News

Isolation of primary human hepatocytes & LiMax-test
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Tissue Engineering (Part C: Methods) accepted our paper entitled "The predictive value of the LiMAx-test for the isolation of primary human hepatocytes".
Authors are R.D. Major, M. Kluge, M. Jara, M. Nösser, R. Horner, J. Gassner, B. Struecker, P. Tang, S. Lippert, A. Reutzel-Selke, D. Geisel, T. Denecke, M. Stockmann, J. Pratschke, I.M. Sauer, and N. Raschzok.

The need for primary human hepatocytes is constantly growing, for basic research as well as for therapeutic applications. However, the isolation outcome strongly depends on the quality of liver tissue, and we are still lacking a preoperative test that allows the prediction of the hepatocyte isolation outcome. Here we evaluated the “maximal liver function capacity test” (LiMAx) as predictive test for the quantitative and qualitative outcome of hepatocyte isolation. This test is already used in clinical routine to measure preoperative and to predict postoperative liver function.
The patient’s preoperative mean LiMAx was obtained from the patient records and preoperative CT and MRI images were used to calculate the whole liver volume in order to adjust the mean LiMAx. The outcome parameters of the hepatocyte isolation procedures were analyzed in correlation with the adjusted mean LiMAx.
Primary human hepatocytes were isolated from partial hepatectomies (n=64).
From these 64 hepatectomies we included 48 to our study and correlated their isolation outcome parameters with volume corrected LiMAx values. From a total of 11 hepatocyte isolation procedures, metabolic parameters (albumin, urea and aspartate aminotransferase) were assessed during the hepatocyte cultivation period of 5 days. The volume adjusted mean LiMAx showed a significant positive correlation with the total cell yield (p= 0.049;r= 0.242;n= 48). The correlations of volume adjusted LiMAx values with viable cell yield and cell viability did not reach statistical significance. A sub-group analysis of isolations from patients with colorectal metastasis revealed a significant correlation between volume adjusted mean LiMAx and total cell yield (p= 0.012;r= 0.488;n= 21) and viable cell yield (p=0.034;r=0.405;n=21). Whereas a sub-group analysis of isolations of patients with carcinoma of the biliary tree showed significant correlations of volume adjusted mean LiMAx with cell viability (r= 0.387;p= 0.046;n=20) and lacked significant correlations with total cell yield (r= - 0.060;p= 0.401;n=20) and viable cell yield (r= 0.012;p= 0.480;n=20). The volume-adjusted mean LiMAx did not show a significant correlation with any of the metabolic parameters. In conclusion, the LiMAx-test might be a useful tool to predict the quantitative outcome of hepatocyte isolation, as long as underlying liver disease is taken into consideration.
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Mixed Reality in Visceral Surgery
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Annals of Surgery accepted our manuscript "Mixed Reality in visceral surgery - Development of a suitable workflow and evaluation of intraoperative use-cases" for publication. The paper evaluates the application of a mixed reality (MR) head-mounted display (HMD) for the visualization of anatomical structures in complex visceral-surgical interventions. A workflow was developed and technical feasibility was evaluated. 
Medical images are still not seamlessly integrated into surgical interventions and thus, remain separated from the surgical procedure. Surgeons need to cognitively relate two-dimensional sectional images to the three-dimensional (3D) during the actual intervention. MR applications simulate 3D images and reduce the offset between working space and visualization allowing for improved spatial-visual approximation of patient and image. The surgeon’s field of vision was superimposed with a 3D-model of the patient’s relevant liver structures displayed on a MR-HMD. This set-up was evaluated during open hepatic surgery. A suitable workflow for segmenting image masks and texture mapping of tumors, hepatic artery, portal vein and the hepatic veins was developed. The 3D model was positioned above the surgical site. Anatomical reassurance was possible simply by looking up. Positioning in the room was stable without drift and minimal jittering. Users reported satisfactory comfort wearing the device without significant impairment of movement. MR technology has high potential to improve the surgeon’s action and perception in open visceral surgery by displaying 3D anatomical models close to the surgical site. Superimposing anatomical structures directly onto the organs within the surgical site remains challenging since the abdominal organs undergo major deformations due to manipulation, respiratory motion and the interaction with the surgical instruments during the intervention. A further application scenario would be intraoperative ultrasound examination displaying the image directly next to the transducer. Displays and sensor-technologies as well as biomechanical modeling and object-recognition algorithms will facilitate the application of MR-HMD in surgery in the near future. Authors are I.M. Sauer, M. Queisner, P. Tang, S. Moosburner, O. Hoepfner, R. Horner, R. Lohmann and J. Pratschke.
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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.
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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.
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BIH Paper of the Month
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Benjamin Strücker, Hendrik Napierala and the rest of the team were awarded with the BIH Paper of the Month for their publication on a new method for developing a transplantable endocrine Neo-Pancreas.

The BIH Paper of the Month is awarded by the BIH Board of Directors to honor a special publication achievement from the joint research space of Charité and MDC. The Paper of the Month is sponsored by the Stiftung Charité as part of its Johanna Quandt Private Excellence Initiative. 

H. Napierala, K.-H. Hillebrandt, N. Haep, P. Tang, M. Tintemann, J. Gassner, M. Noesser, H. Everwien, N. Seiffert, M. Kluge, E. Teegen, D. Polenz, S. Lippert, D. Geisel, A. Reutzel Selke, N. Raschzok, A. Andreou, J. Pratschke, I. M. Sauer & B. Struecker. Engineering an endocrine Neo-Pancreas by repopulation of a decellularized rat pancreas with islets of Langerhans. Scientific Reports 7. Article number: 41777 (2017) doi:10.1038/srep41777
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Engineering an endocrine Neo-Pancreas
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Scientific Reports accepted our latest paper on „Engineering an endocrine Neo-Pancreas by repopulation of a decellularized rat pancreas with islets of Langerhans“. Authors are H. Napierala, K. Hillebrandt, N. Haep, P. Tang, M. Tintemann, J. Gassner, M. Noesser, H. Everwien, N. Seiffert, M. Kluge, E. Teegen, D. Polenz, S. Lippert, D. Geisel, A. Reutzel-Selke, N. Raschzok, A. Andreou, J. Pratschke, I.M. Sauer, and B. Struecker.
Decellularization of pancreata and repopulation of these non-immunogenic matrices with islets and endothelial cells could provide transplantable, endocrine Neo- Pancreata. In this study, rat pancreata were perfusion decellularized and repopulated with intact islets, comparing three perfusion routes (Artery, Portal Vein, Pancreatic Duct). Decellularization effectively removed all cellular components but conserved the pancreas specific extracellular matrix. Digital subtraction angiography of the matrices showed a conserved integrity of the decellularized vascular system but a contrast emersion into the parenchyma via the decellularized pancreatic duct. Islets infused via the pancreatic duct leaked from the ductular system into the peri-ductular decellularized space despite their magnitude. TUNEL staining and Glucose stimulated insulin secretion revealed that islets were viable and functional after the process.
We present the first available protocol for perfusion decellularization of rat pancreata via three different perfusion routes. Furthermore, we provide first proof-of-concept for the repopulation of the decellularized rat pancreata with functional islets of Langerhans. The presented technique can serve as a bioengineering platform to generate implantable and functional endocrine Neo-Pancreata.
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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 livers“ for 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.
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Vascular anatomy of the juvenile Göttingen minipig
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Lab Animal accepted our „Computed tomography-based survey of the vascular anatomy of the juvenile Göttingen minipig“ for publication.

Over the past 50 years, image-guided procedures have been established for a wide range of applications. The development and clinical translation of new treatment regimens necessitate the availability of suitable animal models. The juvenile Göttingen minipig presents a favourable profile as a model for human infants. However, no information can be found regarding the vascular system of juvenile minipigs in the literature. Such information is imperative for planning the accessibility of target structures by catheterization.

We present here a complete mapping of the arterial system of the juvenile minipig based on contrast-enhanced computed tomography. Four female animals weighing 6.13 ± 0.72 kg were used for the analyses. Imaging was performed under anaesthesia, and the measurement of the vascular structures was performed independently by four investigators. Our dataset forms a basis for future interventional studies in juvenile minipigs, and enables planning and refinement of future experiments according to the 3R (replacement, reduction and refinement) principles of animal research.


Authors are J. Siefert, K.H. Hillebrandt, M. Kluge, D. Geisel, P. Podrabsky, T. Denecke, M. Nösser, J. Gassner, A. Reutzel-Selke, B. Strücker, M.H. Morgul, S. Guel-Klein, J.K. Unger, A. Reske, J. Pratschke, I.M. Sauer, and N. Raschzok.
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Monitoring of hepatocyte transplantation by MRI
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A new book on Hepatocyte Transplantation Methods and Protocols, part of the series: Methods in Molecular Biology, Vol. 1506 P. Stock, B. Christ (Eds.), Springer, will be available end of November, 2016. We contributed a chapter on Preclinical swine models for monitoring of hepatocyte transplantation by MRI (authors: Nathanael Raschzok, Ulf Teichgräber, Johann Pratschke, and Igor M. Sauer) and are proud to provide the cover image.

This volume features up-to-date protocols for the isolation, preservation, and validation of various cell sources comprising large and small animal models, examining the impact of cell transplantation on acute and chronic liver diseases. Hepatocyte Transplantation: Methods and Protocols guides readers through laboratory protocols for the generation of humanized livers for the assessment of biological actions in vivo and techniques to monitor cell engraftment after cell transplantation in vivo are described and procedures for computational analyses of hepatocyte transplantation.

Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, Hepatocyte Transplantation: Methods and Protocols is an essential resource for researchers and clinicians to assess the biological as well as the therapeutic potential of hepatocyte transplantation.
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HCC: miRNA profiles of the tumor-surrounding tissue
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The presence of hepatocellular carcinoma (HCC) is a significant complication of cirrhosis because it changes the prognosis and the treatment of the patients. By now, contrast-enhanced CT and MR scans are the most reliable tools for the diagnosis of HCC; however, in some cases, a biopsy of the tumor is necessary for the final diagnosis. The aim of the study was to develop a diagnostic tool using the microRNA (miRNA) profiles of the tissue surrounding the HCC tumor combined with clinical parameters in statistical models. At a transplantation setting, 32 patients with HCC and cirrhosis (B) were compared to 22 patients suffering from cirrhosis only (A). The diagnosis and exclusion of HCC was confirmed following the histopathological examination of the explanted liver.

The HCC patients were significantly older than the patients with cirrhosis only (B: 60.6 and A: 49.9, p<0.001) and showed higher levels of ALT (A: 0.76μkat/l, B: 1.02μkat/, p=0.006) and AFP (A: 5.8ng/ml, B: 70.3ng/ml, p<0.001), whereas the bilirubin levels were higher in the cirrhosis only group (p=0.002). Using age (cut-off 50.23years) and AFP (cut-off 4.2ng/ml) thresholds, the levels of expression of miR-1285-3p and miR-943 differentiated between the patients with HCC and cirrhosis from those with cirrhosis only with an accuracy of 96.3%. This is the first report about the use of stepwise penalized logistic regression and decision tree analyses of miRNA expressions in the tumor-surrounding tissue combined with clinical parameters for the diagnosis of HCC.

Authors are Mehmet Haluk Morgul, Sergej Klunk, Zografia Anastasiadou, Ulrich Gauger, Corinna Dietel, Anja Reutzel-Selke, Philipp Felgendref, Hans-Michael Hau, Hans-Michael Tautenhahn, Rosa Bianca Schmuck, Nathanael Raschzok, Igor Maximillian Sauer, and Michael Bartels. Exp Mol Pathol. 2016 Aug 20;101(2):165-171. doi: 10.1016/j.yexmp.2016.07.014. [Epub ahead of print]
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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.
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Hepatocyte isolation after laparoscopic liver resection
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Tissue Engineering, Part C: Methods accepted our paper entitled "Hepatocyte isolation after laparoscopic liver resection" for publication. Authors are Horner R*, Kluge M*, Gassner J, Nösser M, Major RD, Reutzel-Selke A, Leder AK, Struecker B, Morgul MH, Pratschke J, Sauer IM, Raschzok N (*contributed equally).

Liver tissue obtained from partial hepatectomy is a common source for isolation of primary human hepatocytes. Until now, liver resections were most commonly performed by conventional open surgery. Although the laparoscopic approach is currently emerging in liver surgery, data on the outcome of hepatocyte isolation from laparoscopically resected liver tissue is not available. A total of 22 hepatocyte isolations were performed using the two-step collagenase perfusion technique from October 2015 until March 2016. Liver tissue was obtained from n=15 open liver resections (OLR) and n=7 laparoscopic liver resections (LLR). Isolation parameters (cell yield, viability, percoll survival) were assessed and hepatocyte function (plating efficiency, urea, albumin, and aspartate aminotransferase) was measured over a culture period of 6 days (OLR: n=13; LLR: n=3). Total cell yield (OLR: 36.81 ± 6.77 x106 cells/g vs. LLR 16.84 ± 10.66 x106 cells/g, p=0.0318) as well as viable yield (OLR 31.70 ± 6.05 x106 cells/g vs. LLL 14.70 ± 9.89 x106 cells/g, p=0.0260) were significantly higher in the OLR group. Subgroup analysis revealed that the worse outcome of isolation of laparoscopically resected liver tissue was associated with right-lateral laparoscopic liver resections, while hepatocyte isolation from left-lateral laparoscopic liver resections was as effective as from open surgery. Hepatocyte function did not differ between hepatocytes from openly resected versus left-lateral laparoscopically resected liver tissue. We here present the first data on hepatocyte isolation from laparoscopic liver surgery. While the overall outcome is worse compared to open surgery, our data suggest that liver tissue from laparoscopic resection of the left lobe is an excellent source for primary human hepatocytes.
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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.
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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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Single Pass Albumin Dialysis – Dose finding study
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Artificial Organs accepted our paper „Single Pass Albumin Dialysis (SPAD) – A dose finding study to define optimal albumin concentration and dialysate flow“ for publication.  Authors are R.B. Schmuck, G.-H. Nawrot, P. Fikatas, A. Reutzel-Selke, J. Pratschke, and I.M. Sauer.

Aim of these studies was to define the optimal conditions for SPAD in a standardized experimental set-up. Albumin concentration was adjusted to either 1%, 2%, 3%, or 4%, while the flow rate of the dialysate was kept constant at a speed of 700 ml/h. The flow rate of the dialysate was altered between 350, 500, 700, and 1000 ml/h, whereas the albumin concentration was continuously kept at 3%. 

This study revealed that the detoxification of albumin bound substances could be improved by increasing the concentration of albumin in the dialysate with an optimum at 3%. A further increase of the albumin concentration to 4% did not lead to a significant increase in detoxification. Furthermore, we observed a gradual increase of the detoxification efficiency for albumin bound substances, from 350 ml/h to 700 ml/h (for bilirubin) or 1000 ml/h (for bile acids) of dialysate flow. Water-soluble toxins (ammonia, creatinine, urea, uric acid) were removed almost completely, regardless of albumin concentration or flow rate. 
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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.
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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.
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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.“
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Publication in Jove
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.
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microRNAs in liver tissue engineering
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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.
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