Cryopreservation of Bge Cells
Materials
- 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
Methods
A. Cryopreserving Cells
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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.
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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.
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Add 1 mL of freezing medium (10% DMSO, 20% FBS, 70% Bge medium) to cells such that there are ~1 x 106 cells/mL.
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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.
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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
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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.
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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.