Annals of Clinical & Laboratory Science 34:165-174 (2004)
© 2004 Association of Clinical Scientists
Development of a Protocol for Cryopreservation of Hepatocytes for Use in Bioartificial Liver Systems
Tom D. R. Lloyd,
Samantha Orr,
David P. Berry and
Ashley R. Dennison
Department of Surgery, Leicester General Hospital, Leicester, UK
Address correspondence to Mr. Tom D. R. Lloyd, Department of Surgery, University Hospitals of Leicester, Glenfield General Hospital,, Groby Road, Leicester LE3 9QP, UK; tel 0116 287 1471; fax 0116 287 9852; e-mail: tdlloyd{at}ntlworld.co.uk.
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°C and –80°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 5x106 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.
Keywords: Bioartificial liver, hepatocytes, cryopreservation, hepatocyte culture
Copyright © 2004 by the Association of Clinical Scientists.
