Vero e6 Cells: Making a Frozen Cell Stock, Reconstituting Cells from the Frozen Stock, and Passaging Cells

Everything is done in the Tissue Culture hood

Freezing Vero Cells 20211207

  • Planned on freezing 30 tubes (screw cap cryotubes) with 2mL in each, so a total volume of 60mL
  • Made up 60mL of freezing medium and made it in an autoclaved Erlenmeyer flask
    • 12mL FBS (20% fetal bovine serum) (probably buffers cells)
    • 42mL DMEM (Dulbecco’s modified eagles medium)
    • 6mL DMSO (10% dimethyl sulfoxide) (add last) (protects cells from ice crystals piercing them during freezing)
  • Cover flask with autoclaved foil while not using
  • Make sure all tubes are labeled and there are freezing boxes ready (one for the -80, and one for the LN2 storage)
  • Need to trypsinize the cells (Vero cells are very attached to the flask)
  • Take a flask and pour off the medium into a waste beaker (there were 4 25mm2 flasks)
  • Add 5mL of room temp trypsin (wash the cells first because remainder FBS inhibits trypsin)
  • Right after adding, wash the monolayer quickly without capping/discarding
  • Remove the 5mL trypsin with same serological pipette
  • Add 10mL trypsin to the flask
  • Cap and distribute the trypsin and lay the flask on the hood if there are other flasks to go through. If not, place immediately in the 37 degree incubator
  • Wait ~2 minutes then check on the flask in the incubator. Tip the flask back and forth gently to see the monolayer break apart
  • The quickly put the flask back into the incubator for another 1-2 minutes
  • Take out and check again if the cells are coming apart by tipping back and forth again, if not, put back in the incubator again for a few min. Want all the cells to have come off and gone into the liquid, with no clumps really
  • When all the cells have come off the side of the flask, take the flasks back to the TC hood
  • Get 2 50mL conicals (or whatever tubes needed)
  • Use a 10mL serological pipette to combine all the liquid from the flasks into one flask
  • Use a 50mL serological pipette to split the liquid equally into the 2 50mL conicals
  • Centrifuge 5 minutes at 200rcf
  • Gently pour off the supernatant (it should be clear)
  • Use 5mL of the freezing medium to resuspend the pellet in one of the conicals
  • Then take that 5mL and transfer it to the next conical and resuspend the pellet in there
  • Transfer the resuspended pellets into the flask with the freezing medium
  • Swirl the flask to mix up the cells in the liquid
  • Take a 10mL pipette and fill with freezing medium and cells, then serially pipette 2mL into each cryovial, transferring the cryovial to a freezing box when finished
    • Go slowly and deliberately, it can be tough to unscrew and re-screw the caps of the cryovials with one hand
    • Place the cryovials in the freezing boxes with space between each tube (allows for even freezing)
  • When done, make sure all cryovials are screwed tight
  • Place both boxes in the -80
  • The next day, the LN2 box can be taken to the LN2 storage, and place in the gas phase of the storage

Thaw and Reconstitute Vero Cells 20211208

  • Make medium and let come to room temperature:
    • 2 conicals with
    • 45mL DMEM
    • 5mL FBS
    • 50ul gentamicin
  • Taking 1 2mL tube and spreading it out into 5 25mm2 flasks
  • Thaw 1 cryotube in your hand
  • Get a 15mL tube
  • Once thawed, transfer the 2mL into the 15mL tube
  • Add 8mL of room temp medium to the 15mL tube
  • Centrifuge tube at 400rpm for 3 minutes
  • Pour off the media supernatant into a waste beaker
  • Take a new 35mm2 culture flask with vented caps and fill with 50mL culture medium
  • Take 5mL of that and add to the conical with the cell pellet and resuspend the pellet
  • Transfer the 5mL back into the culture flask with the 50mL
  • Use a 10mL pipette to mix up the medium
  • Transfer 10mL into each of 4 new vented culture flasks, for a total of 5 flasks
  • Lay flasks flat and place in the humidified, 5% CO2, 37 degree incubator
  • The frozen cells were passage 24, so these are passage 25
  • It might take a few days for the cells to recover and need passaging

Passaging Cells 20211213 1:5 Split

  • Make medium and wait until it is at room temp before using:
    • 45mL DMEM
    • 5mL FBS
    • 50ul gentamicin
  • Take 1 flask and pour off the liquid
  • Add 5mL trypsin to the flask and spread out over the flask
  • Immediately pipette off the trypsin into a waste beaker
  • Add 5mL (or probably 10mL) of trypsin to the flask, cap, and distribute onto the flask surface
  • Set the flask in the 37 degree incubator for ~2 minutes
  • Check on the flask: rock it back and forth and look for the monolayer of cells to start coming off
  • Place the flask back into the incubator for a few minutes again
  • Then take out and check again by rocking back and forth seeing the cells come off, if they call come off you can continue, if not then the flask goes back into the incubator for a few minutes
  • Bring the flask back into the TC hood and get a 15mL flask
  • Transfer the liquid in the flask with all the cells to the 15mL conical
  • Centrifuge the 15mL tube for 5 minutes at 200rcf
  • There should be a nice cell pellet present afterwards
  • Pour off the supernatant into the waste beaker
  • Resuspend the pellet in 10mL of culture medium
  • Make up 5 25mm2 flasks with the vented caps, label with the passage (26)
  • Add 40mL of medium to one of the flasks
  • Add the 10mL of resuspended cells to the flask with 40mL and pipette mix
  • Pipette out 10mL of medium with cells to each of the 4 other cell culture flasks
  • Cap, tighten, and lay flasks flat, and place in the 37 degree incubator