Thawing Hepatocytes

Overview

Thawing cells properly is crucial to cell culture health. Follow this protocol to ensure your cryopreserved hepatocytes will have an optimal yield for your experiments. Please note that these hepatocytes cannot be passaged. 

Materials

• Human Hepatocytes
• Hepatocyte Culture Medium
• Hepatocyte Thawing Medium
• Water/Bead Bath (37˚C)
• 70% Ethanol
• Kimwipes
• Biosafety Cabinet (BSC)
• Centrifuge
• Automatic Pipet Filler
• Serological Pipets
• Micropipettes
• Micropipette Tips
• Vacuum Bottle

Methods

1. Warm hepatocyte thawing medium, plating medium and culture medium to 37˚C in the water/bead bath;
2. Remove hepatocyte vial from liquid nitrogen and submerge it in the water/bead bath up to the cap for no longer than 2 minutes;
3. Remove vial when its sides are thawed but its center remains frozen;
4. Spray your vial with 70% Ethanol, wipe it with Kimwipes and move it to a BSC;
5. Spray down all media and pipets and move them into the BSC;
6. Pipette 1 mL of thawing medium into the cell vial;
7. Pipette remaining cells in the 50 mL tube of thawing medium;
8. Rock the tube by hand for a few seconds to suspend cells;
9. Centrifuge tube for 8 minutes at 100 g;
10.  Spray tube down before moving it back into the BSC;
11.  Carefully aspirate supernatant;
12.  If you wish to check for cell viability, follow this protocol (Checking Cell Viability with Trypan Blue) 
13.  Follow the Plating Hepatocytes or Encapsulating Hepatocytes protocol, depending on your final application.