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首页> 外文期刊>Archives of Toxicology >A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes
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A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes

机译：一种简单的方法，用于恢复冷冻保存人肝细胞的分化和功能

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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.
机译：原发性人肝细胞用于肝脏研究的所有方面，从异种症分化的体外研究和对肝功能衰竭的临床管理的临床管理。不幸的是，隔离和冷冻保存期间的细胞应激导致高度不可预测的附着和形成细胞 - 基质和细胞 - 细胞相互作用的能力。推理这一问题可以在后解冻阶段减轻，我们应用了无标签的定量全球蛋白质组学，分析附着和非附带的冷冻保存人肝细胞批次之间的差异。无法附着于胶原基质的肝细胞显示出许多细胞应激迹象，包括糖酵解表型和热休克反应的活化，最终导致凋亡激活。进一步分析活性应激途径揭示了早期细胞凋亡的增加，后解冻后，这表明应激逆转的可能性。因此，我们用旨在通过不同机制降低细胞应激的化合物瞬时处理细胞。短暂暴露于泛胱天冬酶细胞凋亡抑制剂Z-VAD-FMK恢复的附着能力，并促进了分化的形态，以及3D球状体的形成。此外，Z-VAD-FMK处理恢复了代谢和运输功能，对肝毒性损伤保持敏感性。总之，本研究表明，在冷冻保存后，可以恢复次优人肝细胞的分化和功能，从而显着增加了这些珍贵细胞的可用性。

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