Dr. Mathilde Feist and Dr. Paul Ritschl successfully applied for the BIH Charité Clinician Scientist Program. Mathilde Feist will work on "Cytokine-armed oncolytic vaccinia virus for pancreatic cancer therapy". Mentors are Prof. Bahra, Prof. Sauer and Prof. Beling.
Paul Ritschl focusses on "The Impact of Donor Derived Microparticles Following Solid Organ Transplantation". Mentors are Priv.-Doz. Dr. Schmelzle and Priv.-Doz Dr. Öllinger.

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.

Karl Hillebrandt receives one of the 2017 Einstein Center for Regenerative Therapies (ECRT) Kickbox – Junior Scientist Grant. The project is entitled "Fighting liver cirrhosis? Establishment and analysis of decellularized human cirrhotic liver slices as a 3-dimensional model to study cell matrix interactions".

Liver cirrhosis is one of the main indications for liver transplantation. Due to the organ shortage, this therapy option is limited to the minority of patients suffering from cirrhosis. Therefore, there is a need of alternative treatment options.The aim of our project is to establish a decellularization protocol for human cirrhotic livers slices, which preserves the natural extracellular matrix (ECM) of cirrhotic livers. These decellularized liver slices will serve as a 3 dimensional model to study cell matrix interactions. If we are able to establish a protocol which will preserve the ECM, we will conduct in vitro recellularization experiments to study how the cirrhotic ECM will change the genotype and phenotype of different cell types. With this knowledge we aim to modify specific cell types in vivo or vitro for example prior to cell transplantation. Our ambition is to steer the cell matrix interaction via these modified cells after their transplantation and thereby halt or even reverse the progress of liver cirrhosis. This approach may offer an alternative treatment option in the future.

Team : Karl Hillebrandt, Oliver Klein, Ben Strücker, Igor Sauer  

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.

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.

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.