Development of a protocol for cryopreservation of hepatocytes for use in bioartificial liver systems

Porcine hepatocytes are considered the cell type of choice for bioartificial liver and cell transplantation techniques in support of patients with liver failure. A protocol for cryopreservation of hepatocytes that function adequately after thawing would allow on-demand usage and long-term storage. We conducted experiments to evaluate the freeze rate, concentration of hepatocytes during storage, and the effect of a pre-incubation step prior to freezing on cryopreserved hepatocytes isolated from 15 porcine livers. Cell return, attachment, LDH leakage, bilirubin conjugation, and lignocaine metabolism were tested to assess the effects of the interventions. No significant differences were found between a computer-controlled freeze rate, the Nalgene propan-2-ol device, or simply using -20 degrees C and -80 degrees C freezers. Trials at a range of hepatocyte concentrations did not produce significant differences. Pre-incubation did not confer any advantage to the thawed hepatocytes. In conclusion, porcine hepatocytes can be cryopreserved using a simple method of temperature reduction at a concentration of 5x10(6) hepatocytes/ml. Such hepatocytes can potentially be used for bioartificial livers; however, further investigation is required to explain the large cell losses that occur during cryopreservation.