Cryoprservation of the culture cells

pp 25 and 328 , Ian Freshney

Outline:
Grow culture to late log phase, prepare a high conc cell suspension in medium with a cryoprotectant aliquot into ampoules and freeze slowly.

Protocol A : Freezing cells
Materials:

Sterile or aseptically prepared

Culture to be frozen
Monolayer : PBSA and 0.25 % crude trypsin
Growth medium - improves survival after freezing upto 50%. Serum should be washed off after the thawing
Cryoprotectant , free of impurities – DMSO in glaas or PP OR Glycerol in universal container
Syringe 1-5 ml to dispense glycerol
Plastic microfuge 1.2 ml

Non-sterlie :

Haemocytometer or electronic cell counter
Canes or racks for storage (racks may already be placed in freezer)
Insulated container for freezing: polystyrene box lined with cotton wool or plastic foam insulation tube, or controlled rate freezer
Protective gloves, nitrile

Protocol:

Make sure culture satisfies criteria for freezing and check by eye and microscope
Healthy appearance
Morphological characteristics
Phase of growth cycle (should be late log phase before entering plateau)
Freedom from contamination

Grow culture up to late log phase and if you are using monolayer , trypsinize and count cells . if you are using suspension , count and centrifuge cells
Re-suspend at 2X 106 -3X107 cells/ml
Dilute one of cryoprotectants in growth medium to make freezing medium
Add DMS to 10-20 %
Add glycerol 20-30 %
5. Dilute cell suspension 1:1 with freezing medium to give approximately 1X106 - 1X107 cells/ml and add 5-10 % DMSO (or 10-15 % glycerol). It is not advisable to place ampoules on ice In an attempt to minimize deterioration of cells. A delay of upto 30 min at RT is not harmful when using DMSO and beneficial when using glycerol
Dispense cell suspensions into pre-labeled ampoules and cap the ampoules with sufficient torsion to seal ampoule without distorting gasket
Place ampoules on canes for canister storage or leave them loose for drawer storage
Freeze ampoules at 10C/min by one of the method described above. With insulated container methods, this will take a minimum of 4-6 hrs after placing them at –70 oC. if starting from 20 oC or lower , check the freezer record before removing ampoules from –70 oC freezer or controlled rate freezer and identify a suitable location for ampoules
Transfer ampoules to LN2 freezer, preferably not submerged in liquid, placing cane and tube into predetermined canister or individual ampoules into correct spaces In predetermined drawer. This transfer must be done quickly (<2min), as ampoules will reheat at ~10oC /min and cells will deteriorate rapidly if temperature rises above –50oC
When ampoules are safely located in freezer complete appropriate entries in freezer index

Part B : Thawing cells

Materials:

Culture flask
Centrifuge tube
Growth medium
Pipettes 1 ml-10 ml
Syringe at 19-g needle

Non-sterile:
Protective gloves and face mask
Sterile water 37 C, 10 cm deep in a clean alcohol swabbed bucket with lid
Forceps
Alcohol 70 %
Swabs
Napthalene Black (amido black) 1 % or Trypan Blue

Protocol:
Check the index for the location of ampoule to be thawed
Collect all materials, prepare medium and label culture flask
Retrieve the ampoule from freezer, check from the label that it is the correct one and dif it has not been submerged in liquid nitrogen, place it in sterile water at 37oC in a beaker, plastic bucket or water bath. If possible avoid getting water up and dilute to regular seeding conc. This can be made more precise if the cells are counted and an estimate of viability is made, in which case cells can be diluted to regular seeding conc of viable cells.