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Comparative Physiology @ Guelph

Cryogenic Storage of Fibroblasts

Cryoprotectant Media (if making 10mL)

1mL 10% DMSO

2mL 20% DMEM

7mL 70% FBS

* make enough for 1.5mL/cryovial

* use 1 cryovial/2 plates of cells


  • Aspirate media from 90-100% confluent cells and rinse once with PBS/EDTA
  • Add 1mL trypsin in drop-wise fashion, then incubate at 37°C in 5% CO2 until cells dissociate from bottom of plate
  • Stop trypsinization by adding 10mL media (FBS, DMEM, and PS) per plate, then transfer to 15mL tube
  • Centrifuge cells (making sure it’s balanced) for 5 min at 1000rpm, and carefully discard the supernatant
  • Resuspend cells, by aspirating multiple times with a pipette, in 1mL of cryoprotectant media
  • Aliquot cell suspension into labeled cryovials in 1.5-1.8mL volumes
  • Place labeled cryovials into -20°C for 3 hrs, then move to -80°C overnight, and liquid N for long-term storage