Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL.

Metabolic crosstalk of the major nutrients glucose, amino acids and fatty acids (FAs) ensures systemic metabolic homeostasis. The coordination between the supply of glucose and FAs to meet various physiological demands is especially important as improper nutrient levels lead to metabolic disorders, such as diabetes and metabolic dysfunction-associated steatohepatitis (MASH). In response to the oscillations in blood glucose levels, lipolysis is thought to be mainly regulated hormonally to control FA liberation from lipid droplets by insulin, catecholamine and glucagon. However, whether general cell-intrinsic mechanisms exist to directly modulate lipolysis via glucose sensing remains largely unknown. Here we report the identification of such an intrinsic mechanism, which involves Golgi PtdIns4P-mediated regulation of adipose triglyceride lipase (ATGL)-driven lipolysis via intracellular glucose sensing. Mechanistically, depletion of intracellular glucose results in lower Golgi PtdIns4P levels, and thus reduced assembly of the E3 ligase complex CUL7FBXW8 in the Golgi apparatus. Decreased levels of the E3 ligase complex lead to reduced polyubiquitylation of ATGL in the Golgi and enhancement of ATGL-driven lipolysis. This cell-intrinsic mechanism regulates both the pool of intracellular FAs and their extracellular release to meet physiological demands during fasting and glucose deprivation. Moreover, genetic and pharmacological manipulation of the Golgi PtdIns4P-CUL7FBXW8-ATGL axis in mouse models of simple hepatic steatosis and MASH, as well as during ex vivo perfusion of a human steatotic liver graft leads to the amelioration of steatosis, suggesting that this pathway might be a promising target for metabolic dysfunction-associated steatotic liver disease and possibly MASH.

Versatile Mechanically Tunable Hydrogels for Therapeutic Delivery Applications.

Hydrogels provide a versatile platform for biomedical material fabrication that can be structurally and mechanically fine-tuned to various tissues and applications. Applications of hydrogels in biomedicine range from highly dynamic injectable hydrogels that can flow through syringe needles and maintain or recover their structure after extrusion to solid-like wound-healing patches that need to be stretchable while providing a selective physical barrier. In this study, a toolbox is designed using thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) polymeric matrices and nanocelluloses as reinforcing agent to obtain biocompatible hydrogels with altering mechanical properties, from a liquid injectable to a solid-like elastic hydrogel. The liquid hydrogels possess low viscosity and shear-thinning properties at 25 °C, which allows facile injection at room temperature, while they become viscoelastic gels at body temperature. In contrast, the covalently cross-linked solid-like hydrogels exhibit enhanced viscoelasticity. The liquid hydrogels are biocompatible and are able to delay the in vitro release and maintain the bioactivity of model drugs. The antimicrobial agent loaded solid-like hydrogels are effective against typical wound-associated pathogens. This work presents a simple method of tuning hydrogel mechanical strength to easily adapt to applications in different soft tissues and broaden the potential of renewable bio-nanoparticles in hybrid biomaterials with controlled drug release capabilities.

Designed modular protein hydrogels for biofabrication.

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.

Reversible Mechanical Contraception and Endometriosis Treatment Using Stimuli-Responsive Hydrogels.

Female sterilization via fallopian tube ligation is a common procedure; However, after the operation, over 10% of women seek re-fertilization, which is frequently unsuccessful. In addition, there is evidence that fallopian tubes contribute to the spread of endometriotic tissue as they serve as channels for proinflammatory media entering the abdominal cavity via retrograde menstruation. Here, stimuli-degradable hydrogel implants are presented for the functional, biocompatible, and reversible occlusion of fallopian tubes. The hydrogel implants, designed with customized swelling properties, mechanically occlude fallopian tubes in a high-performance manner with burst pressures reaching 255-558 mmHg, exceeding normal abdominal pressures (95 mmHg). Their damage-free removal can be achieved within 30 min using near-visible UV light or a glutathione solution, employing a method akin to standard fallopian tube perfusion diagnostics. Ultrasound-guided implant placement is demonstrated using a clinical hysteroscope in a human-scale uterus model and biocompatibility in a porcine in vivo model. Importantly, the prevention of live sperm as well as endometrial cell passage through blocked fallopian tubes is demonstrated. Overall, a multifunctional system is presented that constitutes a possible means of on-demand, reversible contraception along with the first-ever mechanical approach to abdominal endometriosis prevention and treatment.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

Recovery of Therapeutically Ablated Engineered Blood-Vessel Networks on a Plug-and-Play Platform.

Limiting the availability of key angiogenesis-promoting factors is a successful strategy to ablate tumor-supplying blood vessels or to reduce excessive vasculature in diabetic retinopathy. However, the efficacy of such anti-angiogenic therapies (AATs) varies with tumor type, and regrowth of vessels is observed upon termination of treatment. The ability to understand and develop AATs remains limited by a lack of robust in vitro systems for modeling the recovery of vascular networks. Here, complex 3D micro-capillary networks are engineered by sequentially seeding human bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells (ECs) on a previously established, synthetic plug-and-play hydrogel platform. In the tightly interconnected vascular networks that form this way, the two cell types share a basement membrane-like layer and can be maintained for several days of co-culture. Pre-formed networks degrade in the presence of bevacizumab. Upon treatment termination, vessel structures grow back to their original positions after replenishment with new ECs, which also integrate into unperturbed established networks. The data suggest that this plug-and-play platform enables the screening of drugs with blood-vessel inhibiting functions. It is believed that this platform could be of particular interest in studying resistance or recovery mechanisms to AAT treatment.

Implications of Cellular Mechanical Memory in Bioengineering.

The ability to maintain and differentiate cells in vitro is critical to many advances in the field of bioengineering. However, on traditional, stiff (E ≈ GPa) culture substrates, cells are subjected to sustained mechanical stress that can lead to phenotypic changes. Such changes may remain even after transferring the cells to another scaffold or engrafting them in vivo and bias the outcomes of the biological investigation or clinical treatment. This persistence─or mechanical memory─was initially observed for sustained myofibroblast activation of pulmonary fibroblasts after culturing them on stiff (E ≈ 100 kPa) substrates. Aspects of mechanical memory have now been described in many in vitro contexts. In this Review, we discuss the stiffness-induced effectors of mechanical memory: structural changes in the cytoskeleton and activity of transcription factors and epigenetic modifiers. We then focus on how mechanical memory impacts cell expansion and tissue regeneration outcomes in bioengineering applications relying on prolonged 2D plastic culture, such as stem cell therapies and disease models. We propose that alternatives to traditional cell culture substrates can be used to mitigate or erase mechanical memory and improve the efficiency of downstream cell-based bioengineering applications.

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair.

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.

Serine protease 35 regulates the fibroblast matrisome in response to hyperosmotic stress.

Hyperosmotic stress occurs in several diseases, but its long-term effects are largely unknown. We used sorbitol-treated human fibroblasts in 3D culture to study the consequences of hyperosmotic stress in the skin. Sorbitol regulated many genes, which help cells cope with the stress condition. The most robustly regulated gene encodes serine protease 35 (PRSS35). Its regulation by hyperosmotic stress was dependent on the kinases p38 and JNK and the transcription factors NFAT5 and ATF2. We identified different collagens and collagen-associated proteins as putative PRSS35 binding partners. This is functionally important because PRSS35 affected the extracellular matrix proteome, which limited cell proliferation. The in vivo relevance of these findings is reflected by the coexpression of PRSS35 and its binding partners in human skin wounds, where hyperosmotic stress occurs as a consequence of excessive water loss. These results identify PRSS35 as a key regulator of the matrisome under hyperosmotic stress conditions.

Defatting of Human Livers During Long-Term e x situ Normothermic Perfusion: Novel Strategy to Rescue Discarded Organs for Transplantation.

OBJECTIVE: To develop a protocol for the defatting of steatotic liver grafts during long-term ex situ normothermic machine perfusion. BACKGROUND: Despite the alarming increase in donor organ shortage, the highly prevalent fatty liver grafts are often discarded due to the risk of primary nonfunction. Effective strategies preventing such outcomes are currently lacking. An exciting new avenue is the introduction of ex situ normothermic machine perfusion (NMP), enabling a liver to remain fully functional for up to 2 weeks and providing a unique window of opportunity for defatting before transplantation. METHODS: Over a 5-year period, 23 discarded liver grafts and 28 partial livers from our resection program were tested during ex situ normothermic machine perfusion. The steatosis degree was determined on serial biopsies by expert pathologists, and triglyceride contents were measured simultaneously. RESULTS: Of 51 liver grafts, 20 were steatotic, with up to 85% macrovesicular steatosis, and were perfused for up to 12 days. Ten livers displayed marked (5 of which almost complete) loss of fat, while the other 10 did not respond to long-term perfusion. Successful defatting was related to prolonged perfusion, automated glucose control, circadian nutrition, and L-carnitine/fenofibrate supplementation. Pseudopeliotic steatosis and the associated activation of Kupffer/stellate cells were unexpected processes that might contribute to defatting. Synthetic and metabolic functions remained preserved for most grafts until perfusion ended. CONCLUSION: Ex situ long-term perfusion effectively reduces steatosis while preserving organ viability and may in the future allow transplantation of primarily unusable high-risk grafts, significantly increasing the number of organs available for transplantation.

A Spectrofluorometric Method for Real-Time Graft Assessment and Patient Monitoring.

Biomarkers are powerful clinical diagnostics and predictors of patient outcome. However, robust measurements often require time and expensive laboratory equipment, which is insufficient to track rapid changes and limits direct use in the operating room. Here, this study presents a portable spectrophotometric device for continuous real-time measurements of fluorescent and non-fluorescent biomarkers at the point of care. This study measures the mitochondrial damage biomarker flavin mononucleotide (FMN) in 26 extended criteria human liver grafts undergoing hypothermic oxygenated perfusion to guide clinical graft assessment. Real-time data identified seven organs unsuitable for transplant that are discarded. The remaining grafts are transplanted and FMN values correlated with post-transplant indicators of liver function and patient recovery. Further, this study shows how this device can be used to monitor dialysis patients by measuring creatinine in real-time. Our approach provides a simple method to monitor biomarkers directly within biological fluids to improve organ assessment, patient care, and biomarker discovery.

Control of hydrostatic pressure and osmotic stress in 3D cell culture for mechanobiological studies.

Hydrostatic pressure (HP) and osmotic stress (OS) play an important role in various biological processes, such as cell proliferation and differentiation. In contrast to canonical mechanical signals transmitted through the anchoring points of the cells with the extracellular matrix, the physical and molecular mechanisms that transduce HP and OS into cellular functions remain elusive. Three-dimensional cell cultures show great promise to replicate physiologically relevant signals in well-defined host bioreactors with the goal of shedding light on hidden aspects of the mechanobiology of HP and OS. This review starts by introducing prevalent mechanisms for the generation of HP and OS signals in biological tissues that are subject to pathophysiological mechanical loading. We then revisit various mechanisms in the mechanotransduction of HP and OS, and describe the current state of the art in bioreactors and biomaterials for the control of the corresponding physical signals.

Observations and findings during the development of a subnormothermic/normothermic long-term ex vivo liver perfusion machine.

BACKGROUND: Ex situliver machine perfusion at subnormothermic/normothermic temperature isincreasingly applied in the field of transplantation to store and evaluateorgans on the machine prior transplantation. Currently, various perfusionconcepts are in clinical and preclinical applications. Over the last 6 years ina multidisciplinary team, a novel blood based perfusion technology wasdeveloped to keep a liver alive and metabolically active outside of the bodyfor at least one week. METHODS: Within thismanuscript, we present and compare three scenarios (Group 1, 2 and 3) we werefacing during our research and development (R&D) process, mainly linked tothe measurement of free hemoglobin and lactate in the blood based perfusate. Apartfrom their proven value in liver viability assessment (ex situ), these twoparameters are also helpful in R&D of a long-term liver perfusion machine and moreover supportive in the biomedical engineering process. RESULTS: Group 1 ("good" liver on the perfusion machine) represents the best liver clearance capacity for lactate and free hemoglobin wehave observed. In contrast to Group 2 ("poor" liver on the perfusion machine), that has shown the worst clearance capacity for free hemoglobin. Astonishingly,also for Group 2, lactate is cleared till the first day of perfusion andafterwards, rising lactate values are detected due to the poor quality of theliver. These two perfusate parametersclearly highlight the impact of the organ quality/viability on the perfusion process. Whereas Group 3 is a perfusion utilizing a blood loop only (without a liver). CONCLUSION: Knowing the feasible ranges (upper- and lower bound) and the courseover time of free hemoglobin and lactate is helpful to evaluate the quality ofthe organ perfusion itself and the maturity of the developed perfusion device. Freehemoglobin in the perfusate is linked to the rate of hemolysis that indicates how optimizing (gentle blood handling, minimizing hemolysis) the perfusion machine actually is. Generally, a reduced lactate clearancecapacity can be an indication for technical problems linked to the blood supplyof the liver and therefore helps to monitor the perfusion experiments.Moreover, the possibility is given to compare, evaluate and optimize developed liverperfusion systems based on the given ranges for these two parameters. Otherresearch groups can compare/quantify their perfusate (blood) parameters withthe ones in this manuscript. The presented data, findings and recommendations willfinally support other researchers in developing their own perfusion machine ormodifying commercially availableperfusion devices according to their needs.

Dual carbon sequestration with photosynthetic living materials.

Natural ecosystems offer efficient pathways for carbon sequestration, serving as a resilient approach to remove CO2 from the atmosphere with minimal environmental impact. However, the control of living systems outside of their native environments is often challenging. Here, we engineered a photosynthetic living material for dual CO2 sequestration by immobilizing photosynthetic microorganisms within a printable polymeric network. The carbon concentrating mechanism of the cyanobacteria enabled accumulation of CO2 within the cell, resulting in biomass production. Additionally, the metabolic production of OH- ions in the surrounding medium created an environment for the formation of insoluble carbonates via microbially-induced calcium carbonate precipitation (MICP). Digital design and fabrication of the living material ensured sufficient access to light and nutrient transport of the encapsulated cyanobacteria, which were essential for long-term viability (more than one year) as well as efficient photosynthesis and carbon sequestration. The photosynthetic living materials sequestered approximately 2.5 mg of CO2 per gram of hydrogel material over 30 days via dual carbon sequestration, with 2.2 ± 0.9 mg stored as insoluble carbonates. Over an extended incubation period of 400 days, the living materials sequestered 26 ± 7 mg of CO2 per gram of hydrogel material in the form of stable minerals. These findings highlight the potential of photosynthetic living materials for scalable carbon sequestration, carbon-neutral infrastructure, and green building materials. The simplicity of maintenance, coupled with its scalability nature, suggests broad applications of photosynthetic living materials as a complementary strategy to mitigate CO2 emissions.

Building block properties govern granular hydrogel mechanics through contact deformations.

Granular hydrogels have been increasingly exploited in biomedical applications, including wound healing and cardiac repair. Despite their utility, design guidelines for engineering their macroscale properties remain limited, as we do not understand how the properties of granular hydrogels emerge from collective interactions of their microgel building blocks. In this work, we related building block features (stiffness and size) to the macroscale properties of granular hydrogels using contact mechanics. We investigated the mechanics of the microgel packings through dynamic oscillatory rheology. In addition, we modeled the system as a collection of two-body interactions and applied the Zwanzig and Mountain formula to calculate the plateau modulus and viscosity of the granular hydrogels. The calculations agreed with the dynamic mechanical measurements and described how microgel properties and contact deformations define the rheology of granular hydrogels. These results support a rational design framework for improved engineering of this fascinating class of materials.

Tools for manipulation and positioning of microtissues.

Complex three-dimensional (3D) in vitro models are emerging as a key technology to support research areas in personalised medicine, such as drug development and regenerative medicine. Tools for manipulation and positioning of microtissues play a crucial role in the microtissue life cycle from production to end-point analysis. The ability to precisely locate microtissues can improve the efficiency and reliability of processes and investigations by reducing experimental time and by providing more controlled parameters. To achieve this goal, standardisation of the techniques is of primary importance. Compared to microtissue production, the field of microtissue manipulation and positioning is still in its infancy but is gaining increasing attention in the last few years. Techniques to position microtissues have been classified into four main categories: hydrodynamic techniques, bioprinting, substrate modification, and non-contact active forces. In this paper, we provide a comprehensive review of the different tools for the manipulation and positioning of microtissues that have been reported to date. The working mechanism of each technique is described, and its merits and limitations are discussed. We conclude by evaluating the potential of the different approaches to support progress in personalised medicine.

Solvent Controls Nanoparticle Size during Nanoprecipitation by Limiting Block Copolymer Assembly.

Control of the properties of nanoparticles (NPs), including size, is critical for their application in biomedicine and engineering. Polymeric NPs are commonly produced by nanoprecipitation, where a solvent containing a block copolymer is mixed rapidly with a nonsolvent, such as water. Empirical evidence suggests that the choice of solvent influences NP size; yet, the specific mechanism remains unclear. Here, we show that solvent controls NP size by limiting block copolymer assembly. In the initial stages of mixing, polymers assemble into dynamic aggregates that grow via polymer exchange. At later stages of mixing, further growth is prevented beyond a solvent-specific water fraction. Thus, the solvent sets NP size by controlling the extent of dynamic growth up to growth arrest. An a priori model based on spinodal decomposition corroborates our proposed mechanism, explaining how size scales with the solvent-dependent critical water fraction of growth arrest and enabling more efficient NP engineering.

Thermal stabilization of diverse biologics using reversible hydrogels.

Improving the thermal stability of biologics, including vaccines, is critical to reduce the economic costs and health risks associated with the cold chain. Here, we designed a versatile, safe, and easy-to-use reversible PEG-based hydrogel platform formed via dynamic covalent boronic ester cross-linking for the encapsulation, stabilization, and on-demand release of biologics. Using these reversible hydrogels, we thermally stabilized a wide range of biologics up to 65°C, including model enzymes, heat-sensitive clinical diagnostic enzymes (DNA gyrase and topoisomerase I), protein-based vaccines (H5N1 hemagglutinin), and whole viruses (adenovirus type 5). Our data support a generalized protection mechanism for the thermal stabilization of diverse biologics using direct encapsulation in reversible hydrogels. Furthermore, preliminary toxicology data suggest that the components of our hydrogel are safe for in vivo use. Our reversible hydrogel platform offers a simple material solution to mitigate the costs and risks associated with reliance on a continuous cold chain for biologic transport and storage.

Transplantation of a human liver following 3 days of ex situ normothermic preservation.

Current organ preservation methods provide a narrow window (usually <12 hours) to assess, transport and implant donor grafts for human transplantation. Here we report the transplantation of a human liver discarded by all centers, which could be preserved for several days using ex situ normothermic machine perfusion. The transplanted liver exhibited normal function, with minimal reperfusion injury and the need for only a minimal immunosuppressive regimen. The patient rapidly recovered a normal quality of life without any signs of liver damage, such as rejection or injury to the bile ducts, according to a 1-year follow up. This inaugural clinical success opens new horizons in clinical research and promises an extended time window of up to 10 days for assessment of viability of donor organs as well as converting an urgent and highly demanding surgery into an elective procedure.