Restoring the differentiation and function in cryopreserved hepatocytes

Primary human hepatocytes are used in all areas of liver research, from in vitro studies of toxicity to clinically managing liver failure. The availability of these hepatocytes is, however, limited due to the problem of cellular stress during isolation and cryopreservation, causing a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. A research team led by SciLifeLab researcher Per Artursson have now found what appears to be a solution to the problem.

By applying label-free quantitative global proteomics to analyze the differences between attached and non-attached fractions of the hepatocyte batches, they were able to map the stress pathways of the non-attached hepatocytes. The group discovered many signs of cellular stress, such as a glycolytic phenotype and an activation of the heat shock response, ultimately leading to apoptosis, as well as an increase in early apoptosis immediately post-thawing. As a response to that finding, the group treated the cells with compounds aimed at decreasing the cellular stress. Using brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK, they were able to restore the attachment ability, as well as promote a differentiated morphology and formation of 3D spheroids. The Z-VAD-FMK treatment also 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”, the researchers write in their paper recently published in Archives of Toxicology.

Read the full paper in Archives of Toxicology