Materials

• DMSO (sterile, 5 mL solution)
• FBS (heat-inactivated)
• Bge medium — see Schistosome and Snail Recipes
• 50 or 15 mL sterile conical tubes
• 1 mL pipettes
• 10 mL pipettes
• 1 mL sterile cryopreservation vials

Cryopreserving cells

1. 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.
2. After an accurate estimate of cell numbers is achieved, centrifuge the contents of a flask for 10 minutes at speed 3 in the clinical centrifuge. Remove the supernatant by pipetting, or by decanting medium into a waste container.
3. Add 1 mL of freezing medium (10% DMSO, 20% FBS, 70% Bge medium) to the cells, such that there are ~1 × 10⁶ cells/mL.
4. 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.
5. To freeze, place the vials in a plastic cryopreservation vial box in the -20°C freezer for several hours, then transfer to the -80°C freezer for overnight freezing. The following morning, place the vials in liquid nitrogen.

Thawing cells

1. Remove a tube of cells carefully from the liquid nitrogen and immediately place it in a 30°C water bath. Shake the vial gently to facilitate thawing.
2. 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 and thawing. At this point you can passage them, or decide that the cells are no longer viable.

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Leave cells in the water bath ONLY until they have thawed, and no longer.

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