Studies of Human and Porcine Hepatocyte Cryopreservation and Their Suitability for Use in Bioartificial Liver Development

Abstract: The aim of this thesis was to assess feasibility of using chronically injured liver as a source for hepatocyte isolation, to compare inter-laboratory variability, to establish an efficient method for hepatocyte isolation and cryopreservation and to review whether anti-apoptotic compounds have any effect on cryopreservation outcomes. Method: A two-stage isolation protocol was used, with multiple washes and centrifugation compared with Percoll® purification pre- and post-cryopreservation and pre-incubation. A new cryopreservation media was validated and cells cryopreserved using this with the addition of potential anti-apoptotic agents. Demographic details were collected prospectively and histological samples collected where possible for analysis. Hepatocyte viability, yield per gram, hepatocyte loss, monolayer protein, visual attachment, phase I and II enzyme activity and measures of apoptosis were assessed. Results: Between October 2003-2005, 83 individuals donated tissue to UKHTB, with an additional 140 donors from elsewhere. 175 hepatocyte isolations were performed yielding more than 50 billion cells. Fresh cell culture was universally successful although purification led to significant cell loss. This was exacerbated during cryopreservation with especially poor porcine hepatocytes. None of the adjuncts to improved isolation or cryopreservation demonstrated reproducible improvement in outcome. There was only a weak negative correlation between histological injury and isolation outcome. Normothermic resuscitation prior to isolation conferred benefit across almost all measurable outcomes. Conclusion: There is significant inter-donor variability with regard to the outcomes of hepatocyte isolation and all purification methods cause unacceptable hepatocyte loss. A viable cell will attach and function and this is essential for many of the studies that are subsequently performed. Organs which are turned down for transplantation could be used for hepatocyte isolation.