Woodchuck hepatocytes remain permissive for hepadnavirus infection and mouse liver repopulation after cryopreservation

Abstract

Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to Liver transplantation is a successful and well-established treatment for end-stage liver disease and liver failure. However, donor-organ scarcity is a fundamental limitation of this therapy. The availability of highly differentiated primary liver cells to be used for cell-based therapies, such as hepatocyte transplantation, tissue-engineered organs, or for extracorporeal liver support systems, represents an attractive alternative to whole-organ transplantation. 1,2 Freshly isolated normal adult hepatocytes are already widely used in various research areas of hepatology, pharmacology, and toxicology, and initial clinical trials have also shown their potential for therapeutic applications. 3 Essential prerequisites for therapeutic use of hepatocyte transplantation in humans is that primary liver cells must be promptly available, remain highly differentiated, and maintain their proliferative capabilities within the host liver, because only a limited number of cells can be infused into a patient. 4 In past years, numerous research laboratories established procedures for the isolation of primary hepatocytes from commonly used laboratory animals, rat hepatocytes being the most studied. However, only a few laboratories have the possibility to procure livers and perform the isolation of highly viable hepatocytes from humans and animal species that are scarcely available. Therefore, efficient cryopreservation and banking of hepatocytes would greatly expand and facilitate the use of primary liver cells both for research and therapeutic applications, while decreasing the need of freshly procured livers for the preparation of hepatocytes both from animal species and humans.Persistent infection with hepatitis B virus (HBV) is one of the major causes of liver disease in humans, being associated with various degrees of chronic hepatitis, cirrhosis, and the development of hepatocellular carcinoma. 5,6 Replication of HBV can be successfully achieved by transfecting hepatoma cell lines with cloned HBV-DNA genomes. These systems have significantly contributed to elucidating various asp...

In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice

“…13 Crossing uPA transgenic mice with immunodeficient mice lacking mature T and B lymphocytes permits engraftment and expansion of hepadnavirus-permissive primary hepatocytes. [14][15][16][17] In this study, we used uPA/severe combined immunodeficiency (SCID) chimeric mice to generate primary tupaia hepatocytes (PTHs) chronically infected with woolly monkey hepatitis B virus (WM-HBV), a hepadnavirus closely related to human HBV but infecting tupaia hepatocytes with higher efficiency. Recipient mice were then repopulated with isolated hepadnavirus-infected hepatocytes to investigate the stability and activity of cccDNA in vivo in the setting of liver regeneration.…”

Hepatic Hyperplasia Associated with Discordant Xenogeneic Parenchymal-Nonparenchymal Interactions in Human Hepatocyte-Repopulated Mice

“…2 When transplanted into the uPA-Tg mice, mouse (m) hepatocytes engrafted into the host liver and proliferated, eventually replacing the host hepatocytes with a replacement index (RI) of 80%, 3 where RI represents the ratio of the regions occupied by transplanted hepatocytes in the host liver). The offspring generated by crossing uPA-Tg mice with immunodeficient mice were used as hosts for the xenotransplantation of rat (r), 4 woodchuck, 5 and human (h) hepatocytes.…”

Proliferation of L02 human hepatocytes in tolerized genetically immunocompetent rats

“…The hepatitis virus has species-specificity and, susceptible hosts are confined to higher grade primates, such as humans, macaques and chimpanzees [14] for various reasons. With immunodeficient or induced immunotolerant rats, rat animal models with chimeric human liver infected with hepatitis virus can be established.…”