Dataset Information

Post-thaw restoration of differentiation and function in cryopreserved human hepatocytesPost-thaw restoration of differentiation and function in cryopreserved human hepatocytes

ABSTRACT: Isolated human hepatocytes are used in all facets of liver research, from the clinical management of liver failure to in vitro studies of drug disposition. Cryopreservation provides a reliable source of hepatocytes, but the associated cellular stress causes a highly variable and heterogeneous loss of a differentiated phenotype, manifested by a decreased ability to form cell-matrix and cell-cell interactions. We reasoned that this problem could be mitigated at the post-thawing stage, which would increase the availability of well-functioning human hepatocytes. We applied quantitative global proteomics to analyze the 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, with increased apoptosis activation as the ultimate consequence. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately after thawing, hinting at the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. We found that brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored the ability to attach to collagen, and promoted a differentiated morphology with increased metabolic function. Further, Z-VAD-FMK treatment did not alter hepatocyte protein expression, suggesting that these ‘rescued’ cells would be suitable for applications where hepatocytes of high quality are required. Conclusion: Cryopreserved human hepatocytes are affected by considerable amounts of cellular stress, often resulting in loss of differentiation with a decreased ability to form cell-matrix and cell-cell interactions. This can be alleviated by brief apoptosis inhibition post-thawing, substantially improving key morphological and functional properties of these cells.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Colon Cancer

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R. Wisniewski

PROVIDER: PXD010533 | Pride | 2019-01-02

REPOSITORIES: Pride

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

Ölander Magnus M Wiśniewski Jacek R JR Flörkemeier Inken I Handin Niklas N Urdzik Jozef J Artursson Per P

Archives of toxicology 20181217 3

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 difference ...[more]

PMID: 30560367

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