A new bicornuate model of rat uterus transplantation.

INTRODUCTION: Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. MATERIAL AND METHODS: We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. RESULTS: Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. CONCLUSIONS: Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Tissue-based miRNA mapping in alcoholic liver cirrhosis: different profiles in cirrhosis with or without hepatocellular carcinoma.

Context: Alcoholic liver cirrhosis is a significant risk factor for the development of hepatocellular carcinoma (HCC). The importance of tumour-associated cirrhosis in the development or progression of HCC is not understood. MiRNAs are important regulators for HCC development, but their role in HCC due to alcoholic liver cirrhosis is unclear.Objective: The aim of this study is the detection of miRNA expression in alcoholic liver cirrhosis, tumour-associated cirrhosis, and HCC.Materials and methods: We analysed the differences in the miRNA profiles of HCC, tumour-associated cirrhosis, and cirrhosis without HCC samples from 30 patients who underwent liver transplantation because of alcoholic liver disease.Results: Microarray analyses revealed 40 significantly differentially expressed miRNAs between HCC tissue and tumour-associated cirrhosis tissue. Furthermore, the microarray analysis discovered 56 differentially expressed miRNAs in tumour-associated cirrhosis and cirrhosis without HCC.Discussion: The differences of miRNA profile in alcoholic liver cirrhosis with and without HCC could improve understanding of HCC development, as well as lead to a new diagnostic tool in HCC screening.Conclusion: We were able to show for the first time, the differences of miRNA profile as promising biomarker in HCC, tumour-associated cirrhosis, and cirrhosis without HCC in context of alcoholic liver disease.

Evaluation of Anastomotic Leak after Esophagectomy for Esophageal Cancer: Typical Time Point of Occurrence, Mode of Diagnosis, Value of Routine Radiocontrast Agent Studies and Therapeutic Options.

BACKGROUND: Data on the typical time point of occurrence of anastomotic leak (AL) after esophagectomy for esophageal cancer are currently scarce. Therefore, the usefulness of routine radiocontrast agent studies (RRCS) for testing proper healing of the anastomosis after esophagectomy remains unclear. Furthermore, preferred available tools to diagnose postoperative AL and therapeutic options are still under debate. METHODS: We present a retrospective analysis of 328 consecutive patients who underwent esophagectomy for esophageal cancer between 2005 and 2015. A RRCS has been performed to date in our center on the fifth postoperative day (POD), before returning to normal oral intake. RESULTS: In total, 49 of 328 patients developed AL after esophagectomy (15%). A total of 11 patients (23%) developed AL before the RRCS and 34 patients (69%) after an unremarkable RRCS; and 4 patients (8%) with AL were diagnosed by RRCS, resulting in overall sensitivity of 16%. The median time point of occurrence of AL was POD 9, the majority of AL (84%) occurred between POD 1 and 19. Computed tomography led to the diagnosis of AL in 41% of patients. The most frequent therapy of AL was stenting in 47% of patients. Endoscopic vacuum therapy was used in 4 patients. CONCLUSIONS: The majority of AL occurred within the first 3 weeks after esophagectomy without a typical time point. In our series, RRCS on the fifth POD had a low sensitivity of 16%. Therefore, standardized RRCS and fasting till the examination cannot be generally recommended. In case of clinical suspicion of AL, computed tomography of the chest and abdomen with oral contrast agent should be performed, followed by endoscopy. Endoscopic stent placement remains the standard therapy of AL in our center. Endoscopic vacuum therapy evolves as it is an interesting alternative therapeutic option and can be combined with stenting in selected cases.

Tumor-stromal cross-talk modulating the therapeutic response in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant solid tumor with a dismal prognosis. The stroma component makes up to 90% of the tumor mass and is thought to be one of the main reasons for the tumor's high chemoresistance. Cancer associated fibroblasts (CAFs) have previously been identified to be the key stromal players. This is the first time we provide detailed in vitro experiments investigating tumor-stromal interactions when exposed to three well-known chemotherapeutic agents. METHODS: Monocultures, indirect and direct co-cultures of two PDAC cell lines (AsPC and Panc-1) and six primary patients derived CAFs were treated with gemcitabine, nab-paclitaxel and the γ-secretase-inhibitor (GSI) DAPT. The cell viability of each component was measured with XTT. Finally, IL-6 concentrations of the supernatants were analyzed. RESULTS: On the contrary to PDAC cell lines, CAF monocultures hardly responded to any treatment which suggested that stroma (CAFs) itself is more resistant to standard chemo-treatments than the epithelial cancer cells. Moreover, only a weak chemotherapeutic response was observed in direct co-cultures of cancer cells with CAFs. A change in the morphology of direct co-cultures was accompanied with the chemoresistance. CAFs were observed to build cage-like structures around agglomerates of tumor cells. High levels of IL-6 were also associated with a reduced response to therapy. Indirect co-cultures make the tumor-stromal interaction more complex. CONCLUSIONS: CAFs are highly chemoresistant. Direct cell-cell contact and high levels of IL-6 correlate with a high chemoresistance.

Single Pass Albumin Dialysis-A Dose-Finding Study to Define Optimal Albumin Concentration and Dialysate Flow.

Several artificial liver support concepts have been evaluated both in vitro and clinically. Single pass albumin dialysis (SPAD) has shown to be one of the most simple approaches for removing albumin-bound toxins and water-soluble substances. Being faced with acute liver failure (ALF) in everyday practice encouraged our attempt to define the optimal conditions for SPAD more precisely in a standardized experimental setup. 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. In conclusion, this study confirmed that SPAD is effective in eliminating albumin-bound as well as water-soluble toxins using a simulation of ALF. Furthermore, this project was successful in evaluating the most effective combination of albumin concentration (3%) and dialysate flow (700 mL/h-1000 mL/h) in SPAD for the first time.

The Impact of Obesity on Outcomes Following Resection for Gastric Cancer.

BACKGROUND: Obesity is generally considered to be associated with increased postoperative morbidity and mortality following intraabdominal cancer surgery. However, recent reports showed that overweight patients may have a lower risk for adverse postoperative outcomes and this observation has been described as the 'obesity paradox'. Therefore, we aimed to analyze the impact of obesity on outcomes after resection for gastric cancer. METHODS: Data of patients who underwent resection for gastric cancer between 2005 and 2012 were assessed. Patient characteristics, postoperative outcomes and long-term survivals were compared between patients with body mass index (BMI) ≥30 and <30. RESULTS: Resection for gastric cancer was performed in 249 patients. BMI ≥30 was identified in 49 patients. Obese patients with BMI ≥30 were more frequently diagnosed with diabetes (31 vs. 16%, p = 0.015). Resection for gastric cancer in obese patients was significantly associated with longer duration of surgery (278 vs. 243 min, p < 0.001), longer duration of hospital stay (18 vs. 16 days, p = 0.028), increased postoperative morbidity (49 vs. 33%, p = 0.037), and increased postoperative mortality (10 vs. 3%, p = 0.028). There was no significant difference in overall survival (OS) between patients with BMI ≥30 and patients with BMI <30 (5-year OS rate: 59 vs. 62%, p = 0.587). CONCLUSION: Obesity may complicate resection for gastric cancer increasing the duration of surgical procedure, hospital stay and postoperative morbidity and mortality. However, BMI did not predict OS in our patients. Consequently, BMI may be too simple as a parameter to evaluate sophisticated interactions between different body fat compartments and inflammatory and immune responses and thus to predict long-term oncologic outcomes.

The impact of directly acting antivirals on the enzymatic liver function of liver transplant recipients with recurrent hepatitis C.

BACKGROUND: The new directly acting antivirals (DAAs) enable all-oral interferon-free treatment of chronic hepatitis C virus (HCV) infection. We here investigated the effect of DAAs on the enzymatic liver function of liver transplant recipients with recurrent hepatitis C. METHODS: Twenty-one patients with elevated liver enzymes or advanced fibrosis/compensated cirrhosis caused by recurrent HCV were treated with sofosbuvir either in combination with simeprevir, or in combination with ribavirin or daclatasvir with or without ribavirin for 12 weeks. Biochemical parameters, tacrolimus trough levels, and the maximal liver function capacity (LiMAx) were measured monthly during the treatment and 12 weeks after the end of treatment. RESULTS: All patients achieved sustained virological response 12 weeks after the end of the treatment. The transaminases and cholestasis parameters normalized until week 8 of treatment. The mean LiMAx (normal ranges >315 µg/kg/h) increased from 344±142 µg/kg/h before treatment to 458±170 µg/kg/h (P<.0001) at the 12-week follow-up. In parallel, the tacrolimus trough level to dose ratio decreased from 4.68 down to 2.72 (P=.0004). CONCLUSION: Antiviral treatment with DAAs enabled sustained elimination of recurrent HCV in liver transplant recipients and was associated with a significant improvement of the enzymatic liver function.

Implantation of a Tissue-Engineered Neo-Bile Duct in Domestic Pigs.

BACKGROUND: Extrahepatic bile duct injuries remain severe complications during cholecystectomies and often require reconstruction by bilioenteric anastomosis (i.e., hepaticojejunostomy), which comes with further long-term complications (e.g., recurring ascending cholangitis, secondary biliary cirrhosis). In the case of inherent extrahepatic biliary atresia or during liver transplant, artificial or engineered bile ducts could allow novel surgical strategies without the need for hepaticojejunostomy. METHODS: We present data on the implantation of in vitro-generated neo-bile ducts in 5 domestic pigs. The 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 (aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, creatinine, and leukocytes). Magnetic resonance cholangiopancreatography was performed on postoperative day 14 on 1 pig. Fourteen days after implantation, the pigs were sacrificed and the bile ducts were explanted. RESULTS: All pigs survived the complete study period without severe complications. None of the pigs showed signs of biliary leakage or peritonitis. The neo-bile ducts were infiltrated by neutrophils, and neoangiogenesis was observed around and into the implanted tissue. CONCLUSION: We present a novel strategy for extrahepatic bile duct replacement by implantation of an autologous neo-bile duct generated ex vivo. Whether the presented technique allows the long-term replacement of native bile ducts must be further evaluated.

CD44 and CXCL9 serum protein levels predict the risk of clinically significant allograft rejection after liver transplantation.

The diagnosis of acute cellular rejection (ACR) after liver transplantation is based on histological analysis of biopsies because noninvasive biomarkers for allograft rejection are not yet established for clinical routines. CD31, CD44, and chemokine (C-X-C motif) ligand (CXCL) 9 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 6 months after liver transplantation in a prospective study. The protein levels were measured in 94 patients immediately before transplantation, at postoperative days (PODs) 1, 3, 7, and 14 and when biopsies were performed during episodes of biochemical graft dysfunction. The CD44 serum protein levels were significantly lower at POD 1 in patients who experienced histologically proven ACR in the follow-up compared with patients without ACR (P < 0.001). CXCL9 was significantly higher before transplantation (P = 0.049) and at POD 1 (P < 0.001) in these patients. Low CD44 values (cutoff, <200.5 ng/mL) or high CXCL9 values (cutoff, >2.7 ng/mL) at POD 1 differentiated between rejection and no rejection with a sensitivity of 88% or 60% and a specificity of 61% or 79%, respectively. The combination of both biomarker cutoffs at POD 1 had a positive predictive value of 91% and a negative predictive value of 67% for clinically significant ACR. Moreover, CD44 was significantly lower at the time of ACR (P < 0.001) and differentiated the rejection group from patients with graft dysfunction due to other reasons. Our results suggest that CD44 and CXCL9 may serve as predictive biomarkers to identify liver allograft recipients at risk for clinically significant ACR.

Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization.

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

Toward a 3D Printed Perfusable Islet Embedding Structure: Technical Notes and Preliminary Results.

To date, islet transplantation to treat type 1 diabetes mellitus remains unsuccessful in long-term follow-up, mainly due to failed engraftment and reconstruction of the islet niche. Alternative approaches, such as islet embedding structures (IESs) based on 3D printing have been developed. However, most of them have been implanted subcutaneously and only a few are intended for direct integration into the vascular system through anastomosis. In this study, we 3D printed a proof-of-concept IES using gelatin methacrylate biocompatible ink. This structure consisted of a branched vascular system surrounding both sides of a central cavity dedicated to islets of Langerhans. Furthermore, we designed a bioreactor optimized for these biological structures. This bioreactor allows seeding and perfusion experiments under sterile and physiological conditions. Preliminary experiments aimed to analyze if the vascular channel could successfully be seeded with mature endothelial cells and the central cavity with rat islets. Subsequently, the structures were used for a humanized model seeding human endothelial progenitor cells (huEPC) within the vascular architecture and human islets co-cultured with huEPC within the central cavity. The constructs were tested for hemocompatibility, suture strength, and anastomosability. The 3D printed IES appeared to be hemocompatible and anastomosable using an alternative cuff anastomosis in a simple ex vivo perfusion model. While rat islets alone could not successfully be embedded within the 3D printed structure for 3 days, human islets co-cultivated with huEPC successfully engrafted within the same time. This result emphasizes the importance of co-culture, nursing cells, and islet niche. In conclusion, we constructed a proof-of-concept 3D printed islet embedding device consisting of a vascular channel that is hemocompatible and perspectively anastomosable to clinical scale blood vessels. However, there are numerous limitations in this model that need to be overcome to transfer this technology to the bedside.

Body composition is associated with disease aetiology and prognosis in patients undergoing resection of intrahepatic cholangiocarcinoma.

BACKGROUND: Body composition alterations are frequent in patients with cancer or chronic liver disease, but their prognostic value remains unclear in many cancer entities. OBJECTIVE: We investigated the impact of disease aetiology and body composition after surgery for intrahepatic cholangiocarcinoma (iCCA), a rare and understudied cancer entity in European and North American cohorts. METHODS: Computer tomography-based assessment of body composition at the level of the third lumbar vertebra was performed in 173 patients undergoing curative-intent liver resection for iCCA at the Department of Surgery, Charité - Universitätsmedizin Berlin. Muscle mass and -composition as well as subcutaneous and visceral adipose tissue quantity were determined semi-automatically. (Secondary) sarcopenia, sarcopenic obesity, myosteatosis, visceral and subcutaneous obesity were correlated to clinicopathological data. RESULTS: Sarcopenia was associated with post-operative morbidity (intraoperative transfusions [p = 0.027], Clavien-Dindo ≥ IIIb complications [p = 0.030], post-operative comprehensive complication index, CCI [p < 0.001]). Inferior overall survival was noted in patients with myosteatosis (33 vs. 23 months, p = 0.020). Fifty-eight patients (34%) had metabolic (dysfunction)-associated fatty liver disease (MAFLD) and had a significantly higher incidence of sarcopenic (p = 0.006), visceral (p < 0.001) and subcutaneous obesity (p < 0.001). Patients with MAFLD had longer time-to-recurrence (median: 38 vs. 12 months, p = 0.025, log-rank test). Multivariable cox regression analysis confirmed only clinical, and not body, composition parameters (age > 65, fresh frozen plasma transfusions) as independently prognostic for overall survival. CONCLUSION: This study evidenced a high prevalence of MAFLD in iCCA, suggesting its potential contribution to disease aetiology. Alterations of muscle mass and adipose tissue were more frequent in patients with MAFLD.

Inhibition of Vascular Endothelial Growth Factor Protects against the Development of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome in Wild-Type but Not in CD39-Null Mice.

(1) Background: Sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy is associated with unfavorable outcomes after partial hepatectomy for colorectal liver metastases (CLM). Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), may prevent SOS development. We investigated the impact of VEGF-inhibition on the development of SOS in a murine model. (2) Methods: Male wild-type and CD39-null mice received oxaliplatin, additional anti-VEGF (OxAV), or controls, and were sacrificed or subjected to major partial hepatectomy (MH). Specimen were used for histological analysis of SOS. Liver damage was assessed by plasma transaminases. The VEGF pathway was elucidated by quantitative PCR of liver tissue and protein analysis of plasma. (3) Results: Mice treated with oxaliplatin developed SOS. Concomitant anti-VEGF facilitated a reduced incidence of SOS, but not in CD39-null mice. SOS was associated with increased plasma VEGF-A and decreased hepatocyte growth factor (HGF). After OxAV treatment, VEGF-R2 was upregulated in wild-type but downregulated in CD39-null mice. Oxaliplatin alone was associated with higher liver damage after MH than in mice with concomitant VEGF-inhibition. (4) Conclusions: We established a murine model of oxaliplatin-induced SOS and provided novel evidence on the protective effect of VEGF-inhibition against the development of SOS that may be associated with changes in the pathway of VEGF and its receptor VEGF-R2.

Donor-Specific Antibodies Against Donor Human Leukocyte Antigen are Associated with Graft Inflammation but Not with Fibrosis Long-Term After Liver Transplantation: An Analysis of Protocol Biopsies.

BACKGROUND: Donor-specific antibodies (DSA) against donor human leukocyte antigen after liver transplantation, which are associated with histological changes, have been widely studied with respect to their sustained impact on transplant function. However, their long-term impact after liver transplantation remains unclear. METHODS: We performed a cross-sectional analysis from June 2016 to July 2017 that included all patients who presented themselves for scheduled follow-up after receiving a liver transplantation between September 1989 and December 2016. In addition to a liver protocol biopsy, patients were screened for human leukocyte antigen antibodies (HLAab) and donor-specific antibodies. Subsequently, the association between human leukocyte antigen antibodies, donor-specific antibodies, histologic and clinical features, and immunosuppression was analyzed. RESULTS: Analysis for human leukocyte antigen antibodies and donor-specific antibodies against donor human leukocyte antigen was performed for 291 and 271 patients. A significant association between higher inflammation grades and the presence of human leukocyte antigen antibodies and donor-specific antibodies was detected, while fibrosis stages remained unaffected. These results were confirmed by multivariate logistic regression for inflammation showing a significant increase for presence of human leukocyte antigen antibodies and donor-specific antibodies (OR: 4.43; 95% CI: 1.67-12.6; p=0.0035). Furthermore, the use of everolimus in combination with tacrolimus was significantly associated with the status of negative human leukocyte antigen antibodies and donor-specific antibodies. Viral etiology for liver disease, hepatocellular carcinoma (HCC) and higher steatosis grades of the graft were significantly associated with a lower rate of human leukocyte antigen antibodies. The impact of human leukocyte antigen antibodies and donor-specific antibodies against donor human leukocyte antigen was associated with higher levels of laboratory parameters, such as transaminases and bilirubin. CONCLUSION: Donor-specific antibodies against donor human leukocyte antigen are associated with histological and biochemical graft inflammation after liver transplantation, while fibrosis seems to be unaffected. Future studies should validate these findings for longer observation periods and specific subgroups.

Development of GelMA/PCL and dECM/PCL resins for 3D printing of acellular in vitro tissue scaffolds by stereolithography.

Gelatin methacryloyl (GelMA) is a chemically modified extracellular matrix (ECM)-derived biopolymer that is widely used for 3D fabrication of tissue engineering scaffolds. However, its tendency for physical gelation limits its use in aqueous 3D printing resins to low concentrations, yielding a poor printing resolution in stereolithography (SLA). To obtain a GelMA-based resin that can be printed into high-resolution tissue scaffolds, we formulated resins of fish and porcine-derived GelMA in formamide using GelMA alone or mixed with star-shaped poly(ε-caprolactone) methacrylate (PCL-MA). We identified GelMA resins and GelMA/PCL-MA hybrid resins with a ratio of 70/30 wt-% to yield a suitable viscosity for SLA at 32 °C and demonstrated the resolution of the new resins in SLA by 3D printing acellular human small intestine-mimicking tissue scaffolds. The presence of PCL-MA in the hybrid resins improved the 3D printing fidelity compared to the neat GelMA resins, while GelMA provided the hybrid materials with enhanced swelling and proliferation of seeded cells. We further demonstrated the transferability of our resin formulation to native organ-derived materials by successfully replacing GelMA in the hybrid resin with solubilized, methacryloyl-functionalized decellularized liver ECM (dECM-MA) and by 3D printing multi-layer dECM/PCL-MA hydrogels.

Development of a Rat Liver Machine Perfusion System for Normothermic and Subnormothermic Conditions.

Ex vivo liver machine perfusion (MP) is a promising alternative for preservation of liver grafts from extended criteria donors. Small animal models can be used to evaluate different perfusion conditions. We here describe the development of a miniaturized ex vivo MP system for rat liver grafts, evaluating cell-free and erythrocyte-based perfusion solutions, subnormothermic and normothermic temperatures, and dialysis. A perfusion chamber was designed after a suitable liver position was identified. Normothermic ex vivo liver perfusion (NEVLP) required supplementation of erythrocytes to reduce cell damage. Perfusion with erythrocytes led to rising potassium levels after 12 h (NEVLP, 16.2 mM, interquartile range [IQR]: 5.7 and subnormothermic ex vivo liver perfusion [SNEVLP], 12.8 mM, IQR: 3.5), which were normalized by dialysis using a laboratory dialysis membrane (NEVLP, 6.2 mM, IQR: 0.5 and SNEVLP, 5.3 mM, IQR: 0.1; p = 0.004). Livers treated with NEVLP conditions showed higher bile production (18.52 mg/h/g, IQR: 8.2) compared to livers perfused under SNEVLP conditions (0.4 mg/h/g, IQR: 1.2, p = 0.01). Reducing the perfusion volume from 100 to 50 mL allowed for higher erythrocyte concentrations, leading to significantly lower transaminases (15.75 U/L/mL, IQR: 2.29 vs. 5.97 U/L/mL, IQR: 18.07, p = 0.002). In conclusion, a well-designed perfusion system, appropriate oxygen carriers, dialysis, and miniaturization of the perfusion volume are critical features for successful miniaturized ex vivo liver MP. Impact Statement Ex vivo liver machine perfusion (MP) is an emerging preservation alternative to static cold storage. Even though clinical studies have shown benefits for extended criteria donor grafts, standardized systems for perfusion of rat liver grafts are not available, which are inevitable for large-scaled studies on liver reconditioning by ex vivo MP. We here comprehensively describe the development of an ex vivo rat liver perfusion system that can be used as modular setting in various approaches of liver MP. We describe pitfalls and techniques that might be of interest when establishing such perfusion systems for basic and translational research.

Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen.

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patient's sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

Heterotopic ileal pancreas with lipoma and coexisting fibromatosis associated with a rare case of gastrointestinal bleeding. A case report and review of the literature.

CONTEXT: The development of pancreatic tissue outside the confines of the main gland represents a congenital abnormality referred to as heterotopic pancreas. This is a rare pathological and surgical entity which remains mostly asymptomatic. CASE REPORT: We present the case of a 28-year-old male, who was admitted to hospital because of a history of blood in bowel movements. After a normal gastroscopy and colonoscopy, Tc99m-tagged red blood cells scintigraphy showed enrichment in the right lower abdomen. At double-balloon endoscopy, a intraluminal polypoid mass 8 cm in diameter was revealed 120 cm from the ileocecal valve. The initial macroscopic diagnosis was a gastrointestinal stromal tumor. During surgery, the diagnosis of heterotopic pancreas with lipoma and fibromatosis was made. To our knowledge this is the first case of ileal heterotopic pancreatic tissue and lipoma described to date in the literature. CONCLUSION: Ileal heterotopic pancreas is a rare entity with potentially life-threatening complications, local excision being the appropriate indicated treatment.

Continuously microscopically observed and process-controlled cell culture within the SlideReactor: proof of a new concept for cell characterization.

Certain cell types, especially primary human cells, favor a well-defined culture environment offering continuous supply of nutrients and oxygen and waste product removal. Several bioreactors based on special matrices or hollow fibers have been developed that provide such conditions. However, characterization of matrix re-organization or growth of tissue within these systems is possible only after culture termination. Evaluation of the influence of certain medium additives or culture conditions (e.g., temperature, oxygenation) on cell viability, expansion, and differentiation within these systems remains a challenging task. The SlideReactor, a miniaturized hollow fiber-based bioreactor, was developed to enable the observation of cells during culture. An operation concept offering predefined conditions for various cell types has been designed. For proof of concept, primary human cells (hepatocytes, fibroblasts, keratinocytes) and cell lines (HepG2, HuH7, C3A, WiDr, SkHep1) were cultured and observed. A series of experiments (n=40) showed the feasibility of the set-up; determination of process parameters and continuous observation is possible. The SlideReactor may serve as a simple and cost-efficient tool for cell characterization and optimization of cell-culture conditions.