Somatostatin receptor subtype-2-deficient mice with diet-induced obesity have hyperglycemia, nonfasting hyperglucagonemia, and decreased hepatic glycogen deposition.

Hypersecretion of glucagon contributes to abnormally increased hepatic glucose output in type 2 diabetes. Somatostatin (SST) inhibits murine glucagon secretion from isolated pancreatic islets via somatostatin receptor subtype-2 (sst2). Here, we characterize the role of sst2 in controlling glucose homeostasis in mice with diet-induced obesity. Sst2-deficient (sst2(-/-)) and control mice were fed high-fat diet for 14 wk, and the parameters of glucose homeostasis were monitored. Hepatic glycogen and lipid contents were quantified enzymatically and visualized histomorphologically. Enzymes regulating glycogen and lipid synthesis and breakdown were measured by real-time PCR and/or Western blot. Gluconeogenesis and glycogenolysis were determined from isolated primary hepatocytes and glucagon or insulin secretion from isolated pancreatic islets. Nonfasting glucose, glucagon, and fasting nonesterified fatty acids of sst2(-/-) mice were increased. Inhibition of glucagon secretion from sst2-deficient pancreatic islets by glucose or somatostatin was impaired. Insulin less potently reduced blood glucose concentration in sst2-deficient mice as compared with wild-type mice. Sst2-deficient mice had decreased nonfasting hepatic glycogen and lipid content. The activity/expression of enzymes controlling hepatic glycogen synthesis of sst2(-/-) mice was decreased, whereas enzymes facilitating glycogenolysis and lipolysis were increased. Somatostatin and an sst2-selective agonist decreased glucagon-induced glycogenolysis, without influencing de novo glucose production using cultured primary hepatocytes. This study demonstrates that ablation of sst2 leads to hyperglucagonemia. Increased glucagon concentration is associated with impaired glucose control in sst2(-/-) mice, resulting from decreased hepatic glucose storage, increased glycogen breakdown, and reduced lipid accumulation. Sst2 may constitute a therapeutic target to lower hyperglucagonemia in type 2 diabetes.

Continuously microscopically observed and process-controlled cell culture within the SlideReactor: proof of a new concept for cell characterization.

Certain cell types, especially primary human cells, favor a well-defined culture environment offering continuous supply of nutrients and oxygen and waste product removal. Several bioreactors based on special matrices or hollow fibers have been developed that provide such conditions. However, characterization of matrix re-organization or growth of tissue within these systems is possible only after culture termination. Evaluation of the influence of certain medium additives or culture conditions (e.g., temperature, oxygenation) on cell viability, expansion, and differentiation within these systems remains a challenging task. The SlideReactor, a miniaturized hollow fiber-based bioreactor, was developed to enable the observation of cells during culture. An operation concept offering predefined conditions for various cell types has been designed. For proof of concept, primary human cells (hepatocytes, fibroblasts, keratinocytes) and cell lines (HepG2, HuH7, C3A, WiDr, SkHep1) were cultured and observed. A series of experiments (n=40) showed the feasibility of the set-up; determination of process parameters and continuous observation is possible. The SlideReactor may serve as a simple and cost-efficient tool for cell characterization and optimization of cell-culture conditions.

Effect of matrine on primary human hepatocytes in vitro.

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

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 2.8x10(10) and 6.4x10(10) 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.

Recovery of preservation-injured primary human hepatocytes and nonparenchymal cells to tissuelike structures in large-scale bioreactors for liver support: an initial transmission electron microscopy study.

This study investigated large-scale regeneration and tissue reorganization of adult human liver cells from preservation injured transplant organs. The use of basement membrane protein gels and growth factor enriched culture medium in standard culture flasks promotes liver tissue formation in isolated rat and pig hepatocytes, resulting in prolongation of phenotypic stability and metabolic competence of primary cells in vitro. A special bioreactor construction for high-density three-dimensional cell recovery was developed and isolation of cells from discarded human donor livers was enabled. In vitro regeneration of adult human liver cells isolated from preservation-injured organs took place over a period of 2 weeks in a purpose-built bioreactor. Basement membrane protein and growth factors were avoided. Reorganization of tissue structures was studied using transmission electron microscopy (TEM). This showed regeneration and tissue reorganization of adult human cells from preservation-injured organs by coculture with nonparenchymal cells in the bioreactor. The majority of the aggregated hepatocytes in the bioreactors showed morphological similarities to those in vivo (although not re-formed to hepatocyte plates), exhibiting cell-cell junctions and reconstituted bile canaliculi-like spaces between neighboring hepatocytes. Perfusion channels appeared regularly between cell aggregates. The arrangement of nonparenchymal cells between the hepatocyte aggregates exhibited similarities to liver sinusoids. Endothelial cells often covered the aggregates and formed a borderline to the perfusion channels between the capillaries. Similar to the space of Disse, further nonparenchymal cells were located between the endothelial cells and the parenchymal aggregates. Deposits of biomatrix fibers occurred spontaneously. The regenerated cell mass was close to that of a single liver lobe. In conclusion, the further optimization of bioreactors that enable cell recovery from preservation injury may lead to the utilization of cells from discarded whole or split transplants for extracorporeal temporary liver support therapy or hepatocyte transplantation.

Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions.

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.

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.

Bioartificial liver support systems.

A variety of bioartificial liver support systems were developed to replace some of the liver's function in case of liver failure. Those systems, in contrast to purely artificial systems, incorporate metabolically active cells to contribute synthetic and regulatory functions as well as detoxification. The selection of the ideal cell source and the design of more sophisticated bioreactors are the main issues in this field of research. Several systems were already introduced into clinical studies to prove their safety. This review briefly introduces a cross-section of experimental and clinically applied systems and tries to give an overview on the problems and limitations of bioartificial liver support.

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 hepatocytes 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 fact that liver failure constitutes a life-threatening condition and can, in most cases, only be overcome by orthotopic liver transplantation, lead to the development of various artificial and bioartificial liver support devices. While artificial systems are based on the principles of adsorption and filtration, the more complex concept of bioartificial devices includes the provision of liver cells. Instead of solely focussing on detoxification, these concepts also support the failing organ concerning synthetic and regulative functions.The systems were evaluated in a variety of clinical studies, demonstrating their safety and investigating the impact on the patient's clinical condition. This review gives an overview over the most common artificial and bioartificial liver support devices and summarizes the results of the clinical studies.

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.

Evaluation of primary human liver cells in bioreactor cultures for extracorporeal liver support on the basis of urea production.

Primary human liver cells from donor organs unsuitable for transplantation were cultivated in bioreactors developed for extracorporeal liver support. Because each system contains cells originating from an individual organ, each bioreactor culture must be individually characterized. The objective of this study was to identify suitable decisive parameters for the evaluation of cell culture performance. We analyzed the data from 47 bioreactor cultures containing 437 +/- 110 g of cells. Choosing urea production as the decisive parameter, the bioreactor cultures were divided into high-performance (daily urea production > or = 110 mg per bioreactor between culture days 3 and 14) and low-performance cultures. Comparing the mean courses of the groups revealed a significant distinction in most other investigated biochemical parameters. In conclusion, urea production seems to be an appropriate parameter for evaluating the performance of liver cell cultures in bioreactors because it corresponds to all other evaluated parameters of cell function.

"Blogs" and "wikis" are valuable software tools for communication within research groups.

Appropriate software tools may improve communication and ease access to knowledge for research groups. A weblog is a website which contains periodic, chronologically ordered posts on a common webpage, whereas a wiki is hypertext-based collaborative software that enables documents to be authored collectively using a web browser. Although not primarily intended for use as an intranet-based collaborative knowledge warehouse, both blogs and wikis have the potential to offer all the features of complex and expensive IT solutions. These tools enable the team members to share knowledge simply and quickly-the collective knowledge base of the group can be efficiently managed and navigated.

The SlideReactor--a simple hollow fiber based bioreactor suitable for light microscopy.

Most bioartificial liver support systems are based on hollow fiber capillaries within modified dialysis cartridges or more sophisticated bioreactor constructions. Due to their design microscopic follow-up of reorganization and growth of tissue between the hollow fibers is not possible. The SlideReactor is a simple hollow fiber based bioreactor construction suitable for light microscopy and time-lapse video observation. The SlideReactor offers a cell compartment separated from a medium inflow and outflow compartment. Cell compartment access ports enable easy filling of the cell compartment with cell suspension, as well as fixation of the tissue. For more complex procedures or full access to all the cells, the bioreactor can be opened easily by cutting the silicone seal with a scalpel. Due to its simple design and the utilization of standard materials, it could serve as a suitable, cost-efficient tool to evaluate the behavior of cells cultured between hollow fiber capillaries. The paper describes the production process: similar to open source projects in software engineering, we would like to propose the concept as an open platform to anyone interested in hollow fiber based cell culture.

In vitro evaluation of the transportability of viable primary human liver cells originating from discarded donor organs in bioreactors.

BACKGROUND: The use of primary human liver cells obtained from discarded donor organs is increasingly favored for cell-based extracorporeal liver support systems. However, as cryopreservation of primary human hepatocytes causes a significant loss of metabolic activity, the transport of bioreactors with viable liver cells is required. The aim of this study was to evaluate the impact of two major potential threats to viable cells during transport: temperature changes and mechanical stress. METHODS: In each experiment three hollow fiber-based bioreactors were charged with primary human liver cells originating from the same discarded donor organ and were simultaneously kept under culture conditions for 8 days. In total, 18 bioreactors were evaluated. On the fifth day the bioreactors were exposed to hypothermia (4 degrees C, n = 3), to hyperthermia (42 degrees C, n = 3), or served as normothermic controls (37 degrees C, n = 3). In a second test series bioreactors were exposed to vibration (21 Hz for 20 min, thereafter 7 Hz for 160 min, n = 3), or were operated as control cultures (n = 6). The release of hepatocyte-specific enzymes was determined as an indicator for cell damage. RESULTS: Hypothermic stress resulted in a significant release of transaminases and led to disturbances of the histological integrity, all indicating a high degree of cell damage. When compared with the control cultures, hyperthermia and mechanical stress in terms of vibration had no significant effect on the cells. CONCLUSION: The transport of hollow fiber bioreactors charged with viable primary human liver cells appears to be feasible in transport monitors for perfusion and temperature control.

Extracorporeal liver support based on primary human liver cells and albumin dialysis--treatment of a patient with primary graft non-function.

METHODS: Following liver transplantation, a 26-year old female suffered from primary non-function of the transplant. The patient was subsequently treated with a modular extracorporeal liver support concept until a suitable organ became available. A bioreactor was charged with human liver cells, obtained from a discarded cadaveric graft (470 g, viability: 60%). The bioreactor was integrated into an extracorporeal circuit with continuous single pass albumin dialysis and continuous veno-venuous hemodiafiltration for detoxification and fluid reduction. RESULTS: Over the total system application time of 79 h, a significant reduction of the plasma levels of total bilirubin (21.1 mg/dl at start, 10.1 mg/dl at end of therapy) and ammonia (100 versus 22.7 micromol/l) was achieved. During treatment the patient's neurological status significantly improved from coma stage IV to I permitting extubation. Recovery of kidney function with a urine output of 1325 ml/24 h compared to 45 ml/24 h prior to system application, was noted. Over the treatment period, an improvement of coagulation status was observed. Adverse events were absent. CONCLUSIONS: This first successful clinical treatment of a patient with liver failure suggests that a modular approach combining both primary human liver cell bioreactor technology and detoxification methods is promising.