Prepare the HepaRG® THAWING/PLATING/GENERAL PURPOSE MEDIUM 670
Thaw the HepaRG® Thawing/Plating/General Purpose Medium Supplement (ADD670) by placing the bottle in a +37°C water bath until completely thawed.
Reconstitute the HepaRG® Thawing/Plating/General Purpose Medium 670 by adding the supplement (ADD670) to 100 mL of Base Medium (MIL600 BPI). The ready-to-use media can be stored at +4OC for 1 month.
Pre-warm the HepaRG™ Thaw, Plate & General Purpose Medium in the +37°C water bath.
Pipet 9 mL (per cryovial of HepaRG™ cells to be used) of prewarmed HepaRG™ Thaw, Plate & General Purpose Medium into a sterile 40-mL polystyrene round tube or similar container. Prepare an absorbent paper with 70% ethyl alcohol.
Remove the cryovial from the liquid nitrogen.
Under the laminar flow hood, briefly twist the cap a quarter turn (do not open the cryovial completely) to release the internal pressure, and then close it again.
Quickly transfer the cryovial to the water bath at +37°C. Do not submerge it completely, being careful not to allow water to penetrate into the cap.
While holding the tip of the cryovial, gently agitate the vial for about 2 minutes. Small ice crystal should remain when removed from the water bath.
Wipe the outside of the cryovial with 70% ethyl alcohol on an absorbent paper, and place the cryovial under the laminar flow hood.
Aseptically transfer the "semi"-thawed HepaRG™ cell suspension into the tube containing 9 mL of the pre-warmed HepaRG™ Thaw, Plate & General Purpose Medium (resulting in a 1:10 ratio of cell suspension to total volume).
Rinse out the cryovial once with approximately 1 mL of the HepaRG™ Thaw, Plate & General Purpose Medium and return the resulting suspension to the 40-mL tube.
Centrifuge the differentiated HepaRG™ cell suspension 3 min at 500 × g at room temperature. Do not utilize a traditional hepatocyte centrifugation protocol because HepaRG™ cells are smaller in size and need a longer and faster centrifugation.
Aspirate the supernatant. To avoid aspiration of cells, leave a little bit of medium on the pellet.
Gently resuspend the differentiated HepaRG™ cell pellet in 5 mL of HepaRG™ Thaw, Plate & General Purpose Medium. Do not try to dissociate the bigger clusters.
