wear lab appropriate attire (pants, closed-toe shoes, and lab coat) to prevent personal injury.
  16. Update frozen cell log online.
  17. Return Mr. Frosty to -20C freezer
Adapted from Stratagene AAV Helper-Free System