Igor M. Sauer

Clinical extracorporeal hybrid liver support – phase I study with primary porcine liver cells

Abstract: The objective of this study was to evaluate the feasibility and safety of a hybrid liver support system with extracorporeal plasma separation and bioreactor perfusion in patients with acute liver failure (ALF) who had already fulfilled the criteria for high urgency liver transplantation (LTx). Eight patients (one male, seven female) were treated in terms of bridging to transplantation. The mean age was 36.5 yr (range 20 to 58). Etiology of liver failure was drug‐related in two patients, hepatitis B infection in three patients, and unknown for three patients. The bioreactors were charged with primary liver cells from specific pathogen‐free pigs. Cell viability varied between 91 and 98%. Continuous liver support treatment over a period of 8 to 46 h (mean 27.3 h) was safely performed and well‐tolerated by all patients. No complications associated with the therapy were observed during the follow‐up period. Thrombocytopenia was considered to be an effect of the plasma separation. Subsequently, all patients were transplanted successfully and were observed over at least 3 yr with an organ and patient survival rate of 100%. Screening of patients sera for antibodies specific for porcine endogenous retroviruses (PERVs) showed no reactivity – either prior to application of the system, or after extracorporeal treatment. The results encourage us to continue the development of the technology, and further studies appear to be justified. The bioreactor technology has been integrated into a modular extracorporeal liver support (MELS) system, combining biologic liver support with artificial detoxification technology.

In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD)

The detoxification capacities of single‐pass albumin dialysis (SPAD), the molecular adsorbents recirculation system, (MARS) and continuous veno‐venous hemodiafiltration (CVVHDF) were compared in vitro. In each experiment 4,100 mL of toxin‐loaded human plasma was processed for 6.5 hours. MARS treatment (n = 6) was undertaken in combination with CVVHDF. For SPAD (n = 6) and CVVHDF (n = 6) a high‐flux hollow fiber hemodiafilter (identical to the MARS filter) was used. Levels of ammonia, urea, creatinine, bilirubin, and bile acids were determined. Concentrations before and after application of detoxification procedures were expressed as differences and were compared using the Kruskal‐Wallis test. Post hoc comparisons for pairs of groups were adjusted according to Bonferroni‐Holm. Time, group, and interaction effects were tested using the nonparametric ANOVA model for repeated measurements. SPAD and CVVHDF induced a significantly greater reduction of ammonia levels than MARS. No significant differences were found among SPAD, MARS, and CVVHDF with respect to other water‐soluble substances. SPAD induced a significantly greater reduction in bilirubin levels than MARS. Reductions in bile acid levels were similar for SPAD and MARS. When operating MARS in continuous veno‐venous hemodialysis mode, as recommended by the manufacturer, no significant differences in the removal of bilirubin, bile acids, urea, and creatinine were found. However, MARS in continuous veno‐venous hemodialysis mode was significantly less efficient in removing ammonia than MARS in CVVHDF mode. In conclusion, the detoxification capacity of SPAD is similar to or even greater than that of MARS. (HEPATOLOGY 2004;39:1408–1414.)

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.

Identification of a Novel Human Polyomavirus in Organs of the Gastrointestinal Tract

Polyomaviruses are small, non-enveloped viruses with a circular double-stranded DNA genome. Using a generic polyomavirus PCR targeting the VP1 major structural protein gene, a novel polyomavirus was initially identified in resected human liver tissue and provisionally named Human Polyomavirus 12 (HPyV12). Its 5033 bp genome is predicted to encode large and small T antigens and the 3 structural proteins VP1, VP2 and VP3. Phylogenetic analyses did not reveal a close relationship to any known human or animal polyomavirus. Investigation of organs, body fluids and excretions of diseased individuals and healthy subjects with both HPyV12-specific nested PCR and quantitative real-time PCR revealed additional virus-positive samples of resected liver, cecum and rectum tissues and a positive fecal sample. A capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV12. Seroprevalences of 23% and 17%, respectively, were determined in sera from healthy adults and adolescents and a pediatric group of children. These data indicate that the virus naturally infects humans and that primary infection may already occur in childhood.

Liver support strategies: cutting-edge technologies

The treatment of end-stage liver disease and acute liver failure remains a clinically relevant issue. Although orthotopic liver transplantation is a well-established procedure, whole-organ transplantation is invasive and increasingly limited by the unavailability of suitable donor organs. Artificial and bioartificial liver support systems have been developed to provide an alternative to whole organ transplantation, but despite three decades of scientific efforts, the results are still not convincing with respect to clinical outcome. In this Review, conceptual limitations of clinically available liver support therapy systems are discussed. Furthermore, alternative concepts, such as hepatocyte transplantation, and cutting-edge developments in the field of liver support strategies, including the repopulation of decellularized organs and the biofabrication of entirely new organs by printing techniques or induced organogenesis are analysed with respect to clinical relevance. Whereas hepatocyte transplantation shows promising clinical results, at least for the temporary treatment of inborn metabolic diseases, so far data regarding implantation of engineered hepatic tissue have only emerged from preclinical experiments. However, the evolving techniques presented here raise hope for bioengineered liver support therapies in the future.

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.

Extracorporeal liver perfusion as hepatic assist in acute liver failure: a review of world experience

Background: There are almost no prospective, controlled and randomized clinical trials comparing different approaches towards hepatic assist. In order to create a basis for comparing the value of the existing different hepatic assist methods this article offers a systematic review of the world experience with allogeneic or xenogeneic extracorporeal liver perfusion (ECLP).

Isolation of Primary Human Hepatocytes After Partial Hepatectomy: Criteria for Identification of the Most Promising Liver Specimen

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patients sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

Development of a hybrid liver support system.

Abstract: Hybrid liver systems are being developed as temporary extracorporeal liver support therapy. The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy. In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors. These systems are capable of supporting essential liver functions. Animal experiments verify the possibility of upscaling bioreactors for clinical treatment. However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings. The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure. Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research. For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed. The construction is based on capillaries for hepatocyte aggregate immobilization. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three‐dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. The parallel use of identical units enables easy upscaling. Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising. 1

Graft versus host disease after liver transplantation - a single center experience and review of literature

Graft versus host disease (GvHD) after liver transplantation has an incidence of 0.1–1%. It is an infrequent but severe and mostly lethal complication. Approximately, 80 cases have been reported in literature so far. A single center experience is reported retrospectively. We performed a retrospective analysis of 1815 liver transplants in our center, transplanted over a period of 17 years. Five patients (5/1815 = 0.28%) with histologically diagnosed GvHD were included in the analysis. Onset of GvHD was between postoperative day (POD) 20 and 60. All patients developed skin rash, being the first symptom in four cases; one patient had joint pain as initial symptom. Macrochimerism was confirmed in all patients. Treatment consisted of augmentation of baseline immunosuppression (n = 4), methylprednisolone (n = 4), and T‐cell depleting antibodies (n = 3). One patient received no specific therapy because of her deleterious condition. All patients died because of either haemorrhage or uncontrollable infections. In our experience, GvHD has been an extremely rare, albeit deleterious clinical condition, which was resistant to classical immunosuppressive rescue regimens.