Paul P. C. Poyck

Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver

The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)‐BAL during a 7‐day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2‐fold more ammonia and produced 3‐fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5‐fold higher than MPHs and 6.6‐fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue‐like structures inside the AMC‐BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading. Liver Transpl 13:589–598, 2007.

Bioartificial liver: its pros and cons.

Abstract:  Both the large variety of liver functions for maintaining body homeostasis and the proven effectivity of whole liver transplantation in the therapy of acute liver failure (ALF), are important reasons to presume that cell‐free liver support systems will not be able to adequately support the failing liver. Accordingly, bioartificial liver (BAL) systems have shown their efficacy in experimental ALF models in small and large animals, and have shown to be suitable and safe in phase 1 studies in humans with ALF. However, the optimal BAL system is still under development. Important issues are the source of the cellular component and the configuration of the BAL system with regard to cell attachment, mass transfer characteristics and oxygenation at site. The deficiency of all BAL systems to excrete bile effectively is another important topic for improvement. The great challenge for the future is to develop a well‐functioning and safe human hepatic cell line which can replace the widely used porcine (xenogeneic) hepatocytes. Theoretically, a combination of a cell‐free liver support system and a BAL system might be optimal for the treatment of ALF patients in the near future.

Liver support therapy: an overview of the AMC-bioartificial liver research.

Acute liver failure (ALF) is a disease with a mortality of 60–90% depending on the cause. Only high-urgency liver transplantation is able to increase survival compared to standard intensive care therapy. Liver transplantation is hampered by the increasing shortage of organ donors, resulting in a high incidence of patients with ALF dying on the transplantation waiting list. Amongst a variety of liver assist therapies, bioartificial liver (BAL) therapy is marked as the most promising solution to bridge ALF patients to liver transplantation or to liver regeneration, since several BAL systems showed significant improvement of survival time in experimental animals with irreversible ALF. One of these systems has been developed at the Academic Medical Center in Amsterdam, The Netherlands – the AMC-BAL. This overview describes the development of the AMC-BAL based on porcine hepatocytes which was started 10 years ago. Positive results of in vitrofunctionality and in vivo safety and efficacy led to a successful phase I study in 12 ALF patients in Italy. However, xenotransplantation legislation in many European countries prohibits the use of porcine hepatocytes in clinically applied BAL systems. The future of the BAL, therefore, resides in the development of a human-derived hepatocyte cell line as biocomponent of BAL systems.

Three-dimensional numerical modeling and computational fluid dynamics simulations to analyze and improve oxygen availability in the AMC bioartificial liver

A numerical model to investigate fluid flow and oxygen (O2) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO2) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O2 consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO2 of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL.

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

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.

In vitro comparison of two bioartificial liver support systems : MELS CellModule and AMC-BAL

Clinically applied bioartificial liver (BAL) support systems are difficult to compare with regard to overall hepatocyte-specific function and clinical outcome. We compared two clinically applied BAL systems, the Modular Extracorporeal Liver Support (MELS) CellModule and the AMC-bioartificial liver (AMC-BAL) in an in vitro set-up. Both BAL systems were loaded with 10 billion freshly isolated porcine hepatocytes, cultured for 7 days and tested on days 1, 2, 4 and 7. Average decrease in hepatocyte-specific functions over 7 days was 9.7%. Three parameters differed between both bioreactors: lidocaine elimination at days 1 and 2 was significantly higher in the AMC-BAL, ammonia elimination showed a significantly higher trend for the AMC-BAL over 7 days and LDH release was significantly lower at day 7 for the MELS CellModule. In conclusion, this first in vitro comparison of two clinically applied BAL systems shows comparable functional capacity over a period of 7 days.

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

Background: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL). Methods: Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well. Results: Viable hepatocyte layers of approximately 150 µm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis. Conclusion: Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4α transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the β-catenin signaling pathway.

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes.

Background. Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. Methods. Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37°C. Bioreactors in the transport group were kept at 37°C during day 1, at 15°C during day 2, and again at 37°C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15°C and 26°C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. Results. A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. Conclusions. Mild hypothermic preservation at temperatures as low as 15°C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.

Systematic review and meta‐analysis of skin substitutes in the treatment of diabetic foot ulcers: Highlights of a Cochrane systematic review

Skin substitutes are increasingly used in the treatment of various types of acute and chronic wounds. The aim of this study was to perform a systematic review and meta‐analysis to evaluate the effectiveness of skin substitutes on ulcer healing and limb salvage in the treatment of diabetic foot ulcers. Randomized clinical trials were searched and assessed following the methodology of The Cochrane Collaboration. We included 17 trials, totaling 1655 randomized participants. Risk of bias was variable among included trials. Thirteen trials compared the skin substitutes with standard care. The pooled results showed that that skin substitutes can, in addition to standard care, increase the likelihood of achieving complete ulcer closure compared with standard care alone after 6–16 weeks (risk ratio 1.55, 95% confidence interval [CI] 1.30–1.85). Four of the included trials compared two types of skin substitutes but no particular product showed a superior effect over another. Two trials reported on total incidence of lower limb amputations. Pooling the results of these two trials yielded a statistically significantly lower amputation rate among patients treated with skin substitutes (risk ratio 0.43, 95% CI 0.23–0.81), although the absolute risk difference was small (−0.06, 95% CI −0.10 to −0.01). This systematic review provides evidence that skin substitutes can, in addition to standard care, increase the likelihood of achieving complete ulcer closure compared with standard care alone in the treatment of diabetic foot ulcers. However, effectiveness on the long term, including lower limb salvage and recurrence, is currently lacking and cost‐effectiveness is unclear.