Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells

Diener, Bernd; Traiser, Margarete; Arand, Michael; Leißner, Joachim; Witsch, U; Hohenfellner, R; Fändrich, F.; Vogel, Ingeborg; Utesch, Dietmar; Oesch, Franz

doi:10.1016/0887-2333(94)90105-8

“…Conversely, 17.5% and 20% DMSO appeared to be toxic to the hepatocytes and yielded significantly lower attachment efficiencies and CYP1A1/2 activities and significantly higher LDH leakage than the standard of 10% DMSO. Generally, DMSO is used at a concentration of 10% for hepatocyte cryopreservation 1, 4, 7, 10…”

Section: Discussionmentioning

confidence: 99%

“…A considerable number of human hepatocyte cryopreservation protocols have been reported for experimental use 1‐10. Not surprisingly, one of the main determinants of thawed cell function is the quality of the fresh hepatocytes before cryopreservation.…”

mentioning

confidence: 99%

Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation

Terry

¹

,

Dhawan

²

,

Mitry

³

et al. 2010

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Cryopreservation of human hepatocytes is important for their use in hepatocyte transplantation. On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in comparison with fresh cells. The aim of this study was to modify the different steps in the standard cryopreservation procedure in an attempt to improve the overall outcome. Human hepatocytes with a viability of 69% 6 SD 16% were isolated from donor livers with a collagenase perfusion technique. Different cell densities, concentrations, rates, and methods of addition of dimethyl sulfoxide were tested for the freezing solution. Modified controlled-rate freezer programs were tested to obtain a linear decrease in the temperature. Once they were frozen, the storage time and thawing method for hepatocytes were investigated. The effects on thawed cell viability and attachment, lactate dehydrogenase release, cytochrome P450 1A1/2 activity, and albumin synthesis were determined. The results were used to produce an improved cryopreservation protocol suitable for good manufacturing practice conditions. With a cell density of 10 7 cells/mL in University of Wisconsin solution containing 300 mM glucose, 10% (vol/vol) dimethyl sulfoxide was added dropwise over 5 minutes, and was immediately frozen. Thawing was done rapidly at 37 C, and dilution was performed with Eagle's minimum essential medium containing 300 mM glucose and 4% human serum albumin. Hepatocytes could be stored at À140 C without significant further loss of function for up to 3 years. With this protocol, hepatocytes had a viability of 52% 6 9%, an attachment efficiency of 48% 6 8%, and lactate dehydrogenase leakage of 17% 6 4%. This protocol is currently in use to cryopreserve hepatocytes for use in cell transplantation at our center. Liver Cryopreservation of human hepatocytes is essential for their use in hepatocyte transplantation. Successful cryopreservation allows hepatocytes to be available for the emergency treatment of acute liver failure and also for planned and repeated treatments with cells from the same donor for liver-based metabolic disorders. However, it is well known that cryopreservation affects the viability and metabolic function of hepatocytes on thawing, with the result that the thawed cells are often not suitable for clinical use.A considerable number of human hepatocyte cryopreservation protocols have been reported for experimental use.

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“…An amount of 0.5 ml DMSO was added drop‐wise to each cryovial to give a final DMSO concentration of 10% (v/v), and hepatocytes were then immediately cryopreserved. For both standard and preincubated hepatocytes, cryopreservation was undertaken using a controlled‐rate freezer (Kryo 10, Series III, Planer Products Ltd., Middlesex, UK) and a step‐wise freezing protocol4 with some modifications. Frozen cells were stored at −140°C for 2 weeks until thawing7 using WEM containing 10% FCS as the thawing media.…”

Section: Methodsmentioning

confidence: 99%

“…Successful cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model in toxicity and drug metabolism testing. A number of human hepatocyte cryopreservation protocols are currently in use 1–10. A fundamental limitation to thawed cell function is often the quality of hepatocytes before cryopreservation, which usually determines the quality of hepatocytes obtained upon thawing.…”

mentioning

confidence: 99%

Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing

Terry

¹

,

Dhawan

²

,

Mitry

³

et al. 2005

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Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and ␣-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E 1 , and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, ␣-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100 -300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100 -300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. ␣-lipoic acid preincubation (0.5 -5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM ␣-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation. (Liver Transpl 2005;11:1533-1540.)

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“…An amount of 0.5 ml DMSO was added drop‐wise to each cryovial to give a final DMSO concentration of 10% (v/v), and hepatocytes were then immediately cryopreserved. For both standard and preincubated hepatocytes, cryopreservation was undertaken using a controlled‐rate freezer (Kryo 10, Series III, Planer Products Ltd., Middlesex, UK) and a step‐wise freezing protocol4 with some modifications. Frozen cells were stored at −140°C for 2 weeks until thawing17 using WEM containing 10% FCS as the thawing media.…”

Section: Methodsmentioning

confidence: 99%

Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing

Terry

¹

,

Dhawan

²

,

Mitry

³

et al. 2005

View full text Add to dashboard Cite

Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and ␣-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E 1 , and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, ␣-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100 -300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100 -300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. ␣-lipoic acid preincubation (0.5 -5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM ␣-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation. Liver Transpl 11:1533-1540, 2005.

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Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells

Cited by 17 publications

References 24 publications

Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation

Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation

Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing

Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing

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