Cells isolated from diseased explanted livers

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The International Journal of Artificial Organs (official journal of the European Society for Artificial Organs [ESAO]) published our paper on Isolation, characterization and cold storage of cells isolated from diseased explanted livers. Authors are Belaschk E, Rohn S, Mukiibi R, Reutzel-Selke A, Tang P, Sawitzki B, Pratschke J, Sauer IM and Mogl MT.

Livers discarded after standard organ retrieval are commonly used as a cell source for hepatocyte transplantation. Due to the scarcity of organ donors, this leads to a shortage of suitable cells for transplantation. Here, the isolation of liver cells from diseased livers removed during liver transplantation is studied and compared to the isolation of cells from liver specimens obtained during partial liver resection.
Hepatocytes from 20 diseased explanted livers (Ex-group) were isolated, cultured and stored at 4°C for up to 48 hours, and compared to hepatocytes isolated from the normal liver tissue of 14 liver lobe resections (Rx-group). The nonparenchymal cell fraction (NPC) was analyzed by flow cytometry to identify potential liver progenitor cells, and OptiPrep™ (Sigma-Aldrich) density gradient centrifugation was used to enrich the progenitor cells for immediate transplantation.
There were no differences in viability, cell integrity and metabolic activity in cell culture and survival after cold storage when comparing the hepatocytes from the Rx-group and the Ex-group. In some cases, the latter group showed tendencies of increased resistance to isolation and storage procedures. The NPC of the Ex-group livers contained considerably more EpCAM+ and significantly more CD90+ cells than the Rx-group. Progenitor cell enrichment was not sufficient for clinical application.
Hepatocytes isolated from diseased explanted livers showed the essential characteristics of being adequate for cell transplantation. Increased numbers of liver progenitor cells can be isolated from diseased explanted livers. These results support the feasibility of using diseased explanted livers as a cell source for liver cell transplantation.

Int J Artif Organs. 2017 May 23:0. doi: 10.5301/ijao.5000594. [Epub ahead of print]

Magnetic field and cells labeled with IO particles

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Our paper entitled "The magnetic field of magnetic resonance imaging systems does not affect cells labeled with micrometer-sized iron oxide particles," has been accepted for publication in Tissue Engineering, Part C: Methods. Authors are Martin Kluge, Annekatrin Leder, Karl H. Hillebrandt, Benjamin Struecker, Dominik Geisel, Timm Denecke, Rebeka D. Major, Anja Reutzel-Selke, Peter Tang, Steffen Lippert, Christian Schmidt, Johann Pratschke, Igor M. Sauer, and Nathanael Raschzok.

Labeling using iron oxide particles enables cell tracking via magnetic resonance imaging (MRI). However, the magnetic field can affect the particle-labeled cells. Here, we investigated the effects of a clinical MRI system on primary human hepatocytes labeled using micrometer-sized iron oxide particles (MPIOs).
HuH7 tumor cells were incubated with increasing concentrations of biocompatible, silica-based, micron-sized iron oxide-containing particles (sMPIO; 40 – 160 particles/cell). Primary human hepatocytes were incubated with 100 sMPIOs/cell. The particle-labeled cells and the native cells were imaged using a clinical 3.0-T MRI system, whereas the control groups of the labeled and unlabeled cells were kept at room temperature without exposure to a magnetic field. Viability, formation of reactive oxygen species, aspartate aminotransferase leakage, and urea and albumin synthesis were assessed over a culture period of 5 days.
The dose finding study showed no adverse effects of the sMPIO labeling on HuH7 cells. MRI had no adverse effects on the morphology of the sMPIO-labeled primary human hepatocytes. Imaging using the T1- and T2-weighted sequences did not affect the viability, transaminase leakage, formation of reactive oxygen species, or metabolic activity of the sMPIO-labeled cells or the unlabeled, primary human hepatocytes.
sMPIOs did not induce adverse effects on the labeled cells under the conditions of the magnetic field of a clinical MRI system.

Dr. rer. medic. Dipl.-Ing. Annekatrin Leder

Dr. Leder
Today, Anne Leder successfully defended her thesis "summa cum laude"!

Her work entitled "Entwicklung und Evaluierung eines mikroskaligen, Oligonukleotid-gekoppelten Eisenoxidpartikels zur Stimulation kultivierter humaner Hepatozyten" deals with particle-based delivery systems for therapeutic manipulation and tracking of transplanted cells by magnetic resonance imaging (MRI) based on multifunctional, silica based micron-sized iron oxide-containing particles (sMPIO) that combine fluorescence imaging, MRI tracking, and on-the-spot targeting of specific microRNAs on a particle surface for therapeutic manipulation by RNA interference.

CONGRATULATIONS !

12th Congress of the Cell Transplant Society

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The 12th Congress of the Cell Transplant Society tookplace in Milan, Italy, from July 7 to 11, 2013.
Nathanael Raschzok gave a presentation on "Loco-regional detection and stimulation of transplanted liver cells by particle-based miRNA depletion" and Martina Mogl on "isolation of adult hepatocytes and progenitor cells from explanted diseased human livers“.

Dr. Nora Kammer & Dr. Kirsten Steinz

ESAO2012

On behalf of the
European Society for Artificial Organs (ESAO), Prof. Dr. med. Gustav Steinhoff invites you to the XXXIX. ESAO Congress to be held in Rostock (Germany), September 26th – 29th, 2012. Rostock is a prospering and easy-to-reach hanseatic city directly located at the Baltic Sea coast. The venue of the ESAO 2012 congress is the Academy of Music and Theatre, which resides in an old monastery in the city centre. Rostock is home to one of the oldest universities in the world: founded in 1419. The motto of the ESAO Congress 2012 will be “from replacement to regeneration – from science to clinic”. The meeting will provide a clear program structure by highlighting one special organ system each congress day: heart/cardiovascular, liver and kidney. Above all the congress program integrates aspects of both basic science and clinical development with a clear focus on translation and clinical practice. We intend to prepare an excellent and exciting congress by inviting outstanding experts and by giving young and promising clinicians and scientists the opportunity to present their work. Especially for young scientists there will be one day for yESAO activities. Industry symposia, a poster exhibition and an industrial exhibition will complete the congress program. More information via www.esao2012.org !

Fast dynamic MRI for during liver cell Tx

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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.

CARS microscopy of MPIO

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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.

Tat-peptide modified MPIO

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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.

Imaging of human hepatocytes via MPIO and MRI

Florian Vondran successfully defended his thesis "summa cum laude".
After years of extremely fruitful research in the field of liver support, hepatocyte isolation and cryopreservation he is (co-)author of 7 papers in peer reviewed journals.

CSAAS and MPIO-labelled cells

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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.

Nathanael Raschzok defended thesis "summa cum laude"

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Sehr geehrte Kolleginnen und Kollegen,

die Transplantationsmedizin in Deutschland ist in der Zukunft mit einer Vielzahl von Problemen konfrontiert. Basierend auf hervorragenden Ergebnissen wurden in den letzten Jahren die Indikation zur Transplantation zunehmend großzügiger gestellt. Gleichzeitig führt der andauernde Mangel an Organspendern zu einer steigenden Akzeptanz sogenannter marginaler Organe. Neben diesem zentralen Problem sind sicherlich die steigende Zahl von Patienten zur Retransplantation, Langzeitnebenwirkungen der Immunsuppression sowie die Inzidenz von Malignomen dringende Diskussionpunkte.
Daher lade ich Sie recht herzlich zur 18. Jahrestagung der Deutschen Transplantationsgesellschaft unter dem Motto:’Transplantationsmedizin – Neue Herausforderungen bei gesicherter Routine“ nach Berlin ein.
Neben dem wissenschaftlichen Diskurs möchten wir Sie zu einer Diskussion der aktuellen Probleme und Entwicklungen in der Transplantationsmedizin einladen.
Traditionellerweise wird im Rahmen der Tagung das 13. AKTX-Pflegesymposium am Donnerstag und Freitag stattfinden.
Ich freue mich Sie in Berlin begrüßen zu dürfen, einer Stadt die aufgrund ihrer kulturellen, geschichtlichen und internationalen Präsenz zu einem offenen und zukunftsweisenden Gedankenaustausch einlädt. Neben dem wissenschaftlichen Programm hoffen wir Ihnen auch im Rahmen der Abendveranstaltung etwas von der Attraktivität der Stadt vermitteln zu können.


Ihr Prof. Peter Neuhaus

Haluk Morgül defended thesis "magna cum laude"

N. Raschzok and D. Mücke gave presentations on "Monitoring of liver cell transplantation by MRI" (oral presentation) and " In vitro comparison of iron oxide contrast agents for labelling of human hepatocytes" (poster presentation) at this year's 26th Meeting of the German Association for the Study of the Liver (GASL) in Bonn.

Modified nanoparticles & multimodal imaging

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The XXXVII ESAO Congress will take place in Skopje at the Macedonian Academy of Sciences and Arts on September 8-11, 2010.
Skopje is the capital of Macedonia and the centre of the political, cultural and social life. It is a modern, urban city with a history dating more than 2000 years back. Skopje, once called Skupi, over the history was conquered and ruined several times, but always rebuilt again. It has suffered several natural catastrophes, the last being the earthquake in 1963. Some of the monuments from long ago have still remained: the remainings from the old city, the roman architecture, the fortress, the stone bridge, many churches and monasteries, frescoes and carvings from the middle ages, mosques and amams built by the ottomans in the old bazaar. Old Skopje with its very rich treasures is a very attractive tourist destination. It is very well connected by air and land with the rest of the world.
The Macedonian Academy of Sciences and Arts, as the venue of the ESAO Congress in 2010, will provide the atmosphere for a perfect scientific event. You will enjoy “state of the art” presentations and lectures in some of the most attractive fields of medicine. The ESAO Congress 2010 will bring together distinguished clinicians and scientists of biotechnologies and bioengineering from all over the world. They will discuss results from scientific research in all areas of artificial organs. Our aim is to bring together and to foster exchange and collaboration among scientists, clinicians and industrial partners.
Aleksandar Sikole, the Congress President 2010, cordially welcomes you to Skopje to the ESAO 2010 Annual Congress.
More information via
http://www.esao2010.org.mk/ .

Monitoring cell transplantation in swine model via MRI

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Nora Kammer's paper in Artificial Organs on "Labelling of primary human hepatocytes with micron-sized iron oxide particles in suspension culture suitable for large-scale preparation" is available pre-print. Co-authors are Nils Billecke, Mehmet H. Morgul, Michaela K. Adonopoulou, Martina Mogl, Mao D. Huang, Stefan Florek, Katharina R. L. Schmitt, Nathanael Raschzok and Igor M. Sauer.
Protocols for labelling of hepatocytes with micron-sized iron oxide particles (MPIO) in adhesion culture enable cell detection using clinical Magnetic Resonance equipment. For clinical applications, large numbers of cells must be labelled in a simple and rapid manner, which requires new labelling protocols. The aim of this study was to investigate the feasibility of preparing MPIO-labelled primary human hepatocytes in a temporary suspension culture. Human hepatocytes were isolated from 16 donors and labelled with MPIO in suspension, using the Rotary Cell Culture System. Particle incorporation was investigated by light and electron microscopy. Cells were compared to adhesion culture-labelled and subsequently enzymatically resuspended cells. During a five-day culture period, hepatocyte-specific parameters of cell damage (aspartate aminotransferase and alanine aminotransferase) and metabolic activity (urea and albumin) were analysed. Suspension cultures showed a higher outcome in cell recovery compared to the conventional labelling method. When incubated with 180 particles/cell for four hours, the mean particle uptake was 28.8 particles/cell at a labelling efficiency of 95.1%. Labelling in suspension had no adverse effects on cell integrity or metabolic activity. In conclusion, labelling in suspension is a practicable method for fast and efficient preparation of large numbers of labelled cells that are suitable for clinical applications.

Labelling of hepatocytes in suspension culture

A multicompartment SlideReactor is shown at the exhibition “WeltWissen – World Knowledge”.
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This year, Berlin celebrates 200 years of the Humboldt University, 300 years of the Charité, 300 years since the first statute and first publication by the Academy of the Sciences and, one year later, 100 years of the Max Planck and Kaiser Wilhelm Society and the 350th birthday of the Berlin State Library. The exhibition “WeltWissen – World Knowledge” is the high point of the Berlin Year of Science. The Humboldt University, the Charité, the Berlin-Brandenburg Academy of the Sciences and Humanities and the Max Planck Society have organised the exhibition as a unique joint project. The Technical University, the Berlin State Museums and the Deutsches Museum, Munich are involved as partners. From 24 September 2010 to 9 January 2011, Martin-Gropius-Bau will be host   ing its “WeltWissen“ (World Knowledge) exhibition which takes a look at 300 years of the science in Berlin from an all-embracing perspective that crosses institutions, disciplines and epochs. The exhibition is the high point of the Berlin Year of Science. On an exhibition space of more than 3,200 square metres, visitors are presented with over 1,500 original exhibits, installations and media stations. The Humboldt University, the Charité, the Berlin-Brandenburg Academy of the Sciences and Humanities and the Max Planck Society have organised the exhibition as a unique joint project.
The exhibition correlates sciences in Berlin to the world: only the dynamic interplay of local imprinting and worldwide networking has allowed Berlin since 300 years to generate knowledge and share it with the world. Concrete and highly vivid stories and biographies of objects, researchers and institutions offer exciting insights into the scientific environment. “WeltWissen – World Knowledge” shows how scientists in Berlin work, how they network internationally, how they break down the boundaries of their departments and how they transformed Berlin into a scientific metropolis. 

WeltWissen. 300 Years of Science in Berlin 24 September 2010 – 9 January 2011  Martin-Gropius-Bau, Niederkirchnerstrasse 7, 10963 Berlin
Opening times: Wed - Mo: 10.00 am – 8.00 pm, closed on Tuesdays
Admission: 6 €, reduced 4€ . Free admission for children and adolescents up to an including 16 years of age, two escorts each per kindergarten group or school class as well as recipients of unemployment benefit level II 
Public transport: Underground line 2 (Potsdamer Platz), city train lines 1, 2, 25 (Potsdamer Platz or Anhalter Bahnhof), Buses: M29 (Anhalter Bahnhof) / M41 (Abgeordnetenhaus)
Please find more information at: www.weltwissen-berlin.de, www.gropiusbau.de

Copyright of upper,  large picture:
Roman März