Thawing and Plating Cryopreserved Hepatocytes -- Video Demonstration

Read the thawing and plating protocol thoroughly and set up your biosafety hood properly. Ensure the CHARM thawing medium is warmed to 37°C before beginning the procedure.

Transfer the vial of cryopreserved hepatocytes from cryo storage to the laboratory using a cryogenic container. Keep vials on dry ice or in liquid nitrogen if the storage unit is far away, and transport as quickly as possible to the water bath.

Hold the vial at the capped end and submerge cells in the 37°C water bath. Continuously monitor the vial and swirl or hold steady until all ice melts, which should take less than 2 minutes.

Spray the vial and 50ml CHARM thawing media tube with 70% alcohol, wipe dry, and place in the biosafety cabinet. Pour the vial contents into the tube and rinse the empty vial multiple times with CHARM medium using a wide bore pipette tip, adding all rinses back to the tube. Gently invert the tube at least twice to ensure homogeneous mixture.

Centrifuge the charm tube with cells according to protocol. Carefully remove the tube without disturbing the pellet and pour off the supernatant completely in one fluid motion without shaking. Gently loosen the pellet by flicking the tube bottom, then add approximately 1 ml of media per million cells and gently rock to resuspend.

Perform a trypan blue exclusion count to determine cell yield and viability. Prepare appropriate dilutions, wait 1 minute, gently mix the suspension in the trypan tube, and slowly pipette into the hemocytometer chambers. Load both sides evenly and count using proper technique.

Dilute cells to the recommended seating density based on cell yield counts, referring to the product characterization sheet for optimal density. Using a serological pipet or wide bore pipet, plate hepatocytes slowly while resuspending with each draw to ensure even distribution and reduce well-to-well variability.

Disperse cells evenly across the bottom of culture wells by moving the plate in north-south and east-west motions 2-3 times. Do not disturb the plate for 4-6 hours to allow initial cell attachment, after which hepatocytes are ready for first feeding with serum-free medium.