Effect of cryopreservation on metabolic activities of hepatocytes

Q4 Pharmacology, Toxicology and Pharmaceutics

中国药理学与毒理学杂志 Pub Date : 2008-01-01 DOI:10.3867/J.ISSN.1000-3002.2008.06.011

Q. Pan, Y. Chen, L. Mei, R. Dai

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引用次数: 0

Abstract

AIM To study the effect of cryopreservation on metabolic activities and cytochrome P450(CYP) mRNA expression in hepatocytes and provide support for application of cryopreserved hepatocytes in experimental research.METHODS Freshly isolated rat hepatocytes were cryopreserved with rate-controlled freezer,and thawed after 1 month.Real-time quantitative PCR was used to detect expressions of CYP1A2,CYP2B1 and CYP3A1 mRNA,and LC-MS/MS was used to measure contents of metabolites of midazolam-1′-hydrxylation(OH-Mid),diclofenac-4′-hydroxylation(OH-Dic) and dextromethorphan-O-demethylation(Dex) in hepatocytes,respectively.RESULTS There was no significant difference in cell viability between fresh and cryopreserved hepatocytes.The cryopreserved hepatocytes attached and established extensive cell-cell contact,with round and bright nucleus.CYP1A2 and CYP2B1 mRNA expressions induced by β-naphthoflavone and phenobarbital in cryopreserved hepatocytes were similar to that in the fresh primary cells.However,CYP3A1 mRNA expression did not induced by pregnenolone-16α-carbonitrile in cryopreserved hepatocytes.In cryopreserved hepatocytes,the content of OH-Mid was remained as almost the same as the fresh primary hepatocytes,while contents of OH-Dic decreased approximately as a half,and Dex was double as fresh hepatocytes.CONCLUSION Cryopreservation exerts different effects on metabolic activities of hepatocytes.To acquire objective and appropriate results,it is necessary to consider the different influence of cryopreservation on cell metabolic activity in drug metabolic research.

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低温保存对肝细胞代谢活性的影响

目的研究低温保存对肝细胞代谢活性及细胞色素P450(CYP) mRNA表达的影响，为低温保存肝细胞在实验研究中的应用提供支持。方法新鲜分离的大鼠肝细胞采用速控冷冻机冷冻保存，1个月后解冻。Real-time定量PCR检测CYP1A2、CYP2B1和CYP3A1 mRNA的表达，LC-MS/MS检测肝细胞中咪达唑仑-1′-羟基化(OH-Mid)、双氯芬酸-4′-羟基化(OH-Dic)和右美沙芬- o -去甲基化(Dex)代谢物的含量。结果新鲜肝细胞和冷冻肝细胞的细胞活力无显著差异。冷冻保存的肝细胞附着并建立了广泛的细胞间接触，细胞核圆而明亮。β-萘黄酮和苯巴比妥对低温保存肝细胞中CYP1A2和CYP2B1 mRNA表达的影响与新鲜原代细胞相似。而孕烯醇酮-16α-碳腈在低温保存的肝细胞中没有诱导CYP3A1 mRNA的表达。在冷冻保存的肝细胞中，OH-Mid的含量与新鲜原代肝细胞几乎相同，OH-Dic的含量减少了大约一半，Dex的含量是新鲜肝细胞的两倍。结论低温保存对肝细胞代谢活性有不同的影响。在药物代谢研究中，为了获得客观合理的结果，需要考虑低温保存对细胞代谢活性的不同影响。

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来源期刊

中国药理学与毒理学杂志 Pharmacology, Toxicology and Pharmaceutics-Pharmacology

CiteScore

0.20

自引率

0.00%

发文量

6787

期刊介绍： Chinese Journal of Pharmacology and Toxicology was first published in November 1986. As a journal of high academic value jointly edited by the Chinese Pharmacological Society, Chinese Society of Toxicology and the Academy of Military Medical Sciences (AMMS), it is a dominating one in China’s biomedicine, devoted to publication of research findings, reviews of special topic and new technologies and methods in every branch of experimental toxicology and pharmacology.