Abstract
Background and AimIntraperitoneal 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.MethodsHuman 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.ResultsThe 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 3.5×106 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.ConclusionAn 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.
Highlights
Acute liver failure (ALF) is a devastating condition which causes an abrupt loss of hepatic function leading to encephalopathy, coagulopathy and progressive multiple organ failure
The optimised hepatocyte microbeads (HMBs) were of uniform size (583.5¡3.3 mm) 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
We investigated the influence of cell density on physical integrity, cell viability and function in order to establish the optimal ratio of cell mass per microbead to allow oxygen, nutrients, and metabolite diffusion
Summary
Acute liver failure (ALF) is a devastating condition which causes an abrupt loss of hepatic function leading to encephalopathy, coagulopathy and progressive multiple organ failure. The principle of the microencapsulation technique is that the cells are embedded in a semi-permeable polymerised structure with the aim of protecting cells from host immune attack, while permitting the diffusion of nutrients, oxygen and metabolic products which maintain cell survival and function [6]. This approach allows cell transplantation without using immunosuppression and avoids the risk of bleeding. Microencapsulation could protect hepatocytes from cryoinjury leading to improved cell viability and function [7, 8], allowing cryopreserved cells to be available for emergency transplantation in ALF patients.
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