Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study

Background: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P-450 induction. Aims and Methods: As first stage of this prevalidation study, the purpose of the present work was to set criteria for optimization and harmonization of hepatocyte isolation from human tissue among laboratories to establish a routine procedure. This was achieved by combining and/or comparing the data generated by the two independent European laboratories (France and UK). Results: The results confirmed that surgical waste material is a valuable source for obtaining high quality hepatocytes under certain pre-, intra- and post-operative conditions: cell yield of viable hepatocytes was not significantly affected by age and sex of patients, nor indications for resection, steatosis or cholestasis. Cold ischeamia up to 5 hours did not influence viable cell yield allowing transport of material. Conclusion: The use of biopsy sizes between 50-100 g, cannulation with 2-4 cannulae, digestion with collagenase-containing digestion medium at a flow rate of 25 ml/cannula for 20 minutes, with cut surface being glued in order to reform Glisson's capsule, should optimize the total yield of viable human hepatocytes obtained per preparation of waste liver surgical resections.