Assessment and improvement of liver specific function of the AMC-bioartificial liver.

UNLABELLED: The variety of methods for measuring bioactive mass and functionality of bioartificial livers (BAL) is confusing and prevents accurate comparison of reported data. Here we present a comparison of different hepatocyte quantification methods and propose that estimation of cell pellet volume after centrifugation generates a reliable, useful and fast method. In addition a correlation is made between several function tests performed in 26 bioreactors to assess their predictive value. The ammonia eliminating capacity was found to be most predictive for other liver functions, except for lidocaine elimination as a measure of mixed function oxidase activity, which should therefore be determined separately. The oxygen consumption test proved to be an easy and predictive parameter as well. The first generation of our BAL system needed further development to assure optimal treatment of acute liver failure (ALF) patients. Changes in the porcine hepatocyte isolation method and bioreactor loading as well as changes in bioreactor configuration, including use of different materials, resulted in a significantly improved level and maintenance of in vitro BAL function. A fourfold increase in ammonia eliminating capacity, which is only reduced to 75% after seven days of culturing, offers promising prospects for further clinical application. CONCLUSION: The current second generation of our BAL and improvement of hepatocyte isolation and testing protocols have led to a significant increase in the level as well as the maintenance of hepatocyte specific function in our BAL. Finally, consensus on definition of the bioactive mass to be loaded in the bioreactor and insight in the variation and reliability of the functional and metabolic parameters enhances comparison of the different types of bioartificial livers presented in literature.

Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing.

Due to the scarcity of available human livers, porcine hepatocytes are currently being evaluated as a xenogeneic cell source for extracorporeal bioartificial liver (BAL). Hypothermic storage of isolated porcine hepatocytes could support stocking of cell-loaded bioreactors for BAL use and may provide bioreactors ready to be used at the patient's bedside. For the development of this technology, it is of utmost importance to ensure cell viability and differentiated functions after low-temperature storage and following warm reperfusion. We compared cell viability, functional activity and apoptosis in isolated porcine hepatocytes which were perfused within a radial-flow bioreactor (RFB), stored at 4 degrees C and then reperfused at 37 degrees C. RFBs were loaded with 8 x 10(9), > or = 90% viable hepatocytes at 37 degrees C for 3 h. RFBs were then flushed with 4 degrees C University of Wisconsin solution (UW) and subsequently stored for 24 h or 48 h. RFBs were then reperfused for 8 h with recirculating medium plus serum at 37 degrees C . Cytochrome P450 (CYP) activity was studied before and after cold storage by means of monoethylglycinexylide (MEGX) detection in the effluent medium, after repeated lidocaine injections. After reperfusion experiments, hepatocytes were harvested for total RNA isolation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used in order to amplify specific mRNAs for Bcl-2 and Bax genes, by using appropriate primers; beta-actin primers were used as control. Total RNA was extracted by northern blotting analysis and for Bcl-2, Bax and beta-actin RNA messenger detection, RT-PCR amplification was used. Freshly isolated hepatocytes perfused into the RFB showed a progressive increase of MEGX while a loss in Bax expression was paralleled by an increase in Bcl-2 expression, in comparison to starting hepatocytes. After 4 degrees C storage and warm reperfusion, MEGX production was preserved in 24 h- and 48 h-stored bioreactors as well as a sharp increase of Bcl-2 and a decrease of Bax mRNAs. Our study suggests that refrigeration of hepatocyte-bioreactors is a suitable strategy to maintain both viability and function of isolated hepatocytes, for up to 48 h a time-length that is compatible with long-distance delivery of ready-to-use bioreactors.

Advanced technology for extracorporeal liver support system devices.

Acute Liver Failure (ALF) still presents high mortality rates, and liver transplant is the only treatment with proven efficacy. However transplant is not always possible and systems for Extracorporeal Liver Support (ELS) are being developed which can treat patients with ALF, for whom a transplant is not available, or is delayed. They can also treat patients with chronic liver disease who develop ALF. There are two types of ELS: artificial systems (hemoperfusion, plasmaperfusion, therapeutic plasma exchange, continuous hemodialysis and high volume continuous hemofiltration) and bioartificial systems. These are based on a biological component (animal or human hepatocytes) inserted into a bioreactor, whose main function is to perform the metabolic activity and synthesis that the liver can no longer perform. The results obtained in clinical trials have so far shown that the best results in terms of compensating for lost metabolic function and detoxification are obtained inserting artificial components in the bioartificial circuit.

ALEX (artificial liver for extracorporeal xenoassistance): a new bioreactor containing a porcine autologous biomatrix as hepatocyte support. Preliminary results in an ex vivo experimental model.

Long-term maintenance of viability and expression of differentiated hepatocyte function is crucial for bioartificial liver support. We developed a new bioreactor design (ALEX), associated with a new extracellular autologous hepatocyte biomatrix (Porcine Autologous Biomatrix - PBM) support. To test this new bioreactor, we compared it to a standard BAL (BioArtificial Liver) cartridge in a ex vivo model using human plasma added to bilirubin, ammonium and lidocaine. A pathology study was performed on both bioreactors. The results suggest that ALEX allows a maximal contact between the perfusing plasma and the liver cells and a proper hepatocyte support by a cell-to-matrix attachment. ALEX is a suitable cell support bioreactor, guaranteeing long-term maintenance of the metabolic activity of hepatocytes when compared to a standard BAL cartridge.

Biologic liver support: optimal cell source and mass.

Hepatic support is indicated in acute liver failure (ALF) patients to foster liver regeneration, or until a liver becomes available for orthotopic liver transplantation (OLT), in primary non function of the transplanted liver, and hopefully in chronic liver disease patients affected by ALF episodes, in whom OLT is not a therapeutic option. The concept of bioartificial liver (BAL) is based on the assumption that only the hepatocytes can perform the whole spectrum of biotransformation functions, which are needed to prevent hepatic encephalopathy, coma and cerebral edema. Among others, two important issues are related to BAL development: 1) the choice of the cellular component; 2) the cell mass needed to perform an adequate BAL treatment. Primary hepatocytes, of human or animal origin, should be considered the first choice because they express highly differentiated functions. Accordingly, a minimal cell mass corresponding to 10% of a human adult liver, i.e. 150 grams of freshly isolated, > or = 90% viable hepatocytes should be used. When 4 degrees C cold-stored or cryopreserved hepatocytes are used, the cellular mass should be increased because of a drop in cell viability and function. In case of hepatoma-derived cells, cultured cell lines or engineered cells, an adequate functional cell mass should be used, expressing metabolic and biotransformation activities comparable to those of primary hepatocytes. Finally, the use of porcine hepatocytes or other animal cells in BAL devices should be presently directed only to ALF patients as a bridge treatment to OLT, because of potential transmission of animal retrovirus and prions which may potentially cause major pandemics.

Long-term expression of highly differentiated functions by isolated porcine hepatocytes perfused in a radial-flow bioreactor.

To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry. Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter. During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased. Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study. The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.

[In vitro experimentation of a new model of radial flow bioreactor containing isolated hepatocytes]. / Sperimentazione in vitro di un nuovo modello di bioreattore a flusso radiale contenente epatociti isolati.

Hepatocyte based artificial liver support systems are under investigation to support acute liver failure patients. The main purpose of such systems is to serve as a bridge to liver transplantation, or to promote spontaneous liver recovery. Limitation in mass-transfer capacity makes hollow-fiber bioreactors unsuited for long-term functioning of hybrid devices. We developed a novel radial-flow bioreactor in which the fluid perfuses the module from the center to the periphery, after having diffused through a space occupied by a three-dimensional structure filled with the hepatocytes. Five grams of freshly isolated porcine hepatocytes were seeded into uncoated, woven-non woven, hydrophilic polyester fabric, overlaid by two polyethersulfone membranes. Liver cells were perfused with 37 degrees C-warm, oxygenated, serum-free tissue culture medium, in which NH4Cl and Lidocaine were added at the final concentration of 1 mM and 60 micrograms/ml, respectively. Ammonium chloride removal, urea synthesis, monoethylglycinexylide (MEGX), pO2, pCO2, and pH were measured throughout the 14 day duration of the study. In a separate set of experiments, a scaled-up version of the radial flow bioreactor containing 150 grams of cells was perfused for 7 h with recirculating human plasma and MEGX production was monitored. During the 2 weeks of the study, an increasing production of urea was paralleled by constant ammonium removal. MEGX concentration after Lidocaine addition increased throughout the 14 days of perfusion with tissue culture medium, as well as after 7 hour perfusion with human plasma. Under transmission and scanning electron microscopy cells appeared attached to the polyester and one to each other, displaying ultrastructural features typical of functioning hepatocytes. Our study showed that liver cells were metabolically active when perfused into the radial-flow bioreactor. This configuration allowed close contact between media, or plasma, and cells at a physiological flow rate, by equalizing the concentration of the perfusing components, including O2, throughout the module. Our results suggest a potential use of this system for temporary extracorporeal liver support in acute hepatic failure patients.

Effects of blood flow pulse frequency on mass transfer efficiency of a commercial hollow fibre oxygenator.

The clinical advantages achievable through pulsatile blood perfusion during cardio-pulmonary bypass have recently suggested the design of new pulsatile systems for extracorporeal circulation. Still it is not clear whether current commercial membrane oxygenators could be adopted with such systems, since their behaviour with pulsatile perfusion has not been satisfactorily documented yet. In a previous paper, we assessed that pulsatile perfusion of a widely used hollow fibre oxygenator (Sorin Monolyth) at 60 bpm provides more time-consistent oxygen transfer than steady perfusion. The present work is aimed to evaluate how the pulse frequency influences the gas transfer performance of the same device. The oxygenator was subjected to in vitro trials using a roller pump with pulsatile module (Stockert Instrumente) to generate pulsed flow. Four different pulse frequencies (45, 60, 75 and 90 bpm) were investigated at a fixed blood flow rate (4.0 l/min). The experiments lasting six hours were carried out using bovine blood with inlet conditions according to AAMI standards requirements. Blood samples were withdrawn every hour and O2 and CO2 transfer rates were evaluated. The experimental findings confirm that with pulsatile blood flow no time decay take place during prolonged perfusion. Moreover, when pulse frequency increases, transition levels occur for both O2 and CO2. Over these thresholds gas transfer rates display significant increases (p < 0.05), though of little magnitude (up to 2.5% for oxygen over 60 bpm; up to 3.7% for carbon dioxide over 75 bpm).

Mass transfer efficiency of a commercial hollow fibre oxygenator during six-hour in vitro perfusion with steady and with pulsatile blood flow.

Detailed data about the behaviour of commercial membrane oxygenators with pulsatile blood flow are rarely available. This work deals with an experimental evaluation of the effects induced on gas transfer efficiency by pulsatile perfusion of a hollow fibre oxygenator (Monolyth Sorin Biomedica). The oxygenator was subjected to two in vitro trials both carried out with identical experimental protocols except for the flow type, steady and pulsatile. A roller pump with pulsatile module (Stöckert Instrument) was used to generate both flow types. Three different mean blood flow rates (3.2, 4.0 and 4.8 L/min) were tested. The experiments lasting six hours were carried out using bovine blood with inlet conditions according to AAMI standard requirements. Blood samples were withdrawn every hour and the calculated gas transfer obtained in the two sessions were compared. The device proved to be well-designed for steady flow and to be liable to similar gas transfer performance when used in pulsatile conditions. Furthermore, the use of pulsatile flow rather than steady flow provided more consistent conditions and resulted in a higher eventual oxygen transfer efficiency (final mean difference = 6.2%, p < 0.05), proving to be able to avoid any performance decays.