Section7:Cryopreservation of Cells

From Assay Guidance Wiki

Below is a protocol for the cryopreservation of cells for either the use of cell culture growth and maintenance of cell culture or for use in cell based assays.

1. Cryopreservation
   1. Remove media from flask.
   2. Wash cells gently with 10mls D-PBS and aspirate.
   3. Add 3mls Trypsin-EDTA , let sit on cells for 2-3 minutes.
   4. Tap flasks to detach cells.
   5. Add 7mls of Growth Media (see recipe) to flask. Pipet up and down several times and transfer to a 50ml conical centrifuge tube. Take a 100ul aliquot of the cell suspension and add to 20mls Isoflow/Isoton and count using Coulter Counter. Count cells three times.
      
      (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl) e.g. 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million
      
      Divide by the number of cells you want to freeze down and add that final number in mls of freezing solution.
      
      Example: 25.11 million ÷ 5 million = 5.022 (number of mls of freezing solution to add to get 5 X 10e6 cells per vial)
   6. Spin cells in centrifuge at 1500rpms for 3 minutes.
   7. Remove supernatant and resuspend cells in freezing medium (see below) @ a concentration of 5 X 10e6 cells per ml in a 1.5ml Cryo vial.
   8. Label vials with cell line, passage #, freeze down date, notebook number if possible and number of cells frozen.
   9. Place in a freezing container and place in a -80°C freezer for ~ 2 to 4 hours.
   10. Remove and place in liquid nitrogen tank for long term storage.
       
       Freezing Solution
       • 10% DMSO + 90% Characterized FBS (You want to use the FBS used in the Growth media for making up this solution)
2. Thawing of Cryopreserved Cells
   • Centrifugation Method:
     1. Remove cells from liquid nitrogen storage and thaw quickly in a 37°C water bath.
     2. Remove cells from cryo vial and place in 50ml conical tube.
     3. Add ~20 to 30mls of cool Growth medium slowly to the tube.
     4. Centrifuge cells @ 1500rpms for 5 minutes.
     5. Discard supernatant.
     6. Resuspend cells in Growth media and count cells and seed flasks.
   • Direct Plating Method:
     1. Remove cells from liquid nitrogen storage and thaw quickly in a 37°C water bath.
     2. Plate cells directly with Growth medium. Use 15-20mls of Growth Medium/ 1ml frozen cells @ 3 X 10e5.
     3. Culture cells 6-8 hours. Replace with fresh Growth Medium to remove the cryopreservative.