A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes

Abstract

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.

Characterization of Human Induced Pluripotent Stem Cell-Derived Hepatocytes with Mature Features and Potential for Modeling Metabolic Diseases

“…Interestingly, in the DEGs between hiPS-HEP and hphep d0 and d1 "Attenuation phase" and "HSP1-dependent transactivation" are over-represented pathways which are related to cellular response to heat stress. It has been reported previously that cryopreservation and re-plating of hphep results in reduced ability to form cell-matrix and cell-cell interactions which triggers stress and heat shock responses [45]. Our observation that DEGs involved in these pathways (HSPA1A, HSPA1B, and DNAJB1) are upregulated in hphep d0 and d1 compared to hiPS-HEP is in line with these reports.…”

Characterization Of Human Pluripotent Stem Cell-Derived Hepatocytes With Adult Features And Potential For Modeling Metabolic Diseases

“…It has been reported previously that cryopreservation and re-plating of hphep results in reduced ability to form cell-matrix and cell-cell interactions which triggers stress and heat shock responses [47]. Our observation that DEGs involved in these pathways (HSPA1A, HSPA1B, and DNAJB1) are upregulated in hphep d0 and d1 compared to hiPS-HEP is in line with these reports.…”

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids

“…Total viabilities of the cell preparations after thawing ranged between 90–95% for hepatocytes from all donors, except for the TM6SF2 E167K-1 donor (84%). Since lower total cell viability is frequently associated with reduced spheroid formation capability, brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK was used to support spheroid formation, as described recently 46 . The same treatment was done for the WT-3 donor to allow for direct comparisons of both preparations (Fig.…”