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

Abstract

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.

Evaluating the Effects of Dithiothreitol and Fructose on Cell Viability and Function of Cryopreserved Primary Rat Hepatocytes and HepG2 Cell Line

“…This finding is in line with the study performed by Terry et al reporting 45% viability after thawing of the control group (33). In the current study, Williams’ culture medium E was used in freezing medium, while in the literature, University of Wisconsin solution (UW solution) has been recommended (13, 32, 33). The study results showed that using fructose improves the viability after thawing of the cells (both primary cells and cell line).…”

“…After preincubation with DTT (250 and 500 μM), the cell viability, attachment efficacy and function were significantly increased. Terry et al have shown that incubation of hepatocytes with glucose, fructose, or α-lipoic acid prior to freezing improves the post thaw viability and function of the hepatocytes (33). Fructose can protect the hepatocytes against apoptosis by forming nicotinamide adenine dinucleotide phosphate (NADPH) to regenerate the reduced glutathione (GSH).…”

“…This event reduces the generation of reactive oxygen species and affects the survival (34). Using fructose during hepatocyte isolation improves the recovery of energy by cells after ischemia reperfusion injury and cell isolation stress (33). GSH as an important component of cellular antioxidant defense mechanism, plays a crucial role in protecting cells against oxidative stress, and is essential for cell functions and survival.…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…There is also evidence by a few research groups that shows that a pre-incubation of hepatocytes as a suspension culture after their isolation greatly improves their recovery after cryopreservation (Darr and Hubel 2001; Hubel et al 2000; Gomez-Lechón et al 2006). Furthermore, several groups investigated the beneficial effect of anti-oxidants within this pre-incubation medium and concluded that the presence of anti-oxidant agents greatly helps the recovery of hepatocytes following thawing (Gomez-Lechón et al 2008; Terry et al 2006; Silva et al 1999). In most studies, hepatocytes are cryopreserved at a concentration ranging between 10 6 and 10 7 cells/ml and it has been demonstrated previously that the use of the UW solution supplemented with 10 % human albumin or fetal calf serum as a freezing medium has a beneficial effect on hepatocytes after thawing (Terry et al 2010; Arikura et al 2002; Adams et al 1995).…”

Current development of bioreactors for extracorporeal bioartificial liver (Review)

“…The strategies currently taken include ͑1͒ refinement of the isolation technology, and incubation of the isolated cells with medium containing antioxidants, at low temperature, before freezing. [42][43][44] This can effectively reduce the isolation damage to the cells and improve their quality prefreezing. ͑2͒ Modification of the formula of the cryopreserving medium and the protective agent.…”