Cryopreservation of Bge Cells
- DMSO (sterile, 5 mL solution)
- FBS (Heat-inactivated)
- Bge Medium (see: Schistosome Recipes)
- 50 or 15 mL sterile conical tubes
- 1 mL pipettes
- 10 mL pipettes
- 1 mL sterile cryopreservation vials
A. Cryopreserving Cells
Counting the number of cells prior to centrifugation is optional for the Bge cell line. However, the cell line needs to be in log growth phase before freezing is attempted.
After an accurate estimate of cell numbers is achieved, centrifuge the contents of a flask for 10 m. at speed 3 in the clinical centrifuge. Remove the supernatant by pipetting or by decanting medium into a waste container.
Add 1 mL of freezing medium (10% DMSO, 20% FBS, 70% Bge medium) to cells such that there are ~1 x 106 cells/mL.
Carefully re-suspend the cells in the medium, then transfer the 1 mL to a sterile cryopreservation vial. Label with the cell line, date frozen and concentration of cells.
To freeze the cells, place the vials in a plastic cryopreservation vial box in the -20ºC freezer for several hours, then transfer the cells to -80ºC freezer for overnight freezing. The following morning, place the vials in liquid nitrogen.
B. Thawing Cells
Remove a tube of cells carefully from the liquid nitrogen and immediately place tube in a 30ºC water bath. Shake vials gently to facilitate thawing. Leave cells in the water bath ONLY UNTIL THE CELLS HAVE THAWED AND NO LONGER.
In the BSC, add cells to culture medium already present in a 75 cm2 flask. Allow the cells several days to recover from the effects of freezing/thawing. At this point you can passage or decided that the cells are no longer viable.