Cell therapy: Liver and pancreatic cell therapy research
Research on liver and pancreatic cell therapy are carried out at the Leicester General Hospital with ongoing local projects and national and international collaborations. Cell therapy is another clinical approach used for treating numerous diseases, for this reason liver and pancreatic cells could be used in the treatment of diseases affecting the liver and pancreatic function.
Liver cell therapy
Our main objective is to develop a liver support system (bioartificial liver: BAL) that will allow post-operative support of patients with hepatic failure. A BAL would also support patients who need large resections of liver tumour. A BAL would also support patients who need large volume resections of liver tumours and who are left with small remnants of liver after surgical intervention unable to maintain enough hepatic function.
The incorporation of human liver cells (hepatocytes) is by far the most common approach to a BAL. Isolated hepatocytes incorporated in a BAL could allow a transient mainstay of the diseased or partially resected liver, in order to detoxify the organism. The use of hepatocytes in a BAL bridged to a patient, during the time course of liver regeneration could avoid liver transplantation.
Isolated hepatocytes are also used for numerous other applications than BAL. Human hepatocytes are useful in pharmacotoxicology for the evaluation of new drugs and in cell transplantation for the treatment of liver failure. Human hepatocytes could be directly transplanted in patients, since hepatocyte transplantation for the treatment of metabolic disorders has been proven.
We routinely isolate hepatocytes from surgical specimens where there is normal liver beyond the resected tumour that would otherwise be discarded (Figure 1a and 1b).
Figure 1a: Diagram of a liver tumour resection
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Figure 1b: Normal liver tissue that is usually discarded
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Viable intact hepatocytes are obtained by a cell isolation method consisting of the perfusion of a collagenase enzyme solution through the vessels of the liver tissue (Figure 2). Once the collagenase has digested the extracellular matrix tissue (essentially made of collagen proteins), hepatocytes can be released and suspended in a specific medium before culture and/or cryopreservation.
Figure 2: Human liver perfused with a collagenase solution
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Isolated hepatocytes are cultured immediately after isolation to establish the optimal culture conditions and to maintain liver function in vitro for as long as possible (Figure 3).
Figure 3: Human hepatocyte monolayer culture (magnification x200)
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Freshly isolated hepatocytes can also be cryopreserved (frozen at extremely low temperature and stored in liquid nitrogen i.e. -196°C) to constitute an available source of human hepatocytes for a bioartificial liver (Figure 4).
Figure 4: Cryopreserved human hepatocytes in liquid nitrogen
Cryopreservation, the process of freezing cells at a temperature below –150°C, has been successfully used for the long-term storage of different cell types. Nevertheless, cryopreservation methods appear to significantly decrease hepatocyte viability and function once thawed, hence the need to improve freezing methods for long-term storage of human hepatocytes. Techniques for improving the effective isolation, culture and cryopreservation of highly functional human hepatocytes are currently optimised in our laboratory to develop a BAL.
A rigorous evaluation of hepatocyte function is required. Amongst numerous liver functions, cellular capacities of in vitro detoxification are the most important ones and are evaluated specifically on hepatocytes by measuring P450 detoxifying enzyme activities. These studies are carried out in collaboration with the pharmaceutical company AstraZeneca in Charnwood and with DeMonfort University Laboratories in Leicester. The evaluation of the efficacy of human hepatocytes in the treatment of liver failure needs to be carried out as well in experimental models of hepatocyte transplantation which mimick human acute toxic fulminant hepatitis, acute liver failure following large resection of liver tumours or chronic cirrhosis. Two international collaborative research centres in Australia (University of Adelaide Department of Surgery, Adelaide) and in France (Fondation Transplantation in Strasbourg and University of Besançon, Besançon) have extensive experience in hepatocyte transplantation and are also participating in the experimental evaluation of hepatocytes for BAL development. We are also collaborating with UMIST (University of Manchester Institute of Science & Technology) in Manchester to develop a suitable membrane and bioreactor for the maintenance of hepatocytes in a 3D configuration (Figure 5), which is crucial for the survival of the cells and for the development of a BAL.
Figure 5: Human hepatocyte 3D culture (magnification x200)
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Figure 6: Poster presented at the Hepatocyte Users Group meeting, Stansted, 3-4 November 2006. “Does curcumin prevent oxidative damage during human hepatocyte isolation, culture or cryopreservation?”
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Figure 7: Poster presented at the Human Hepatocyte Transplantation meeting, Valencia, Spain, 10-11 November 2006.
“Human hepatocyte spheroid culture for use in bioartificial liver systems”
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Figure 8: Presentation at the Society of Academy & Research Surgery meeting, Cambridge, 10-12 January 2006.
“Can human hepatocyte spheroids be used in bioartificial liver systems?”
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