Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

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. </jats:p

Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure

Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF) providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use.Human hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.The optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10(6) cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.An optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation

Intraperitoneal transplantation of rat hepatocyte microbeads in rats with ALF.

<p>Representative images of (A) microbeads 7 days post-transplantation distributed throughout abdominal cavity [black arrow] without any signs of inflammation or adhesion; and (B) recovered microbeads, EMBs (left) partly covered by host cells [black arrow] and RMBs (right) with no evidence of cell adhesion; and (C) viable cells in RMBs demonstrated by FDA/PI staining.</p

Effect of intraperitoneal transplantation of rat hepatocyte microbeads on biochemical indexes of liver injury.

<p>Liver injury was evaluated by (A) prothrombin time (PT), (B) blood ammonia level, (C) serum aspartate aminotransferase (AST) level, (D) serum alanine aminotransferase (ALT) level, (E) serum bilirubin level and renal impairment assessed by (F) serum creatinine. Data are expressed as median and range; statistical significance*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.</p

Distribution of human hepatocytes in alginate-hepatocyte microbeads.

<p>Representative image of HMBs produced with different cell densities; 2.0, 2.5, 3.0 and 3.5×10⁶ cells/ml alginate (A, B, C, and D respectively) under light microscopy (left) and fluorescence microscopy (right). Representative confocal microscopy images used in 3D reconstruction to demonstrate (E & F) cell distribution and viability across the microbead, and (G) viability of cells within outer half vs inner half of same microbead. Green; viable and red; dead cells.</p