Research - Human hepatocyte studies and their suitability for use in bioartificial liver development
Background
Interest in external liver support including both artificial and bioartificial systems (BAL) began in the 1950's and continues to develop. Originally a concept for the treatment of fulminant hepatic failure it potentially has wider applications. These include the concept of "bridging" patients with hepatic failure until a donor organ is available for liver transplantation: currently the only long-term treatment (1). Donor organ shortages and the length of time patients wait for a transplant organ (2) have made the need for an artificial system capable of supporting patients increasingly acute.
Advances in surgery and anaesthesia have led to dramatic advances in the field of liver surgery. Primary and secondary malignancies can be treated by major resections of up to 80% by volume of functional liver tissue (3). Larger resections are not currently feasible as the patients' remaining liver is unable to cope with the metabolic demands placed upon it. It is hoped that a functioning BAL will aid such patients while the liver remnant recovers sufficiently, therefore allowing more extensive resections to take place. A resection of 90% leaving just the caudate lobe, is theoretically possible and would approximately quadruple the number of potential surgical candidates.
If a human storage bank were to be successfully established it would abolish the requirement for a porcine cell BAL where safety concerns have been expressed (4). These include the possibility of hypersensitivity reactions and the potential for humoral sensitisation with the production of antibodies cross-reacting with human antigens (including HLA), although this is currently unfounded (5). Additionally there is further concern that the exposure of porcine tissue to the human will allow cross-species infection, in particular the pig endogenous retrovirus (PERV) (6). This virus has been transmitted to human cells in vivo (7), but their potential for infection and morbidity in vitro is not fully understood.
The Bioartificial Liver
Early attempts at constructing a BAL concentrated on detoxifying the blood by the utilisation of plasma exchange, plasmapheresis, blood exchange, haemodialysis, haemofiltration, cross-circulation, cross-haemodialysis and the use of charcoal (8,9). These measures generally had little impact in the long-term prognosis of patients and it was realised that the complexities of acute liver failure were not going to be resolved by these methods. Hepatocytes were therefore required to not only detoxify, but to maintain metabolic function, synthesis of protein and macromolecular structures and to partake in immune and hormonal pathways (10).
In 1969, Berry and Friend developed a method for isolating fresh hepatocytes that enabled the study of hepatocytes in an extra-corporeal bioreactor. This has lead to the production of a new generation of bioreactors with their successful application being demonstrated in animal models (11,12) and more recently in human trials (13).
The basic design of most BAL systems consists of a hollow fibre cartridge housing active hepatocytes in culture. Detoxification is achieved by the flow of plasma or whole blood across the surface of the structure. This allows diffusion of toxins and hepatocyte products across the interface. The different designs currently under evaluation have been reviewed (10). The array of models being developed clearly illustrates the complexity and difficulty the scientific community have had in developing this device.
One of the rate-limiting factors in BAL development is the availability and source of fresh hepatocytes. The methods for isolating and maintaining hepatocytes in culture are well developed and cells have been successfully extracted from the livers of various animal species (rat, porcine, rabbit) (14) and humans (15). Extraction of fresh human hepatocytes for use in any BAL system relies on the availability of liver tissue either from liver resections or multiple organ donors. Unfortunately this tissue is in short supply and with the limited time that cells function once isolated, there is the need to develop protocols for the long-term storage of functioning hepatocytes.
Cryopreservation
Cryopreservation has long been considered the answer to the storage and supply of hepatocytes in the intermediate term, but has proven more difficult than anticipated. With other cell types the parameters for routine cell cryopreservation have led to advances in animal cell technology. Principally, the protocols are based on the slow freeze and fast thaw of healthy cells in the presence of high protein concentrations and an agent that increases cell membrane permeability. After 20 years of experimentation with preservation of sperm, it was Polge et al (16) who made the observation that glycol enhanced the survival of fowl sperm at –79˚ C. The technique was then applied to several other cell types, including erythrocytes (17). The next step involved the discovery of the cryoprotective properties of dimethyl sulphoxide (DMSO) (18). It was not until the work of Mazur (19) that the mechanism of cryopreservation was better understood. He showed that cells, cooled slowly in the presence of a cryoprotectant, shrink and do not contain intracellular ice. Each cell type has its own optimum cooling rate, which is determined by its water permeability and the role of DMSO is to increase that permeability.
Nevertheless the last two decades has seen a significant increase in the interest in hepatocyte cryopreservation and research is underway in a number of units world-wide. Progress has been hindered by the poor availability of human cells and consequently initial work has concentrated on small animals and pigs. Clearly this makes comparison between the various studies complicated. Not only are different species used, but centres have used different experimental protocols. No standard cryopreservation protocol has as yet been internationally accepted although some have been suggested (20).
Over the last five years there has been a significant amount of work undertaken locally with both human and porcine hepatocytes. A number of variables that may impact on human hepatocyte yield and viability have been reviewed. These include donor factors (21), freeze rates and pre-incubation (22) and the storage concentration of hepatocytes (23). Despite promising results, "research should continue to improve the cryopreservation procedures…" (24).
Whilst hepatocyte viability declines over time after isolation, the extent to which we can achieve the goal of a clinically significant BAL depends on maintaining the function of these cells. There is consequently increasing interest in the use of anti-apoptotic and anti-necrotic agents within the field of cryopreservation. Apoptosis contributes to cell death in banked hepatocytes and therefore strategies to prevent anoikis (apoptosis occurring due to cell detachment from the extracellular matrix) are required to increase viability. Agents have been developed which demonstrate anti-apoptotic activity in cultured hepatocytes and include benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVAD-fmk) (25), caspase I inhibitor V (CryoStor CS 5N®) (26) and glucose (27). Further effort aimed at modulating apoptosis could significantly improve pre-existing methodology by improving viability.
The successful utilisation of cryopreserved hepatocytes and the membranes required to construct a bioartificial liver for organ support is vital to the function of that reactor (28). Realistically, a successful bioartificial liver system will be a hybrid system: function will be a result of a combination of filtration and the action of isolated hepatocytes. For cells to succeed they require the support of a membrane to allow high cell density and 3D cell arrangements to promote function, improve cell surface area and more closely resemble the original organ. Membrane type, pore size and configuration are still debated and it is this that needs further assessment.
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