Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Abstract

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.

Natural Flavonol, Myricetin, Enhances the Function and Survival of Cryopreserved Hepatocytes In Vitro and In Vivo

“…Recently, novel methods for the cryopreservation of hepatocytes have been reported to improve the viability after freezing and thawing, i.e., alginate gel encapsulation [24,25], and vitrification [26]. Surprisingly, the combination of cryoprotective supplements such as anti-apoptotic pan-caspase inhibitor, iron chelator, and human serum albumin and alginate gel encapsulation increased albumin production of cryopreserved hepatocytes rather than freshly isolated hepatocytes [24]. The cryopreservation of a large mass of encapsulated hepatocytes has also been reported [25].…”

Human hepatocyte transplantation for liver disease: current status and future perspectives

“…The technique has been proven successful in a rat model of acute liver injury (35), and has shown encouraging results when used in children with ALF (unpublished). Furthermore, a recent study has shown that alginate microbeads containing hepatocytes can be cryopreserved with some maintenance of hepatic functions after thawing, figuring the possibility of an off-the-shelf product available in an emergency setting typical of fulminant liver failure (86).…”

Clinical Hepatocyte Transplantation: What Is Next?

“…One strategy involves the encapsulation of the donor cells in microbeads which provide an immune-protective coating while enabling the maintenance of necessary metabolic functions (75, 79–82). This approach is more amenable to the management of acute liver failure and recent work establishing animal models and developing mechanisms to create banked, cryopreserved microbeads for emergency use are potential advancements (83, 84). Additional techniques used to evade the host immune system include tissue-engineering strategies whereby liver mass constructs are assembled ex vivo and then inserted (usually into the peritoneal cavity) to provide a functional support system (85–87).…”