Cell Transplantation have been treated with hepatocyte transplantation. In principle, the procedures for islet isolation, culture and transplantation may also be applied for hepatocytes. The liver tissue is digested with a two-step collagenase perfusion, followed by continuous-density Ficoll gradient purification in a refrigerated COBE 2991 centrifuge. Hepatocyte preparations are maintained in culture medium supplemented with ABO-identical serum. Data confirm that isolation of hepatocytes from the liver tissue is easier to perform than islet isolation and the isolation technique produces large quantities of viable hepatocytes. Thus, islet isolation facilities can be used also for hepatocyte isolation. Routes of hepatocyte transplantation As in islet transplantation, hepatocytes may be transplanted into the liver via the portal vein and it has been demonstrated that hepatocytes then translocate from the portal pedicle into the space of Disse by disrupting the sinusoidal endothelium and then join with adjacent host hepatocytes. Hepatocytes are regularly well engrafted in the liver (better than islets). Other potential implantation sites are striated musculature, peritoneal cavity, mesenteric leaves, the spleen, lung parenchyma, under the kidney capsule, and in the subcutaneous space. Therapeutic hepatocyte mass Hepatocytes occupy almost 80% of the total liver volume. The cell mass required to restore adequate liver function in a patient is dependent on the underlying disease. The cell mass needed to correct a single enzymatic defect e.g. familial hypercholesterolemia, is significantly less than for treatment of either acute or chronic liver failure. Thus, it has been suggested that 1- 5% of <strong>no</strong>rmal liver mass may be sufficient to restore adequate function of a single enzymatic liver defect, while a considerably larger hepatocyte mass is required for the treatment end stage liver failure. 10% of the liver mass (150 g) can be transplanted safely as hepatocytes via the intraportal route into the diseased liver. Since both fulminant and chronic liver failure require replacement of more than 10% of functional hepatocytes into the liver, additional ectopic sites such as the peritoneal cavity or musculature, may provide alternative sites for hepatocyte implantation. To correct enzymatic defects or to bridge a patient to Ltx or liver recovery 10 % functional hepatocytes from a <strong>no</strong>rmal liver may sufficient. An important distinction from islets is that there exists a wide range of endoge<strong>no</strong>us hepatic growth stimulators which increase the mitotic activity of transplanted hepatocytes and thus increases total hepatic mass. Several growth factors such as insulin and hepatocyte growth factor (HGF) can be injected and thereby could speed up the mitotic activity further. Interestingly, it has been shown that low molecular weight dextran sulfate (LMW-DS), a newly developed drug (within the Nordic Network for Islet Transplantation) used to counteract islet-blood interactions in clinical islet transplantation, induces a100-fold increase in the patient’s level of HGF. Interaction between foreign material and blood is a universal reaction following infusion, thus the inflammatory response elicited by hepatocyte-blood interactions may probably be diminished by LMW-DS, as it does in islet transplantation. Thus, the potential of LMW-DS in hepatocyte transplantation is promising. These hypotheses need further investigation. Sources of hepatocytes A limiting factor of clinical hepatocyte transplantation is shortage of mature functioning human hepatocytes, which currently are obtained from livers rejected for Ltx. The most common cause for rejecting a do<strong>no</strong>r liver for Ltx is excessive steatosis. Other potential sources of hepatocytes are part of livers after reduced sized Ltx, fetal livers and livers from patients transplanted for other causes than disordered hepatocytes such as polycystic livers and livers explanted due to hemangiomas. An other important source of hepatocytes could be livers from PSC patients (without cirrhosis) transplanted due to risk of developing malignancy in the liver. PSC is the most common cause of Ltx in Scandinavia, comprising more than 20% of all Ltxs. The suitability of such livers for hepatocyte transplantation should be explored, and if suitable, a significant number of livers will be available for hepatocyte transplantation in the Nordic countries. Furthermore, hepatocytes derived from bone marrow stem cells, immortalized hepatocyte cell lines and xe<strong>no</strong>genic hepatocytes should be investigated further. Banking of hepatocytes The most important indications for hepatocyte transplantation beyond enzymatic defects in children, are acute fulminant hepatic failure and liver failure following too extensive liver resections in patients with liver malignancy. In these acute situations, stored hepatocytes should be available. Therefore, in order to achieve this goal, a functional freezing protocol needs to be developed for maintenance of hepatocyte viability and metabolic capacity for a long period of time. In this aspect several cryopreservation techniques are developed but need further refinement. 36
Conclusions The major factor limiting hepatocyte transplantation <strong>research</strong> at present is the lack of availability of good quality human hepatocytes. However, PSC livers without cirrhosis and malignancy may represent an important source. Ultimately, as in islet transplantation, an unlimited supply of cells may be established through the creation of immortalized hepatocyte banks or the use of stem cells. 37
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