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Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution 被引量:2
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作者 Mohammed Saliem Frida Holm +5 位作者 Rosita Bergstrm Tengzelius Carl Jorns Lisa-Mari Nilsson bo-gran ericzon Ewa Ellis Outi Hovatta 《World Journal of Hepatology》 CAS 2012年第5期176-183,共8页
AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cry... AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation. 展开更多
关键词 Human HEPATOCYTES VIABILITY CYTOCHROME P540 DIMETHYLSULPHOXIDE CRYOPROTECTANT CRYOPRESERVATION
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Bile acid formation in primary human hepatocytes 被引量:1
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作者 Curt Einarsson Ewa Ellis +3 位作者 Anna Abrahamsson bo-gran ericzon Ingemar Bjrkhem Magnus Axelson 《World Journal of Gastroenterology》 SCIE CAS CSCD 2000年第4期522-525,共4页
AIM To evaluate a culture system for bile acidformation in primary human hepatocytes incomparison with HepG2 cells.METHODS Hepatocytes were isolated fromnormal human liver tissue and were cultured inserum-free William... AIM To evaluate a culture system for bile acidformation in primary human hepatocytes incomparison with HepG2 cells.METHODS Hepatocytes were isolated fromnormal human liver tissue and were cultured inserum-free William’s E medium.The medium wascollected and renewed every 24 h.Bile acids andtheir precursors in media were finally analysed bygas chromatography-mass spectrometry.RESULTS Cholic acid(CA)andchenodeoxycholic acid(CDCA)conjugated withglycine or taurine accounted for 70% and 25% oftotal steroids.A third of CDCA was alsoconjugated with sulphuric acid.Dexamethasoneand thyroid hormone alone or in combination didnot significantly effect bile acid formation.Theaddition of cyclosporin A(10 μmol/L)inhibited thesynthesis of CA and CDCA by about 13% and30%,respectively.CONCLUSION Isolated human hepatocytes inprimary culture behave as in the intact liver byconverting cholesterol to conjugated CA andCDCA.This is in contrast to cultured HepG2 cells,which release large amounts of bile acidprecursors and unconjugated bile acids into themedium. 展开更多
关键词 BILE acid FORMATION cell culture CHOLESTEROL METABOLISM CYCLOSPORIN human HEPATOCYTES
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