Studies into the isolation and cryopreservation of human and porcine hepatocytes and their suitability for use in bioartificial liver development

Introduction, Clinical bioartificial liver development for the treatment of liver failure has been actively researched for many years. The development of an easily accessible cell source that also provides an adequate range of functions has proved a major block to its development. The aim of these studies was to evaluate and develop methods for the successful cryopreservation of human and porcine hepatocytes for potential use within a bioartificial liver. Methods. Both porcine and human hepatocytes were studied. Following informed patient consent, human liver tissue was extracted from discarded specimens removed during liver resection. Porcine tissue was sourced from an abattoir. The hepatocytes were then isolated from the tissue, and investigations into cryopreservation parameters performed. Measurements of cellular return, LDH leakage, attachment, bilirubin conjugation and lignocaine metabolism were performed on the thawed hepatocyte cultures and compared to untreated hepatocyte controls. Patient, operative and isolation parameters were reviewed to determine their influence on pre- and post-hepatocyte function and quality. Results, The processing of surgically resected liver tissue provided consistently viable and functionally high quality hepatocytes. Large tissue samples and methods of cannulation influenced the level of cell viability following isolation. No other pre-operative factors were identified as being of any influence. The temperature reduction method had no significant affect on the function of the hepatocytes contrary to reports in the literature. The concentration of hepatocytes, and the pre-incubation of hepatocytes did not significantly affect the quality of the hepatocytes following post cryopreservation culture. Hepatocyte functional data proved similar for hepatocytes cultured following isolation and post-cryopreservation. Conclusions, Human and porcine hepatocytes can be successfully cryopreserved using a gradual temperature reduction at an optimal concentration of 5 x 106. There are minimal differences within the functional parameters between fresh and post-cryopreservation with human or porcine hepatocytes. A reduction of approximately 70% of viable hepatocytes was found following cryopreservation. Further method optimisation will be required to reduce this loss.