P11: Targeted modulation of hepatocellular stress pathways during biopreservation affects storage outcome

Abstract

Primary hepatocytes are central to a number of in vitro applications including hepatotoxicity testing for drug development, potential uses in cell therapy, and as research tools for improving organ transplantation. Further, hepatocyte derivatives such as the human hepatoma cell line C3A serve important roles in other technologies such as bioartificial liver support systems. While it is well characterized that molecular stress pathways are activated in response to biopreservation associated stresses thereby resulting in cell death and system failure, it remains unclear whether the specific molecular pathways activated are universal or cell specific. We hypothesized that given the vastly different characteristics of hepatocyte cell systems, cell type specific stress pathways may be activated under different storage regimes. To this end, we compared the responses of two hepatic cell types following both hypothermic and hypoxic normothermic storage and examined storage related stress signaling. Normal hepatocytes (NHEP) and C3A were stored under either hypothermic (4 °C) conditions for 18hrs to 3d or hypoxic normothermic (37 °C) conditions for 1–14 d. Respective growth media, HBSS (Balanced Salt Solution), and ViaSpan (UW solution) were utilized as storage media with and without the addition of salubrinal or resveratrol to examine targeted molecular pathway modulation. Viability was assessed at 0, 24 and 48 h post-storage. Apoptotic and necrotic populations were assessed via flow cytometry and fluorescent microscopy post-storage. Protein immunoblotting was performed to evaluate alterations in ER stress pathway proteins and apoptotic signaling proteins. Viability studies demonstrated a significant difference in modulation study outcomes for the two cell types. This was noted as salubrinal addition to HBSS stored C3A yielded significant improvement in viability (∼60%) while having no significant impact on NHEPs. Conversely, resveratrol addition to HBSS resulted in a significant decrease in C3A viability (∼80%) whereas it conferred a marked improvement on NHEP survival (∼25%). Interestingly, when the modulators were utilized under a hypoxic normothermic regime a differential response was observed as salubrinal negatively impacted C3A viability (∼50% decrease) while yielding improvement to NHEP survival (∼30% increase) and resveratrol yielded decreased viability in both cell types. Investigations into cell death populations demonstrated marked changes in the number of cells undergoing apoptosis and necrosis that was influenced by the use of these molecular modulators. Further, immunoblot analyses revealed alterations to protein levels associated with apoptotic activation (pro-caspase cleavage) as well as implications of ER stress induced cell death signaling (calnexin cleavage). These data demonstrate a differential response in hepatic cell systems that is dependent upon storage temperature, basal media and modulator addition. This is important for the processing and storage of biologics as it demonstrates that universal protocols may not be effective for all cell types and conditions and a more specific and directed approach may be necessary for optimal efficacy.