Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CL int ) in the liver. However, the CL int values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kp uu ) effects on the CL int were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CL int . Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CL int with CYP amounts and Kp uu could only partly explain the discordance in absolute values of CL int for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Background Perforations related to endoscopic retrograde cholangiopancreatography (ERCP) are rare but feared adverse events with highly reported morbidity and mortality rates. The aim was to evaluate the incidence and outcome of ERCPrelated perforations and to identify risk factors for death due to perforations in a population-based study. Methods Between May 2005 and December 2013, a total of 52,140 ERCPs were registered in GallRiks, a Swedish nationwide, population-based registry. A total of 376 (0.72%) were registered as perforations or extravasation of contrast during ERCP or as perforation in the 30-day follow-up. The patients with perforation were divided into fatal and non-fatal groups and analyzed for mortality risk factors. The case volume of centers and endoscopists were divided into the upper quartile (Q4) and the lower three quartile (Q1-3) groups. Furthermore, fatal group patients' records were reviewed. Results Death within 90 days after ERCP-related perforations or at the index hospitalization occurred in 20% (75 out of 376) for all perforations and 0.1% (75 out of 52,140) for all ERCPs. The independent risk factors for death after perforation were malignancy (OR 11.2, 95% CI 5.8-21.6), age over 80 years (OR 3.8, 95% CI 2.0-7.4), and sphincterotomy in the pancreatic duct (OR 2.8, 95% CI 1.1-7.5). In Q4 centers, the mortality was similar with or without pancreatic duct sphincterotomy (14% vs. 13%, p = 1.0), but in Q1-3 centers mortality was higher (45% vs. 21%, p = 0.024). Conclusions ERCP-related perforations are severe adverse events with low incidence (0.7%) and high mortality rate up to 20%. Malignancy, age over 80 years, and sphincterotomy in the pancreatic duct increase the risk to die after a perforation. The risk of a fatal outcome in perforations after pancreatic duct sphincterotomy was reduced when occurred at a Q4-center. In the case of a complicated perforation a transfer to a Q4-center may be considered.