Gesine Pless

Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study.

Introduction. The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support. Methods. Primary liver cells were isolated from 54 discarded organs (donor age 56.7±13.2 years). Between 2.8×1010 and 6.4×1010 parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36). The metabolic activity of the cells was regularly determined during culture. The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks. Results. Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days. Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures. Vascular cavities (CD 31 immunoreactivity [IR]) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed. Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver. Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected. Conclusion. The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy. In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

Cryopreservation of primary human hepatocytes: The benefit of trehalose as an additional cryoprotective agent

Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 ± 13 vs. 46.9 ± 11%, P < 0.01) and plating efficiency (41.5 ± 18 vs. 17.6 ± 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation. Liver Transpl, 2007.

Improvement of the cold storage of isolated human hepatocytes.

Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation results in severe cryoinjury, short-term storage is currently performed at 2–8°C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes. The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography.

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepa‐tocytes during and following cell application and the ability of a timely response to potential complications, a non‐invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron‐sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose‐finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5‐day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO‐labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO‐labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.

Artificial and Bioartificial Liver Support

The liver is a complex organ with various vital functions in synthesis, detoxification and regulation; its failure therefore constitutes a life threatening condition1. Liver failure (LF) can either occur without preceding liver disease (acute liver failure, ALF), usually caused either by intoxication (Amanita phalloides, acetaminophen, methylendioxymethamphteamine) or as acute decompensation of chronic liver-related illness (acute-on-chronic liver failure, AoCLF). In both cases, its symptoms include icterus, hepatic encephalopathy and impairment of coagulation status and may result in multi organ failure. Exceptionally, liver failure may also be triggered by certain diseases (Budd-Chiari-syndrome, Morbus Wilson) or pregnancy. The only long-term therapy in most cases is orthotopic liver transplantation, unless the liver is able to regenerate. Many patients, especially those who are not listed for high urgency transplantation, may not survive until a suitable donor organ is available, since donor organs are rare. In other cases, contraindications do not permit liver transplantation. For these indications, extracorporeal liver assist devices have been developed in order to either bridge the patient to transplantation or temporarily support the failing organ until it is able to regenerate.

Network Analysis of the Kinetics of Amino Acid Metabolism in a Liver Cell Bioreactor

The correlation of the kinetics of 18 amino acids, ammonia and urea in 18 liver cell bioreactor runs was analyzed and described by network structures. Three kinds of networks were investigated: i) correlation networks, ii) Bayesian networks, and iii) dynamic networks that obtain their structure from systems of differential equations. Three groups of liver cell bioreactor runs with low, medium and high performance, respectively, were investigated. The aim of this study was to identify patterns and structures of the amino acid metabolism that can characterize different performance levels of the bioreactor.

Dynamic model of amino acid and carbohydrate metabolism in primary human liver cells

Human liver cell bioreactors are used in extracorporeal liver support therapy. To optimize bioreactor operation with respect to clinical application an understanding of the central metabolism is desired. A two-compartment model consisting of a system of 48 differential equations was fitted to time series data of the concentrations of 18 amino acids, ammonia, urea, glucose, galactose, sorbitol and lactate, measured in the medium outflow of seven liver cell bioreactor runs. Using the presented model, the authors predict an amino acid secretion from proteolytic activities during the first day after inoculation of the bioreactor with primary liver cells. Furthermore, gluconeogenetic activites from amino acids and/or protein were predicted.

Prediction of the performance of human liver cell bioreactors by donor organ data

Human liver cell bioreactors are used in extracorporeal liver support therapy. To optimize bioreactor operation with respect to clinical application an early prediction of the long-term bioreactor culture performance is of interest. Data from 70 liver cell bioreactor runs labeled by low (n=18), medium (n=34) and high (n=18) performance were analyzed by statistical and machine learning methods. 25 variables characterizing donor organ properties, organ preservation, cell isolation and cell inoculation prior to bioreactor operation were analyzed with respect to their importance to bioreactor performance prediction. Results obtained were compared and assessed with respect to their robustness. The inoculated volume of liver cells was found to be the most relevant variable allowing the prediction of low versus medium/high bioreactor performance with an accuracy of 84 %.

Extracorporeal Liver Support

In patients with liver failure, the accumulation of lipophilic, albumin-bound toxins occurs which cannot be eliminated by standard hemodialysis and hemofiltration techniques. For this purpose (artificial and bioartificial) liver support systems were developed. Extracorporeal systems for artificial liver support include: Molecular adsorbents recirculating system (MARS) Single-pass albumin dialysis (SPAD) Fractionated plasma separation and adsorption (Prometheus) Selective plasma exchange therapy (SEPET) Extracorporeal systems for bioartificial liver support include: Extracorporeal liver perfusion (ECLP) HepatAssist Extracorporeal liver-assist device (ELAD) Modular extracorporeal liver support system (MELS) Bioartificial liver of the Academisch Medisch Centrum (AMC-BAL) The MARS, SPAD, and Prometheus systems are available for clinical use and are mostly employed either for bridging the patient to transplant or else for bridging to recovery of liver function. Positive data exist regarding biochemical efficacy and clinical improvement of certain end points such as hepatic encephalopathy, but adequately powered clinical trials evaluating survival rates are at present lacking.