• Determine the viability and cell count of the population for freezing using Protocol 3.8.
• Calculate the volume of diluent needed to resuspend the cells at 5 x 106/ml.
• Prepare this volume of a 10% solution of DMSO in culture medium and mix well.
• Label the appropriate number of freezing vials clearly with the date, pass number and cell type using the black marker pen.
• Centrifuge the cell suspension at 1000 rpm for 5 min, discard the supernatant and resuspend the cells in the required volume of 10% DMSO. Mix well.
• Dispense the cell suspension as 1.8 ml aliquots into the freezer tubes using a micropipette and cap them securely.
• For programmable freezing the vials can be transferred directly to the freezer and the freezing cycle started (see manufacturer's instructions).
• Transfer the vials to the long-term storage facility when freezing is complete.
• For freezing with a device for the neck of the cell bank or storage vessel, try the following as a starting point: Keep the filled freezing vials at 4 °C for 60 min, transfer to the freezing device and keep in vapour phase overnight, then transfer them to the long-term storage facility.
• For freezing with a polystyrene box, try the following as a starting point:
Keep the filled freezing vials at 4 °C for 60 min, transfer to the box and hold the box at -20 °C for 60 min, transfer the box to -80 ° C overnight, then transfer them to the long-term storage facility. (Note: Frozen cells can be stored at -80 °C for several months.)
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