ECRT Kickbox – Advanced Scientist Grant

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PD Dr. Nathanael Raschzok receives one of the 2017 Einstein Center for Regenerative Therapies (ECRT) Kickbox – Advanced Scientist Grant.
Einstein Center for Regenerative Therapies Kickbox – advanced scientist grant. The project is entitled „Overcoming steatotic compromise – Reconstitution of endogenous repair in severely steatotic liver grafts by metabolic reconditioning“. The project will be conducted by Nathanael Raschzok, Angelika Kusch, Duska Dragun, and Igor M. Sauer.

In order to stimulate excellent and creative research ideas that might take regenerative therapies a vital step forward, the Einstein Center offers a special two-stage funding scheme.
At first, the Kickbox seed grant provides a great framework to investigate initial ideas and to develop sound research concepts. Subsequently, the flexible funds enable the realisation of projects that evolved from the Kickbox initiation phase in order to reach the scientific goals of the Einstein Center.

Congratulations!

Poster_Programm

Recellularization of rat livers: morphology and function

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The Journal of Tissue Engineering and Regenerative Medicine accepted our paper Evolution of graft morphology and function after recellularization of decellularized rat liversfor publication.

Decellularization of livers is a well-established procedure. Data on different reseeding techniques or the functional evolution and re-organization processes of repopulated grafts remains limited.
We established a proprietary, customized bioreactor to repopulate decellularized rat livers (n=21) with primary rat hepatocytes (150 x 106 cells) via the hepatic artery and to subsequently evaluate graft morphology and function during seven days of ex vivo perfusion. Grafts were analyzed at 1h, 6h, 12h, 24h, 3d, 5d and 7d after recellularization (all n=3) by immunohistologic evaluation, hepatocyte-related enzyme (AST, ALT, LDH) and albumin measurement in the perfusate.
To the best of our knowledge, this is the first available protocol for repopulation of rat livers via the hepatic artery. Within the first 24 hours after repopulation, the hepatocytes seemed to migrate out of the vascular network and form clusters in the parenchymal space around the vessels. Graft function increased for the first 24 hours after repopulation with a significantly higher function compared to standard 2D culture after 24 hours. Thereafter, graft function constantly decreased with significantly lower values after six and seven days of perfusion, although histologically viable hepatocytes were found even after this period. Our data suggests that due to a constant loss of function, repopulated grafts should potentially be implanted as soon as cell engraftment and graft re-organization are completed.

Authors are Antje Butter, Khalid Aliyev, Karl-Herbert Hillebrandt, Nathanael Raschzok, Martin Kluge, Nicolai Seiffert, Peter Tang, Hendrik Napierala, Muhammad Imtiaz Ashraf, Anja Reutzel-Selke, Andreas Andreou, Johann Pratschke, Igor Maximilian Sauer, and Benjamin Struecker.

Bile: miRNA Pattern post OLT

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BIOMARKERS accepted our latest paper on "Bile: miRNA Pattern and Protein Based Biomarkers May Predict Acute Cellular Rejection after Liver Transplantation" for publication. Authors are Rosa Bianca Schmuck, Anja Reutzel-Selke, Nathanael Raschzok, Mehmet Haluk Morgul, Benjamin Struecker, Steffen Lippert, Cynthia de Carvalho Fischer, Moritz Schmelzle, Sabine Boas-Knoop, Marcus Bahra, Andreas Pascher, Johann Pratschke, and Igor M. Sauer.

Bile rather than blood depicts the local inflammation in the liver and may improve prediction and diagnosis of acute cellular rejection (ACR) after liver transplantation (OLT). Secretome and miRNAs were analyzed during the first two weeks and on clinical suspicion of ACR in the bile of 45 OLT recipients. Levels of CD44, CXCL9, miR-122, miR-133a, miR-148a and miR-194 were significantly higher in bile of patients who developed ACR within the first 6 months after OLT and during ACR. Analysis of secretome and miRNA in bile could further our understanding of the local inflammatory process during rejection.

Biomarkers. 2016 Aug 5:1-9. [Epub ahead of print]

NeoHybrid liver graft – proof of concept

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Cells Tissues Organs accepted our latest paper on "Allogeneic liver transplantation and subsequent syngeneic hepatocyte transplantation in a rat model – proof of concept for in vivo tissue engineering" for publication.
Authors are Susanne Rohn, Jan Schroeder, Henriette Riedel, Dietrich Polenz, Katarina Stanko, Anja Reutzel-Selke, Peter Tang, Lydia Brusendorf, Nathanael Raschzok, Peter Neuhaus, Johann Pratschke, Birgit Sawitzki, Igor M. Sauer, and Martina T. Mogl.

Aim of the project was the evaluation of a new concept for in vivo tissue engineering using autologous primary human hepatocytes and hepatic progenitor cells isolated from diseased livers explanted during orthotopic liver transplantation (LTx). Cells will be isolated and infused into the spleen for repopulation of the allogeneic liver graft. The latter is serving as biological matrix for the engraftment of autologous cells. Once these cells have engrafted, it is assumed that autologous cells will repopulate the allogeneic liver, since they should have a selective advantage due to their autologous origin. It is postulated that this will lead to a neo-hybrid liver graft, reducing immunogenicity and inducing immunoregulation thus minimizing the need for extensive immunosuppression and eventually inducing operational tolerance.
We therefore developed a new rat model for combined liver and liver cell transplantation under stable immunosuppression. Immunohistochemistry demonstrated the engraftment of transplanted cells, as confirmed by fluorescence in-situ hybridization, showing repopulation of the liver graft with 15.6 % male cells (± 1.8 SEM) at day 90. The quantitative PCR revealed 14.15 % (mean ± 5.09 SEM) male DNA at day 90. Engraftment of transplanted autologous cells after combined liver and cell transplantation was achieved for up to 90 days under immunosuppression. Immunohistochemistry indicated cell proliferation, and the fluorescence in-situ hybridization results were partly confirmed by quantitative RT-PCR. This new protocol in rats appears feasible to address long-term function and eventually induction of operational tolerance in the future.

LTx – microRNA signatures in peripheral blood ?

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BIOMARKERS accepted our latest paper on „microRNA signatures in peripheral blood fail to detect acute cellular rejection after liver transplantation“ for publication. Authors are N. Raschzok, A. Reutzel-Selke, R. Schmuck, L. Tannus, M. Morgul, C. Dietel, A. Leder, B. Struecker, S. Lippert, H. Sallmon, M. Schmelzle, M. Bartels, S. Jonas, J. Pratschke, and I.M. Sauer.

We investigated whether microRNA signatures in whole blood samples are associated with acute cellular rejection (ACR) after liver transplantation. Blood samples were collected using Paxgene technology and analyzed by microarrays and qRT-PCR.
microRNA signatures failed to distinguish between 19 patients with ACR and 16 controls. Let-7b-5p and let-7c were up-regulated in a subgroup of patients with ACR during the 6th and 7th postoperative day but failed in an independent validation of 20 patients.
microRNA signatures in whole blood processed by Paxgene technology are not suited for detection of ACR after liver transplantation.

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Although we do hate Facebook, feel kind of robbed and dislike the music –
We have to admit: 7,201,027 clicks (as of January 25th, 2016) are impressive…

Feature in Berliner Zeitung on Tissue Engineering

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Recellularized liver created in our lab featured in Berliner Zeitung.

ESOT | YPT – Interview with Karl Hillebrandt

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Being the winner of the Rising Stars Video Session organised by the YPT Committee at the ESOT2015 Brussels Congress Karl Hillebrandt gave an interview for the ESOT | YPT webpage.
His abstract "Optimized decellularization of rat livers byarterial and portal venous perfusion underoscillating pressure conditions" and the accompanying video were the most voted at the Rising Stars Session, where the audience voted live for the best video abstract. You can read Karl's abstract in the special ESOT2015 issue of Transplant International.

Implantation of a Neo Bile Duct in domestic pigs

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European Surgical Research accepted our latest paper entitled "Implantation of a tissue engineered Neo Bile Duct in domestic pigs" for publication. Authors are B. Struecker, K. Hillebrandt, N. Raschzok, K. Jöhrens, A. Butter, P. Tang, A. Andreou, H. Napierala, D. Polenz, A. Reutzel-Selke, T. Denecke, J. Pratschke, and I.M Sauer.

Extrahepatic bile duct injuries remain severe complications during cholecystectomy and often require reconstruction by bilioenteric anastomosis (i.e. hepatico-jejunostomy), which comes along with further long-term complications (e.g. recurring ascending cholangitis, secondary biliary cirrhosis). Furthermore, in case of inherent extrahepatic biliary atresia or during liver transplant artificial or engineered bile ducts could enable novel surgical strategies without the need for hepatico-jejunostomy. We present data on the implantation of in vitro generated Neo Bile Ducts in five domestic pigs. Neo Bile Ducts were engineered through decellularization of allogeneic blood vessels and recellularization with autologous cholangiocytes.On postoperative days 0, 1, 7 and 14 blood samples were taken and analyzed (AST, ALT, Bilirubin, Alkaline Phosphatase, Creatinine and Leukocytes). An magnetic resonance cholangiography was performed on postoperative day 14 with one pig. 14 days after implantation pigs were sacrificed and bile ducts were explanted. All pigs survived the complete study period without severe complications. None of the pigs showed signs of biliary leakage or peritonitis. Neo Bile Ducts were infiltrated by neutrophils and neo-angiogenesis was observed around and into the implanted tissue. Whether the presented technique enables the long-term replacement of native bile ducts has to be further evaluated.

Human hepatocyte isolation – new paper

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Tissue Engineering, Part C: Methods accepted our paper „Human hepatocyte isolation: Does portal vein embolization affect the outcome?“ Authors are Martin Kluge, Anja Reutzel-Selke, Hendrik Napierala, Karl H. Hillebrandt, Rebeka D. Major, Benjamin Struecker, Annekatrin Leder, Jeffrey Siefert, Peter Tang, Steffen Lippert, Daniel Seehofer, Johann Pratschke, Igor M. Sauer und Nathanael Raschzok.

Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n=190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE prior to surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, indication for surgery), isolation parameters (liver weight, cold ischemic time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16±2.03x106 cells/g vs. 9.70±0.73 x106 cells/g, p=0.499). The initial viability was slightly better in the PVE-group (77.8 ±2.03% vs. 74.4 ±1.06%). The mean viable cell yield (p=0.819) and the mean viability (p=0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic activity of cultured hepatocytes.
Although PVE leads to drastic metabolic alterations and changes in hepatic blood flow, embolized liver tissue is a suitable source for the isolation of primary human hepatocytes and is equivalent to untreated liver tissue in regard to cell yield and viability.

Publication in Jove

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„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.

Dr. Jan Schröder

Today, Jan Schröder successfully defended his thesis (summa cum laude) entitled "Vergleichende in vivo und in vitro Analysen im Rahmen der Etablierung der kombinierten Leber- und Leberzelltransplantation im Rattenmodell" !

Congratulations !

The Morning After

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.“ See Liver Transplantation, Volume 21, Issue 9, pages 1120–1122, September 2015

CD44 and CXCL9 predicting rejection after LTx

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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 transplantationhas 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.

Rebeka Major: BIH-Promotionsstipendium

Rebeka Major successfully applied for the BIH-Promotionsstipendium grant. Her work will focus in the role of the INDY („I’m Not Dead Yet“) gene in liver regeneration – a project in cooperation with Prof. A. Birkenfeld, Universitätsklinikum Carl Gustav Carus in Dresden.
In the Berlin Institute of Health (BIH), the Max Delbrück Center for Molecular Medicine (MDC) and the Charité - Universitätsmedizin Berlin have joined forces. The core idea is to combine translational research with an overarching systems medicine approach to bridge the gap between basic research and clinical application.

Ben Strücker: Charité Clinical Scientist

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Dr. med Benjamin Strücker successfully applied for the Charité Clinical Scientist 2015 program.

His project is entitled „Humanized Porcine Liver““. Clinical mentor is Prof. Dr. Johann Pratschke, scientific mentors is Priv.-Doz. Dr. med Igor M. Sauer.

The program is supported by Stiftung Charité which was endowed by the entrepreneur Johanna Quandt in order to promote biomedical "knowledge entrepreneurs" that is, change makers in biomedicine at the Charité. The goal of this program is to develop new career paths in clinical specialist medical training. The focus of the training program "Clinical Scientist" is translational research ("bench-to-bedside") which will be realized by a reduction in clinical routine and an improved curriculum with defined goals.

Sham Laparotomy and microRNA Expression in Rats

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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.

Decellularization of porcine livers

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Ben Strücker’s paper entitled „Porcine liver decellularization under oscillating pressure conditions – A technical refinement to improve the homogeneity of the decellularization process“ has been accepted for publication in Tissue Engineering, Part C: Methods.
Co-authors are K. Hillebrandt, R. Voitl, A. Butter, R.B. Schmuck, A. Reutzel-Selke, D. Geisel, K. Joehrens, P.A. Pickerodt, N. Raschzok, G. Puhl, P. Neuhaus, J. Pratschke, and I.M. Sauer.

Decellularization and recellularization of parenchymal organs may facilitate the generation of autologous functional liver organoids by repopulation of decellularized porcine liver matrices with induced liver cells. We present an accelerated (7 h overall perfusion time) and effective protocol for human scale liver decellularization by pressure-controlled perfusion with 1% Triton X-100 and 1% SDS via the hepatic artery (120 mmHg) and portal vein (60 mmHg). In addition, we analyzed the effect of oscillating pressure conditions on pig liver decellularization (n=12). The proprietary perfusion device used to generate these pressure conditions mimics intra-abdominal conditions during respiration to optimize microperfusion within livers and thus optimize the homogeneity of the decellularization process. The efficiency of perfusion decellularization was analyzed by macroscopic observation, histological staining (H&E, Sirius red, Alcian blue), immunohistochemical staining (collagen IV, laminin, fibronectin) and biochemical assessment (DNA, collagen, glycosaminoglycans) of decellularized liver matrices. The integrity of the extracellular matrix post-decellularization was visualized by corrosion casting and three-dimensional CT scanning. We found that livers perfused under oscillating pressure conditions (P+) showed a more homogenous course of decellularization and contained less DNA compared to livers perfused without oscillating pressure conditions (P-). Microscopically, livers from the (P-) group showed remnant cell clusters, while no cells were found in livers from the (P+) group. The grade of disruption of the ECM was higher in livers from the (P-) group, although the perfusion rates and pressure did not significantly differ. Immunohistochemical staining revealed that important matrix components were still present after decellularization. Corrosion casting showed an intact vascular (portal vein and hepatic artery) and biliary framework. In summary, the presented protocol for pig liver decellularization is quick (7 h) and effective. The application of oscillating pressure conditions improves the homogeneity of perfusion and thus the outcome of the decellularization process.

Decellularization and oscillating pressure conditions

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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. 

Decellularization of rat liver – New time lapse video