Referring to our paper „CD44 and CXCL9 serum protein levels predict the risk of clinically significant allograft rejection after liver transplantation“ Geoffrey W. McCaughan, Patrick Bertolino and David G. Bowen wrote an interesting editorial entitled „Could The Morning After liver transplant be immunologically interesting?“ 

They conclude, „that our study urges us to study the immune system response in liver allograft recipients during the very early phases after liver transplantation and to explore how events in immune organs and the allograft are reflected within the serum. Whether the patterns observed truly represent early detection of ACR versus tolerance, or a combination of both, requires further study and experimentation, including the identification of the cellular sources of these and other potential markers of immune outcome. It seems that despite significant levels of immunosuppressive drugs, immune activation and engagement occurs very early after human liver transplant, within the first 24 hours, in a manner that may have similarities with experimental animal models. Thus, the morning after effect could be an exciting window to longer-term immune outcomes, rather than just being preoccupied with observing important routine outcomes and detecting early complications.“

„Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device“ is available in J Vis Exp. 2015 Aug 10;(102). doi: 10.3791/53029 – accessible via the JOVE servers. 

Authors are K. Hillebrandt, D. Polenz, A. Butter, P. Tang, A. Reutzel-Selke, A. Andreou, H. Napierala, N. Raschzok, J. Pratschke, I.M. Sauer, and B. Struecker . The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Our paper "microRNAs in liver tissue engineering - New promises for failing organs"was accepted for publication in Advanced Drug Delivery Reviews (IF: 15.038). Authors are Nathanael Raschzok, Hannes Sallmon, Johann Pratschke and Igor M. Sauer.

miRNA-based technologies provide attractive tools for several liver tissue engineering approaches. Herein, we review the current state of miRNA applications in liver tissue engineering. Several miRNAs have been implicated in hepatic disease and proper hepatocyte function. However, the clinical translation of these findings into tissue engineering has just begun. miRNAs have been successfully used to induce proliferation of mature hepatocytes and improve the differentiation of hepatic precursor cells. Nonetheless, miRNA-based approaches beyond cell generation have not yet entered preclinical or clinical investigations. Moreover, miRNA-based concepts for the biliary tree have yet to be developed. Further research on miRNA based modifications, however, holds the promise of enabling significant improvements to liver tissue engineering approaches due to their ability to regulate and fine-tune all biological processes relevant to hepatic tissue engineering, such as proliferation, differentiation, growth, and cell function.

Based on a fruitful collaboration with the department of Visceral, Transplantation, Thoracic, and Vascular Surgery at the University of Leipzig our paper on „CD44 and CXCL9 serum protein levels predict the risk of clinically significant allograft rejection after liver transplantation“ has been accepted for publication in Liver Transplantation. 

Authors are Nathanael Raschzok, Anja Reutzel-Selke, Rosa Bianca Schmuck, Mehmet Haluk Morgul, Ulrich Gauger, Kukuh Aji Prabowo, Laura-Marie Tannus, Annekatrin Leder, Benjamin Struecker, Sabine Boas-Knoop, Michael Bartels, Sven Jonas, Christian Lojewski, Gero Puhl, Daniel Seehofer, Marcus Bahra, Andreas Pascher, Johann Pratschke, and Igor Maximilian Sauer.

The diagnosis of acute cellular rejection (ACR) after liver transplantation is based on histological analysis of biopsies because non-invasive biomarkers for allograft rejection are not yet established for clinical routines. CD31, CD44, and CXCL9 have previously been described as biomarkers for cross-organ allograft rejection. Here, we assessed the predictive and diagnostic value of these proteins as serum biomarkers for clinically significant ACR in the first six months after liver transplantation in a prospective study. The protein levels were measured in 94 patients immediately prior to transplantation, at postoperative days (POD) 1, 3, 7, and 14, and when biopsies were performed during episodes of biochemical graft dysfunction. Our results suggest that CD44 and CXCL9 may serve as predictive biomarkers to identify liver allograft recipients at risk for clinically significant ACR.

BMC Research Notes accepted our latest paper on „Independent Effects of Sham Laparotomy and Anesthesia on Hepatic microRNA Expression in Rats“ for publication.

Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly down-regulated following anesthesia and surgery. We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery.

Authors are W. Werner, H. Sallmon, A. Leder, S. Lippert, A. Reutzel-Selke, M.H. Morgül, S. Jonas, S. Dame, P. Neuhaus, J. Iacomini, S.G. Tullius, I.M. Sauer and N. Raschzok.

Brought to you by the world’s leading transplant clinicians, Textbook of Organ Transplantation provides a complete and comprehensive overview of modern transplantation in all its complexity, from basic science to gold-standard surgical techniques to post-operative care, and from likely outcomes to considerations for transplant program administration, bioethics and health policy.
Beautifully produced in full color throughout, and with over 600 high-quality illustrations, it successfully
- Provides a solid overview of what transplant clinicians/surgeons do, and with topics presented in an order that a clinician will encounter them.
- Presents a holistic look at transplantation, foregrounding the interrelationships between transplant team members and non-surgical clinicians in the subspecialties relevant to pre- and post-operative patient care, such as gastroenterology, nephrology, and cardiology.
- Offers a focused look at pediatric transplantation, and identifies the ways in which it significantly differs from transplantation in adults.
- Includes coverage of essential non-clinical topics such as transplant program management and administration; research design and data collection; transplant policy and bioethical issues.

Editors are Allan D. Kirk, Stuart J. Knechtle, Christian P. Larsen, Joren C. Madsen, Thomas C. Pearson, and Steven A. Webber.
I.M. Sauer, N. Raschzok und P. Neuhaus contributed chapter 47: „Artificial Liver, In Vivo Tissue Engineering, and Organ Printing – Solutions for Organ Scarcity“

The Wiley-Blackwell book (ISBN: 978-1-118-87014-3, 1880 pages) is available here.

Pluripotent stem cells have been an important tool for researches in hepatology, starting with mouse embryonic stem cells and transgenesis. Together, with other animal models, they largely contributed to our current knowledge in hepatic cells development and disease modeling. Therefore, with the possibility to manipulate human embryonic stem cells and more recently human induced pluripotent stem cells, there was an existing substratum to study these processes in a human environment, which contributed to the tremendous explosion of the emerging Liver Regenerative Medicine field. The use of such cells in the last few years has been already at the origin of numerous breakthroughs in disease modeling, host-pathogen interactions studies, or liver bioengineering and many are to come.

Investigators are invited to participate in a special issue on „Human Pluripotent Stem Cells in Hepatic Development, Liver Reconstruction and Disease Modeling“ through original research articles at the forefront of this fast pace moving field as well as reviews describing current and forthcoming challenges.

Lead Guest Editor is Karim Si-Tayeb (Institute of the Thorax, Nantes). Guest Editors are Gareth Sullivan (Institute of Basic Medical Sciences, University of Oslo), Robert Schwartz (Weill Cornell Medical College, Cornell University, New York), Nathanael Raschzok (Charité – Universitaetsmedizin Berlin), and Tamir Rashid (King's College London).

Manuscript Due: Friday, 23 January 2015
First Round of Reviews: Friday, 17 April 2015
Publication Date: Friday, 12 June 2015

The Journal of Tissue Engineering and Regenerative Medicine accepted our latest paper on „Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions“ for publication. Authors are B. Struecker, A. Butter, K. Hillebrandt, D. Polenz , A. Reutzel-Selke, P. Tang, S. Lippert, A. Leder, S. Rohn, D. Geisel, T. Denecke, K. Aliyev, K. Jöhrens, N. Raschzok, P. Neuhaus, J. Pratschke and I.M. Sauer.

One approach of regenerative medicine to generate functional hepatic tissue in vitro is de- and recellularization and several protocols for the decellularization of different species have been published. To the best of our knowledge this is the first report on rat liver decellularization by perfusion via the hepatic artery under oscillating pressure conditions, intending to optimize microperfusion and minimize damage to the ECM. Four decellularization protocols were compared: perfusion via the portal vein (PV) or the hepatic artery (HA), with (+P) or without (-P) oscillating pressure conditions. All rat livers (n=24) were perfused with 1% Triton X-100 and 1% SDS, each for 90 minutes with a perfusion rate of 5ml/min. Perfusion decellularization was observed macroscopically and the decellularized liver matrices (DLMs) were analyzed by histology and biochemical analyses (e.g., levels of DNA, glycosaminoglycans (GAG), and hepatocyte growth factor (HGF). Livers decellularized via the hepatic artery and under oscillating pressure showed a more homogenous decellularization and less remaining DNA, compared to livers of the other experimental groups. The novel decellularization method described is effective, quick (3 hours) and gentle to the ECM and thus represents an improvement of existing methodology.

Nature Reviews Gastroenterology and Hepatology invited us to provide a review on liver support strategies.

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.


Update: The review „Liver support strategies: cutting-edge technologies“ (authors: Benjamin Struecker, Nathanael Raschzok & Igor M. Sauer) is now available.

Our latest review on „Noninvasive monitoring of liver cell transplantation“ (authors are N. Raschzok, H.M. Morgül, L. Stelter & I.M. Sauer) is available in Imaging in Medicine, 2013; 5: 47-61:
Liver cell transplantation was developed as a therapeutic alternative to solid liver transplantation in the management of liver-based metabolic disorders and may be useful for the treatment of acute or chronic liver failure. While clinical studies have demonstrated temporal amelioration of the symptoms of metabolic liver disorders by transplanted liver cells, the long-term outcome of liver cell transplantation is still insufficient. A major limitation for improving liver cell transplantation is the inability to track the fate of cells once they have been infused. Radionuclide-based imaging, MRI and optical methods have been investigated as methods for noninvasive monitoring of liver cell transplantation. This article summarizes and critically discusses these approaches, with a special focus on MRI-based tracking of transplanted liver cells and provides an outlook on possible clinical applications for the near future.

Nathanael Raschzok’s latest paper on „Feasibility of fast dynamic MRI for noninvasive monitoring during ectopic liver cell transplantation to the spleen in a porcine model“ is now available in AJR Am J Roentgenol. 2012 Jun;198(6):1417-23.
Liver cell transplantation is a promising approach for the treatment of metabolic liver disorders. However, a method for noninvasive monitoring during liver cell transplantation is not available clinically. The aim of this study was to investigate the feasibility of fast dynamic MRI monitoring during liver cell infusion to the spleen, which is considered an ectopic implantation site for liver cell transplantation. Porcine liver cells were labeled with micron-sized iron oxide particles and infused to the spleens of pigs (n = 5) via the lineal artery. MRI was performed using a 3-T MR scanner. Initially, T1- and T2-weighted pulse sequences were tested. Thereafter, fast dynamic MRI was performed during cell infusion. MR findings were verified by immunohistological examinations.

Images from static MRI (TR/TE, 2500/105.2) showed significantly lower signal intensity and signal-to-noise ratio after cell infusion compared with pretransplant images. T2-weighted fast dynamic MRI enabled visualization of signal decrease of the spleen during cell infusion. When cells were infused systemically, no signal changes in the spleen were observed. This study shows that fast dynamic MRI can enable noninvasive monitoring during liver cell transplantation to the spleen. This approach could be useful for preclinical studies and for quality control of clinical liver cell transplantation.

Following a successful project sponsored by the BMBF G. Pless, I.M. Sauer and U. Rauen report on the "Improvement of the cold storage of isolated human hepatocytes" (Cell Transplant. 2011 Jun 7. [Epub ahead of print]).
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 2-8º 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 pre-culture 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 one 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 two 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, glucacon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.

Micrometer-sized iron oxide particles (MPIOs) attract increasing interest as contrast agents for cellular tracking by clinical Magnetic Resonance Imaging (MRI). Despite the great potential of MPIOs for in vivo imaging of labeled cells, little is known on the intracellular localization of these particles following uptake due to the lack of techniques with the ability to monitor the particle uptake in vivo at single-cell level. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy enables non-invasive, label-free imaging of MPIOs in living cells with sub-micron resolution in three dimensions. CARS allows simultaneous visualization of the cell framework and the MPIOs, where the particles can be readily distinguished from other cellular components of comparable dimensions, and localized inside the cell.
The fruitful cooperation with the FOM Institute AMOLF in Masterdam resulted in the paper "CARS microscopy for the visualization of micrometer-sized iron oxide MRI contrast agents in living cells" (Rago G, Langer CM, Brackman C, Day JP, Domke KF, Raschzok N, Schmidt C, Sauer IM, Enejder A, Mogl MT, Bonn M.) published in Biomed Opt Express. 2011 Sep 1;2(9):2470-83.

As a first result of our latest projects concerning the role of miRNA in liver regeneration the American Journal of Physiology - Regulatory, Integrative and Comparative Physiology has accepted our paper "Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy": The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small non-coding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 hours - 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. 2D-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 hours after resection. The temporal dynamics of liver regeneration were characterized by BrdU, PCNA, IL-6, and HGF. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 hours after resection. Expression of thirteen miRNAs was significantly reduced 12-48 hours after resection (> 25% change), ouf of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 hours, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration. Authors are N. Raschzok, W. Werner, H. Sallmon, N. Billecke, C. Dame, P. Neuhaus and I.M. Sauer.

Our latest paper on "Monitoring of liver cell transplantation in a preclinical swine model using magnetic resonance imaging" has been accepted for publication in CELL Medicine (Part B of CELL TRANSPLANTATION). Authors are Nathanael Raschzok, Ulf Teichgräber, Nils Billecke, Anja Zielinski, Kirsten Steinz, Nora N. Kammer, Mehmet H. Morgul, Sarah Schmeisser, Michaela K. Adonopoulou, Lars Morawietz, Bernhard Hiebl, Ruth Schwartlander, Wolfgang Rüdinger, Bernd Hamm, Peter Neuhaus and Igor M. Sauer. The study was based on the excellent colaboration with the department of Radiology and the Institute of Pathology, both Charité - Campus Mitte, Universitätsmedizin Berlin, Berlin, Germany, the Centre for Biomaterial Development and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Institute for Polymer Research, GKSS Research Centre Geesthacht GmbH, Teltow, Germany, the Department of Materials, ETH Zurich, Zurich, C Switzerland, and Cytonet GmbH, Weinheim, Germany.
Liver cell transplantation (LCT) is a promising treatment approach for certain liver diseases, but clinical implementation requires methods for non-invasive follow-up. Labeling with superparamagnetic iron oxide particles can enable the detection of cells with magnetic resonance imaging (MRI). We investigated the feasibility of monitoring transplanted liver cells by MRI in a preclinical swine model and used this approach to evaluate different routes for cell application. Liver cells were isolated from landrace piglets and labeled with micron-sized iron oxide particles (MPIO) in adhesion. Labeled cells (n = 10), native cells (n = 3) or pure particles (n = 4) were transplanted to minipigs via intraportal infusion into the liver, direct injection into the splenic parenchyma, or intra-arterial infusion to the spleen. Recipients were investigated by repeated 3.0 Tesla MRI and computed tomography angiography up to 8 weeks after transplantation. Labeling with MPIO, which are known to have a strong effect on the magnetic field, enabled non-invasive detection of cell aggregates by MRI. Following intraportal application, which is commonly applied for clinical LCT, MRI was able to visualize the microembolization of transplanted cells in the liver that were not detected by conventional imaging modalities. Cells directly injected into the spleen were retained, whereas cell infusions intraarterially into the spleen led to translocation and engraftment of transplanted cells in the liver, with significantly fewer microembolisms compared to intraportal application. These findings demonstrate that MRI can be a valuable tool for non-invasive elucidation of cellular processes of LCT and - if clinically applicable MPIO are available - for monitoring of LCT under clinical conditions. Moreover, the results clarify mechanisms relevant for clinical practice of LCT, suggesting that the intra-arterial route to the spleen deserves further evaluation.

The European Journal of Neuroscience published the paper "Mechanisms of hypothermia-induced cell protection mediated by microglial cells in vitro" First author is Antje Diestel from Katharina Schmitt's group - co-authors are Silke Troeller, Nils Billecke, Igor M. Sauer, Felix Berger and Katharina R. L. Schmitt.
Despite the widespread interest in the clinical applications of hypothermia, the cellular mechanisms of hypothermia-induced neuroprotection have not yet been clearly understood. Therefore, the aim of this study was to elucidate the cellular effects of clinically relevant hypothermia and rewarming on the morphological and functional characteristics of microglia. Microglial cells were exposed to a dynamic cooling and rewarming protocol. For stimulation, microglial cells were treated with 1 μg/mL lipopolysaccharide (LPS). We found that hypothermia led to morphological changes from ramified to ameboid cell shapes. At 2 h after hypothermia and rewarming, microglial cells were again ramified with extended branches. Moreover, we found enhanced cell activation after rewarming, accompanied by increased phagocytosis and adenosine triphosphate consumption. Interestingly, hypothermia and rewarming led to a time-dependent significant up-regulation of the anti-inflammatory cytokines interleukin-10 and interleukin-1 receptor antagonist in stimulated microglial cells. This is in line with the reduced proliferation and time-dependent down-regulation of the pro-inflammatory cytokines tumor necrosis factor-alpha and monocyte chemotactic protein-1 in comparison to normothermic control cells after LPS stimulation. Furthermore, degradation of the inhibitor of the nuclear transcription factor-kappaB (IkappaB-alpha) was diminished and delayed under conditions of cooling and rewarming in LPS-stimulated microglial cells. Thus, our results show that hypothermia and rewarming activate microglial cells, increase phagocytosis and shift the balance of cytokine release in stimulated microglial cells towards the anti-inflammatory cytokines. This could be a new cellular mechanism of hypothermia-induced neuroprotection mediated by activated microglial cells.
European Journal of Neuroscience, 2010; 31: 779-787

Lars Stelter's studies on In vitro and in vivo detectability of modified superparamagnetic nanoparticles for multimodal imaging using fluorescence microscopy, 3T MRI and animal PET are published in the latest issue of Molecular Imaging & Biology (Mol Imaging Biol. 2010 Jan-Feb;12(1):25-34). Co-authors are Jens Pinkernelle, Roger Michel, Ruth Schwartländer, Nathanael Raschzok, Mehmet H. Morgul, Martin Koch, Timm Denecke, Holger Amthauer, Juri Ruf, Andreas Jordan, Bernd Hamm, Igor M. Sauer, Ulf Teichgräber.
Cell transplantation is a major field in regenerative medicine and a promising alternative to whole organ transplantation. However, the process of cell engraftment is not yet fully understood and the hitherto achieved clinical outcome is limited. The aim of our study was to modify an aminosilan-coated nanoparticle for cell labeling and make it applicable for multimodal imaging using MRI, PET and fluorescent imaging. HIV-1 tat, linked FITC, and Gallium-68 were covalently bound to the particle and injected into Wistar rats. Animal-PET imaging was performed followed by MRI at 3.0T. Hepatic accumulation of the particles was proven by radionuclide distribution after 10 minutes in PET as well as in MRI over a 24 hour-period. Histological workup of the liver also revealed content of iron oxide particles in the reticuloendothelial system. Adjacent in vitro studies incubating hepatogenic HuH7 cells with the particles showed a rapid intracellular accumulation, clearly detectable by fluorescence microscopy and MRI. In conclusion our modified nanoparticle is stable under in vitro and in vivo conditions and is applicable for multimodal molecular imaging. Cellular labeling with this particle is possible and might help to get new insights into understanding the process of cell transplantation.

Tanja Deurholt's paper on "Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes" is published in BMC Biotechnology. Co-authors are Niek P. van Til, Aniska A. Chhatta, Lysbeth ten Bloemendaal, Ruth Schwartlander, Catherine Payne, John N. Plevris, Igor M. Sauer, Robert A.F.M. Chamuleau, Ronald P.J. Oude Elferink, Jurgen Seppen, and Ruurdtje Hoekstra.
A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. The article describes the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.

Antje Diestel's manuscript entitled "Tacrolimus and methylprednisolone prevent hypothermia induced endothelial dysfunction" has been accepted for publication in the Journal of Heart and Lung Transplantation. Co-authors are Nils Billecke, Joerg Roessler, Boris Schmitt, Silke Troeller, Ruth Schwartlander, Felix Berger, Igor Maximilian Sauer and Katharina Rose Luise Schmitt.
Hypothermia is used to preserve organs for transplant and it is the oldest method to protect organs during complex pediatric cardiac surgery. Loss of tissue function and tissue edema are common complications in children undergoing cardiac surgery and heart transplantation. The present study was designed to examine the effects of methylprednisolone (MP) and Tacrolimus (TAC) on endothelial cell function and morphology after deep hypothermia and rewarming. Human umbilical vein endothelial cells (HUVECs) were pretreated with MP and/orTAC and incubated either within a specially designed bioreactor or in monolayers. They were then exposed to a dynamic cooling and rewarming protocol. Immunocytochemistry, time lapse video microscopy within the SlideReactor bioreactor system, cell permeability and adherence assays and western blot analysis were performed. Confluent endothelial cells exposed to hypothermia displayed elongated cell shapes with intercellular gap formation, increased endothelial cell-layer permeability and loss in adherence. Upon rewarming, however, endothelial cell integrity was restored. Opening and closing of intercellular gaps was dependent on ERK 1/2 activation and connexin 43 (Cx43) expression. The combined treatment with MP and TAC inhibited these hypothermia-induced changes. These results suggest that MP and TAC inhibit hypothermia induced endothelial gap formation via pERK 1/2 inhibition and connexin 43 stabilization. Application of combined drugs that affect multiple targets may therefore be considered as a possible new therapeutic strategy to prevent endothelial dysfunction after hypothermia and rewarming.

As a result of the fruitful collaboration with the Institute for Analytical Sciences Berlin Nathanael Raschzok's paper on "Quantification of Cell Labelling with Micron-Sized Iron Oxide Particles Using Continuum Source Atomic Absorption Spectrometry" has been accepted by Tissue Engineering for publication. Co-authors are Nils Billecke, Nora N. Kammer, Mehmet H. Morgul, Michaela K. Adonopoulou, Igor M. Sauer, Stefan Florek, Helmut Becker-Ross, and Mao-Dong Huang.

Detection of cells after transplantation is necessary for quality control in regenerative medicine. Labelling with micron-sized iron oxide particles (MPIOs) enables non-invasive detection of single cells by magnetic resonance imaging. However, techniques for evaluation of the particle uptake are challenging. The aim of this study was to investigate continuum source atomic absorption spectrometry (CSAAS) for this purpose. Porcine liver cells were labelled with MPIOs and the iron concentration of the cell samples was investigated by a CSAAS spectrometer equipped with a Perkin-Elmer THGA graphite furnace. The weak iron line at 305.754 nm provides only about 1/600 sensitivity of the iron resonance line at 248.327 nm and was used for CSAAS measurements. Iron concentrations measured from labelled cells ranged from (5.8 ± 0.3) to (25.8 ± 0.9) pg Fe/cell, correlating to an uptake of (8.2 ± 0.5) to (25.7 ± 0.8) particles/cell. The results were verified by standardised morphometric evaluation. CSAAS enabled rapid quantification of particle load from small quantities of cells without extensive preparation steps. Thereby, CSAAS could be used for quality control in a clinical setting of cell transplantation.

Nathanel Raschzok's and Haluk Morgül's manuscript entitled "Imaging of Primary Human Hepatocytes Using Micron-Sized Iron Oxide Particles and Clinical Magnetic Resonance Tomography" has been accepted for publication in the Journal of Cellular and Molecular Medicine (impact factor: 6,55). Authors are Nathanael Raschzok, Mehmet H. Morgul, Jens Pinkernelle, Florian W.R. Vondran, Nils Billecke, Nora N. Kammer, Gesine Pless, Michaela K. Adonopoulou, Christian Leist, Lars Stelter, Ulf Teichgraber, Ruth Schwartlander and Igor M. Sauer. Nathanael Raschzok and Mehmet Haluk Morgul contributed equally to this work. The contribution of Ruth Schwartländer has to be emphasised as well. Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For visualisation of hepatocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging of 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 labelling experiments. Following dose finding studies, hepatocytes were incubated with 30 particles/cell for 4 hours in 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. Hepatocytes were enzymatically resuspended and analysed during a five-day reculture period for viability, total protein, enzyme leakage (AST, LDH) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and 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 viability, enzyme leakage and metabolic activity of human hepatocytes. Conclusion: 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.