Quality assessment by bile composition in normothermic machine perfusion of rat livers.

BACKGROUND: The persistent challenge of organ scarcity in liver transplantation leads to an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) is used for improved preservation. Due to the mimicked in vivo conditions during normothermic machine perfusion, the liver is metabolic active, which allows quality assessment during perfusion. Bile seems to be of rising interest in clinical studies since it is easily collectible for analysis. As there is currently no data on biliary bile acids during NMP, the primary objective of this study was to use our experimental rodent NMP model to assess changes in bile composition through organ damage during perfusion to inform clinical evaluation of donor organs during NMP. METHODS: 30 livers from male Sprague Dawley rats in five groups and underwent 6 hours of NMP using either erythrocyte-supplemented DMEM or Steen solution, with or without 30min of warm ischemia time (WIT). We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at three-hour intervals. Bile samples were analyzed for biliary pH, LDH and GGT as well as biliary bile acids via mass spectrometry and UHPLC. RESULTS: Compared to regular livers, liver injury parameters were significantly higher in our donation after circulatory death (DCD) model. Bile production was significantly reduced in livers exposed to WIT, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers experiencing WIT. However, regular livers produced a higher total amount of biliary bile acids during perfusion. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced during perfusion due to the missing enterohepatic circulation. CONCLUSIONS: WIT-induced liver injury affects bile composition within our small animal NMP model. We hypothesize this phenomenon to be attributed to the energy-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile. Our results may inform clinical studies, in which biliary bile acids might have a potential as a quantifiable viability marker in human NMP liver transplantation studies.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

On the relationship between viscoelasticity and water diffusion in soft biological tissues.

Magnetic resonance elastography (MRE) and diffusion-weighted imaging (DWI) are complementary imaging techniques that detect disease based on viscoelasticity and water mobility, respectively. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering the clinical translation of combined DWI-MRE markers. We used DWI-MRE to study 129 biomaterial samples including native and cross-linked collagen, glycosaminoglycans (GAGs) with different sulfation levels, and decellularized specimens of pancreas and liver, all with different proportions of solid tissue, or solid fractions. We developed a theoretical framework of the relationship between mechanical loss and tissue-water mobility based on two parameters, solid and fluid viscosity. These parameters revealed distinct DWI-MRE property clusters characterizing weak, moderate, and strong water-network interactions. Sparse networks interacting weakly with water, such as collagen or diluted decellularized tissue, resulted in marginal changes in water diffusion over increasing solid viscosity. In contrast, dense networks with larger solid fractions exhibited both free and hindered water diffusion depending on the polarity of the solid components. For example, polar and highly sulfated GAGs as well as native soft tissues hindered water diffusion despite relatively low solid viscosity. Our results suggest that two fundamental properties of tissue networks, solid fraction and network polarity, critically influence solid and fluid viscosity in biological tissues. Since clinical DWI and MRE are sensitive to these viscosity parameters, the framework we present here can be used to detect tissue remodeling and architectural changes in the setting of diagnostic imaging. STATEMENT OF SIGNIFICANCE: The viscoelastic properties of biological tissues provide a wealth of information on the vital state of cells and host matrix. Combined measurement of viscoelasticity and water diffusion by medical imaging is sensitive to tissue microarchitecture. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering full exploitation of these properties as a combined clinical biomarker. Therefore, we analyzed the parameter space accessible by diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE) and developed a theoretical framework for the relationship between water mobility and mechanical parameters in biomaterials. Our theory of solid material properties related to particle motion can be translated to clinical radiology using clinically established MRE and DWI.

From morbidity reduction to cost-effectiveness: Enhanced recovery after surgery (ERAS) society recommendations in minimal invasive liver surgery.

PURPOSE: Minimal-invasive liver surgery (MILS) reduces surgical trauma and is associated with fewer postoperative complications. To amplify these benefits, perioperative multimodal concepts like Enhanced Recovery after Surgery (ERAS), can play a crucial role. We aimed to evaluate the cost-effectiveness for MILS in an ERAS program, considering the necessary additional workforce and associated expenses. METHODS: A prospective observational study comparing surgical approach in patients within an ERAS program compared to standard care from 2018-2022 at the Charité - Universitätsmedizin Berlin. Cost data were provided by the medical controlling office. ERAS items were applied according to the ERAS society recommendations. RESULTS: 537 patients underwent liver surgery (46% laparoscopic, 26% robotic assisted, 28% open surgery) and 487 were managed by the ERAS protocol. Implementation of ERAS reduced overall postoperative complications in the MILS group (18% vs. 32%, p = 0.048). Complications greater than Clavien-Dindo grade II incurred the highest costs (€ 31,093) compared to minor (€ 17,510) and no complications (€13,893; p < 0.001). In the event of major complications, profit margins were reduced by a median of € 6,640. CONCLUSIONS: Embracing the ERAS society recommendations in liver surgery leads to a significant reduction of complications. This outcome justifies the higher cost associated with a well-structured ERAS protocol, as it effectively offsets the expenses of complications.

Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome.

BACKGROUND: Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess potential metabolic changes in response to differing enrichment levels. METHODS: We conducted a six-month study involving 24 male Sprague Dawley rats, randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an activity focused rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed. RESULTS: Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p = 0.007). We observed 1871 distinct proteins in liver tissue, with 77% being common across groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p = 0.024 and p = 0.009). Rats in the intervention groups with weekly playtime displayed the least amount of reported distress during inspection or upon room entry and were less prone to accepting treats. Removing animals from their enclosure was most effortless for those in the large cage group. Over time, there was a decrease in conflicts among rats that interacted only twice weekly during playpen time. DISCUSSION: In summary, refining husbandry practices for aging rats is both simple and budget-friendly, with no apparent adverse effects on stress levels, animal development, or relevant metabolic changes in the liver.

Improved outcomes after hypothermic oxygenated machine perfusion in liver transplantation-Long-term follow-up of a multicenter randomized controlled trial.

BACKGROUND: While 4 randomized controlled clinical trials confirmed the early benefits of hypothermic oxygenated machine perfusion (HOPE), high-level evidence regarding long-term clinical outcomes is lacking. The aim of this follow-up study from the HOPE-ECD-DBD trial was to compare long-term outcomes in patients who underwent liver transplantation using extended criteria donor allografts from donation after brain death (ECD-DBD), randomized to either HOPE or static cold storage (SCS). METHODS: Between September 2017 and September 2020, recipients of liver transplantation from 4 European centers receiving extended criteria donor-donation after brain death allografts were randomly assigned to HOPE or SCS (1:1). Follow-up data were available for all patients. Analyzed endpoints included the incidence of late-onset complications (occurring later than 6 months and graded according to the Clavien-Dindo Classification and the Comprehensive Complication Index) and long-term graft survival and patient survival. RESULTS: A total of 46 patients were randomized, 23 in both arms. The median follow-up was 48 months (95% CI: 41-55). After excluding early perioperative morbidity, a significant reduction in late-onset morbidity was observed in the HOPE group (median reduction of 23 Comprehensive Complication Index-points [p=0.003] and lower incidence of major complications [Clavien-Dindo ≥3, 43% vs. 85%, p=0.009]). Primary graft loss occurred in 13 patients (HOPE n=3 vs. SCS n=10), resulting in a significantly lower overall graft survival (p=0.029) and adverse 1-, 3-, and 5-year survival probabilities in the SCS group, which did not reach the level of significance (HOPE 0.913, 0.869, 0.869 vs. SCS 0.783, 0.606, 0.519, respectively). CONCLUSIONS: Our exploratory findings indicate that HOPE reduces late-onset morbidity and improves long-term graft survival providing clinical evidence to further support the broad implementation of HOPE in human liver transplantation.

Proteomic analysis of decellularized mice liver and kidney extracellular matrices.

BACKGROUND: The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. RESULTS: In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. CONCLUSION: The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell-matrix interactions and their contribution to a tissue-specific microenvironment.

miRNA as potential biomarkers after liver transplantation: A systematic review.

BACKGROUND: Liver transplantation is a life-saving therapy for end-stage liver disease patients, but acute cellular rejection (ACR) and graft complications remain significant postoperative challenges. Early and accurate diagnosis is crucial for timely intervention and improved patient outcomes, but their diagnosis rely currently on invasive biopsy sampling, thus prompting the search for non-invasive Biomarkers. MicroRNA (miRNA) have emerged as promising biomarkers in various pathological conditions, and their potential utility in diagnosing acute cellular rejection after liver transplantation has gained significant interest. METHODS: This systematic review of PubMed, Web of Science, and the ClinicalTrials.gov registry analyzes studies exploring miRNA as biomarkers for ACR and graft dysfunction in liver transplantation (PROSPERO ID CRD42023465278). The Cochrane Collaboration tool for assessing risk of bias was employed. Population data, identified miRNA and their dynamic regulation, as well as event prediction were compared. Data extraction and quality assessment were performed independently by two reviewers. RESULTS: Thirteen studies were included in this systematic review. Various investigated miRNAs were upregulated in association with acute cellular rejection, like miR-122, miR-155, miR-181, miR-483-3p, and miR-885-5p, demonstrating great biomarker potential. Additionally, several studies conducted target gene analysis, revealing insights into cellular mechanisms linked to ACR. Moreover, various miRNA were also capable of predicting different organ complications following transplantation, expanding their versatility. Remaining challenges include the standardization of miRNA profiling, the need for functional validation, and the necessity for long-term studies. CONCLUSION: The results highlight the potential of miRNA as specific, non-invasive biomarkers for ACR and graft dysfunction following liver transplantation. However, further research is needed to validate these findings and establish standardized diagnostic panels to incorporate them into clinical practice and explore miRNA-based therapies in the future.

Minimal Invasive Versus Open Surgery for Colorectal Liver Metastases: A Multicenter German StuDoQ|Liver Registry-Based Cohort Analysis in Germany.

Objective: To compare the outcome of minimally invasive liver surgery (MILS) to open liver surgery (OLS) for resection of colorectal liver metastases (CRLM) on a nationwide level. Background: Colorectal cancer is the third most common malignancy worldwide. Up to 50% of all patients with colorectal cancer develop CRLM. MILS represents an attractive alternative to OLS for treatment of CRLM. Methods: Retrospective cohort study using the prospectively recorded German Quality management registry for liver surgery. Propensity-score matching was performed to account for variance in the extent of resection and patient demographics. Results: In total, 1037 patients underwent liver resection for CRLM from 2019 to 2021. MILS was performed in 31%. Operative time was significantly longer in MILS (234 vs 222 minutes, P = 0.02) compared with OLS. After MILS, median length of hospital stay (LOS) was significantly shorter (7 vs 10 days; P < 0.001). Despite 76% of major resections being OLS, postoperative complications and 90-day morbidity and mortality did not differ. The Pringle maneuver was more frequently used in MILS (48% vs 40%, P = 0.048). After propensity-score matching for age, body mass index, Eastern Cooperative Oncology Group, and extent of resection, LOS remained shorter in the MILS cohort (6 vs 10 days, P < 0.001) and operative time did not differ significantly (P = 0.2). Conclusion: MILS is not the standard for resection of CRLM in Germany. Drawbacks, such as a longer operative time remain. However, if technically possible, MILS is a reasonable alternative to OLS for resection of CRLM, with comparable postoperative complications, reduced LOS, and equal oncological radicality.

Moderate LMWH Anticoagulation Improves Success Rate of Hind Limb Allotransplantation in Mice.

Background: The mouse hind limb model represents a powerful research tool in vascularized composite tissue allotransplantation, but its applicability is limited due to poor graft survival (62%-83%). Vascular thrombosis and massive hemorrhage are the major causes for these drop-outs. We hypothesize that because of better anticoagulation effect and lower risk of thrombocytopenia, application of low molecular weight heparin (LMWH) will minimize vascular complications and enhance graft and animal survival. Methods: Fifty allogeneic hind limb transplantations were performed (C57BL/6 to DBA/2 mice) using five different anticoagulation protocols. Bleeding and thromboembolic events were recorded macroscopically by postoperative hemorrhage and livid discoloration of the graft, respectively. Graft perfusion and survival were monitored daily by capillary-refill-time of graft toes within 2-3 seconds. Vascular congestion and tissue necrosis were examined by histological evaluation of hematoxylin-eosin-stained tissue sections. Results: All transplantations were technically successful. Increase in thromboembolic events and a concomitant decrease in bleeding events were observed with the decreasing concentration of heparin in the perfusion solution. Although treatment of donor and recipient with low dose of LMWH could not reduce thromboembolic events, moderate dose effectively reduced these events. Compared with the poor outcome of graft perfusion with heparin alone, additional treatment of donor and recipient with low dose of LMWH improved graft and animal survival by 18%. Interestingly, animals treated with moderate dose of LMWH demonstrated 100% graft and animal survival. Conclusions: Treatment of donor and recipient mice with a moderate dose of LMWH prevents vascular complications and improves the outcome of murine hind limb transplants.

Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins.

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs. Notably, this discussion also covers photocrosslinkable dECM bioresins, which are particularly attractive due to their ability to provide precise spatiotemporal control over the gelation in bioprinting. Both in extrusion printing and vat photopolymerization, there is a need for more standardized protocols to fully harness the unique properties of dECM-derived materials. In addition to mammalian tissues, the most recent bioprinting approaches involve the use of microbial extracellular polymeric substances in bioprinting of bacteria. This presents similar challenges as those encountered in mammalian cell printing and represents a fascinating frontier in bioprinting technology.

Development and systematic evaluation of decellularization protocols in different application models for diaphragmatic tissue engineering.

BACKGROUND: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. METHODS: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy. RESULTS: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. CONCLUSION: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.

Advancing Perfusion Models: Dual-Vessel Ex Vivo Rat Liver Perfusion Based on a Clinical Setup.

Normothermic ex vivo liver machine perfusion (NEVLP) has been developed to address the increasing organ shortage in liver transplantation, through optimal preservation, assessment, and conditioning of grafts from extended criteria donors. There remains a need to establish simple and standardized animal models that simulate clinical NEVLP to test novel therapies. Liver grafts from 36 Sprague-Dawley rats were perfused for 6 h in a dual-vessel NEVLP system with a Dulbecco's modified Eagles medium-based perfusate supplemented with rat plasma and erythrocytes. Varying doses of the clinically used vasodilator epoprostenol, Kupffer cell inhibitor glycine, and a Steen™-based perfusate were assessed. Perfusion pressures and bile production were recorded, and perfusate was analyzed for transaminase secretion. Tissue samples were evaluated histologically, and levels of cytokines and 8-Isoprostane were measured. Increasing levels of epoprostenol and the addition of glycine resulted in a stepwise decrease of transaminase secretion and improved bile production. Steen further decreased transaminase release and interleukin 1 beta levels. Liver grafts perfused with the optimized Steen-based protocol exhibited lowest levels of oxidative stress and best-preserved liver integrity. In conclusion, epoprostenol seemed to ameliorate liver function and prevent cellular damage beyond its vasodilatory effect, with glycine acting synergistically. The anti-inflammatory and antioxidative properties of Steen further improved the outcome of perfusion. Our rodent NEVLP system may be used to rapidly test new agents for the pharmacologic conditioning of livers and help translate findings from bench-to-bedside.

Validation of risk scores for allograft failure after liver transplantation in Germany: a retrospective cohort analysis.

A growing number of clinical risk scores have been proposed to predict allograft failure after liver transplantation. However, validation of currently available scores in the Eurotransplant region is still lacking. We aimed to analyze all clinically relevant donor and recipient risk scores on a large German liver transplantation data set and performed a retrospective cohort analysis of liver transplantations performed at the Charité-Universitätsmedizin Berlin from January 2007 until December 2021 with organs from donation after brain death. We analyzed 9 previously published scores in 906 liver transplantations [Eurotransplant donor risk index (ET-DRI/DRI), donor age and model for end-stage liver disease (D-MELD), balance of risk (BAR), early allograft dysfunction (EAD), model for early allograft function (MEAF), liver graft assessment following transplantation (L-GrAFT7), early allograft failure simplified estimation (EASE), and a score by Rhu and colleagues). The EASE score had the best predictive value for 3-month, 6-month, and 12-month graft survival with a c-statistic of 0.8, 0.77, and 0.78, respectively. In subgroup analyses, the EASE score was suited best for male recipients with a high-MELD (>25) and an EAD organ. Scores only based on pretransplant data performed worse compared to scores including postoperative data (eg, ET-DRI vs. EAD, p<0.001 at 3-month graft survival). Out of these, the BAR score performed best with a c-statistic of 0.6. This a comprehensive comparison of the clinical utility of risk scores after liver transplantation. The EASE score sufficiently predicted 12-month graft and patient survival. Despite a relatively complex calculation, the EASE score provides significant prognostic value for patients and health care professionals in the Eurotransplant region.

Effect of Preoperative Chemotherapy on the Isolation Outcome of Primary Human Hepatocytes.

Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pretreated and untreated donor. Furthermore, the transcription levels of cytochrome P450 (CYP) 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens before isolation seem to perform as well as hepatocytes without preoperative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies. Impact statement Isolated primary human hepatocytes are an essential resource for pharmacological and toxicological studies. Our results present further evidence that isolated hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies-especially when fluoropyrimidine-based regimens are used.

Ex Vivo Liver Machine Perfusion: Comprehensive Review of Common Animal Models.

The most common preservation technique for liver grafts is static cold storage. Due to the organ shortage for liver transplantation (LT), extended criteria donor (ECD) allografts are increasingly used-despite the higher risk of inferior outcome after transplantation. Ex vivo liver machine perfusion (MP) has been developed to improve the outcome of transplantation, especially with ECD grafts, and is currently under evaluation in clinical trials. We performed a literature search on PubMed and ISI Web of Science to assemble an overview of rodent and porcine animal models of ex vivo liver MP for transplantation, which is essential for the present and future development of clinical liver MP. Hypothermic, subnormothermic, and normothermic MP systems have been successfully used for rat and pig LT. In comparison with hypothermic systems, normothermic perfusion often incorporates a dialysis unit. Moreover, it enables metabolic assessment of liver grafts. Allografts experiencing warm ischemic time have a superior survival rate after MP compared with cold storage alone, irrespective of the temperature used for perfusion. Furthermore, ex vivo MP improves the outcome of regular and ECD liver grafts in animal models. Small and large animal models of ex vivo liver MP are available to foster the further development of this new technology. Impact Statement Ex vivo machine perfusion is an important part of current research in the field of liver transplantation. While evidence for improve storage is constantly rising, the development of future applications such as quality assessment and therapeutic interventions necessitates robust animal models. This review is intended to provide an overview of this technology in common large and small animal models and to give an outlook on future applications. Moreover, we describe developmental steps that can be followed by others, and which can help to decrease the number of animals used for experiments based on the replace, reduce, refine concept.

Solid fraction determines stiffness and viscosity in decellularized pancreatic tissues.

The role of extracellular matrix (ECM) composition and turnover in mechano-signaling and the metamorphic fate of cells seeded into decellularized tissue can be elucidated by recent developments in non-invasive imaging and biotechnological analysis methods. Because these methods allow accurate quantification of the composition and structural integrity of the ECM, they can be critical in establishing standardized decellularization protocols. This study proposes quantification of the solid fraction, the single-component fraction and the viscoelasticity of decellularized pancreatic tissues using compact multifrequency magnetic resonance elastography (MRE) to assess the efficiency and quality of decellularization protocols. MRE of native and decellularized pancreatic tissues showed that viscoelasticity parameters depend according to a power law on the solid fraction of the decellularized matrix. The parameters can thus be used as highly sensitive markers of the mechanical integrity of soft tissues. Compact MRE allows consistent and noninvasive quantification of the viscoelastic properties of decellularized tissue. Such a method is urgently needed for the standardized monitoring of decellularization processes, evaluation of mechanical ECM properties, and quantification of the integrity of solid structural elements remaining in the decellularized tissue matrix.

The influence of the COVID-19 pandemic on surgical therapy and care: a cross-sectional study.

BACKGROUND: Due to the COVID-19 pandemic, an extensive reorganisation of healthcare resources was necessary-with a particular impact on surgical care across all disciplines. However, the direct and indirect consequences of this redistribution of resources on surgical therapy and care are largely unknown. METHODS: We analysed our prospectively collected standardised digital quality management document for all surgical cases in 2020 and compared them to the years 2018 and 2019. Periods with high COVID-19 burdens were compared with the reference periods in 2018 and 2019. RESULTS: From 2018 to 2020, 10,723 patients underwent surgical treatment at our centres. We observed a decrease in treated patients and a change in the overall patient health status. Patient age and length of hospital stay increased during the COVID-19 pandemic (p = 0.004 and p = 0.002). Furthermore, the distribution of indications for surgical treatment changed in favour of oncological cases and less elective cases such as hernia repairs (p < 0.001). Postoperative thromboembolic and pulmonary complications increased slightly during the COVID-19 pandemic. There were slight differences for postoperative overall complications according to Clavien-Dindo, with a significant increase of postoperative mortality (p = 0.01). CONCLUSION: During the COVID-19 pandemic we did not see an increase in the occurrence, or the severity of postoperative complications. Despite a slightly higher rate of mortality and specific complications being more prevalent, the biggest change was in indication for surgery, resulting in a higher proportion of older and sicker patients with corresponding comorbidities. Further research is warranted to analyse how this changed demographic will influence long-term patient care.

Clonality and timing of relapsing colorectal cancer metastasis revealed through whole-genome single-cell sequencing.

Recurrence of tumor cells following local and systemic therapy is a significant hurdle in cancer. Most patients with metastatic colorectal cancer (mCRC) will relapse, despite resection of the metastatic lesions. A better understanding of the evolutionary history of recurrent lesions is required to identify the spatial and temporal patterns of metastatic progression and expose the genetic and evolutionary determinants of therapeutic resistance. With this goal in mind, here we leveraged a unique single-cell whole-genome sequencing dataset from recurrent hepatic lesions of an mCRC patient. Our phylogenetic analysis confirms that the treatment induced a severe demographic bottleneck in the liver metastasis but also that a previously diverged lineage survived this surgery, possibly after migration to a different site in the liver. This lineage evolved very slowly for two years under adjuvant drug therapy and diversified again in a very short period. We identified several non-silent mutations specific to this lineage and inferred a substantial contribution of chemotherapy to the overall, genome-wide mutational burden. All in all, our study suggests that mCRC subclones can migrate locally and evade resection, keep evolving despite rounds of chemotherapy, and re-expand explosively.