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Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

World Journal of Hepatology 2012年第5期4卷 176-183页

作者： mohammed saliem Frida Holm Rosita Bergstrm Tengzelius Carl Jorns Lisa-Mari Nilsson Bo-Gran Ericzon Ewa Ellis Outi Hovatta Division of Obstetrics and Gynecology Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden Division of Transplantation Surgery Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden Division of Pediatrics Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden

AIM:To optimize a xeno-free cryopreservation protocol for primary human ***:The demand for cryopreserved hepatocytes is increasing for both clinical and research *** several hepatocyte cryopreservation protocols being available,improvements are urgently *** first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the *** 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) *** 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 *** 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 ***: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 *** 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 ***,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were *** of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte ***,it may be a better alternative to the standard DMSO-UW *** CYP activities did not seem to be a relevant way to compare hepatocyte function

关键词： Human hepatocytes Viability Cytochrome P540 Dimethylsulphoxide Cryoprotectant Cryopreservation

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