• 3D in vitro liver models
• Liver fibrosis
• Organoids

• 22A0204L1 Ministry of Science and ICT, South Korea
• RS-2024-00356692 Ministry of Food and Drug Safety

• Biliary reconstruction
• Bioengineered liver
• Decellularization
• Recellularization
• Reendothelialization

• 3D bioprinting
• bioink
• biomaterials
• decellularized extracellular matrix (dECM)
• organoid

• Humans
• Printing, Three-Dimensional
• Bioprinting/methods
• Liver/metabolism
• Liver/cytology
• Tissue Engineering/methods
• Animals
• Decellularized Extracellular Matrix/chemistry
• Decellularized Extracellular Matrix/metabolism
• Tissue Scaffolds/chemistry
• Hydrogels/chemistry
• Extracellular Matrix/metabolism
• Extracellular Matrix/chemistry
• Biocompatible Materials/chemistry

• CCl4
• decellularized liver matrix
• hepatocyte growth factor/heparin–complex
• liver fibrosis
• liver regeneration

• Animals
• Carbon Tetrachloride/toxicity
• Liver
• Hepatocyte Growth Factor/metabolism
• Heparin/chemistry
• Hepatocytes/drug effects
• Male
• Extracellular Matrix/metabolism
• Extracellular Matrix/chemistry
• Tissue Engineering/methods
• Mice
• Rats
• Liver Cirrhosis/therapy
• Chemical and Drug Induced Liver Injury/metabolism
• Humans
• Tissue Scaffolds/chemistry
• Rats, Sprague-Dawley

• 112-UN0076 National Taiwan University Hospital and National Taiwan University
• NTU-CC-110L892506 National Taiwan University
• NTU-CC-111L891906 National Taiwan University
• NTU-CC-112L890806 National Taiwan University

• 3D bioprinting
• 3D scaffolds
• bioink
• hepatic organoids
• hydrogels

• SERB Start-up research
• SERB POWER grant

• decellularization
• liver
• recellularization
• scaffolds
• tissue and organoids

• 3D bioprinting
• bioink
• cornea
• decellularization
• recellularization
• slaughterhouse waste
• xeno-keratoplasty

• 3D scaffold
• caprine pancreas
• collagen
• decellularization
• extracellular matrix (ECM)
• sulfated glycosaminoglycans (sGAG)
• tissue engineering

• biological cues
• decellularized extracellular matrices
• matrices
• mechanical properties
• organoids

Liver transplantation is the only curative therapy for end stage liver disease, but is limited by the organ shortage, and is associated with the adverse consequences of immunosuppression. Repopulation of decellularised whole organ scaffolds with appropriate cells of recipient origin offers a theoretically attractive solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Decellularisation methodologies vary widely but seek to address the conflicting objectives of removing the cellular component of tissues whilst keeping the 3D structure of the extra-cellular matrix intact, as well as retaining the instructive cell fate determining biochemicals contained therein. Liver scaffold recellularisation has progressed from small rodent in vitro studies to large animal in vivo perfusion models, using a wide range of cell types including primary cells, cell lines, foetal stem cells, and induced pluripotent stem cells. Within these models, a limited but measurable degree of physiologically significant hepatocyte function has been reported with demonstrable ammonia metabolism in vivo. Biliary repopulation and function have been restricted by challenges relating to the culture and propagations of cholangiocytes, though advances in organoid culture may help address this. Hepatic vasculature repopulation has enabled sustainable blood perfusion in vivo, but with cell types that would limit clinical applications, and which have not been shown to have the specific functions of liver sinusoidal endothelial cells. Minority cell groups such as Kupffer cells and stellate cells have not been repopulated. Bioengineering by repopulation of decellularised scaffolds has significantly progressed, but there remain significant experimental challenges to be addressed before therapeutic applications may be envisaged.

• Regenerative
• Bioengineering
• Scaffolds
• Liver
• Decellularisation
• Recellularisation

• liver transplantation
• machine preservation
• repair and regeneration medicine

Acellular adipose matrix (AAM) has received increasing attention for soft tissue reconstruction, due to its abundant source, high long-term retention rate and in vivo adipogenic induction ability. However, the current decellularization methods inevitably affect native extracellular matrix (ECM) properties, and the residual antigens can trigger adverse immune reactions after transplantation. The behavior of host inflammatory cells mainly decides the regeneration of AAM after transplantation. In this review, recent knowledge of inflammatory cells for acellular matrix regeneration will be discussed. These advancements will inform further development of AAM products with better properties.

• Cell migration
• Decellularization
• Immune responses
• Regenerative medicine
• Tissue engineering

Extracellular matrix (ECM) plays a multitude of roles, including supporting cells through structural and biochemical interactions. ECM is damaged in the process of isolating human islets for clinical transplantation and basic research. A platform in which islets can be cultured in contact with natural pancreatic ECM is desirable to better understand and support islet health, and to recapitulate the native islet environment. Our study demonstrates the derivation of a practical and durable hydrogel from decellularized human pancreas that supports human islet survival and function. Islets embedded in this hydrogel show increased glucose- and KCl-stimulated insulin secretion, and improved mitochondrial function compared to islets cultured without pancreatic matrix. In extended culture, hydrogel co-culture significantly reduced levels of apoptosis compared to suspension culture and preserved controlled glucose-responsive function. Isolated islets displayed altered endocrine and non-endocrine cell arrangement compared to in situ islets; hydrogel preserved an islet architecture more similar to that observed in situ. RNA sequencing confirmed that gene expression differences between islets cultured in suspension and hydrogel largely fell within gene ontology terms related to extracellular signaling and adhesion. Natural pancreatic ECM improves the survival and physiology of isolated human islets.

Liver transplantation is currently the only effective treatment for patients with end-stage liver disease; however, donor liver scarcity is a notable concern. As a result, extensive endeavors have been made to diversify the source of donor livers. For example, the use of a decellularized scaffold in liver engineering has gained considerable attention in recent years. The decellularized scaffold preserves the original orchestral structure and bioactive chemicals of the liver, and has the potential to create a de novo liver that is fit for transplantation after recellularization. The structure of the liver and hepatic extracellular matrix, decellularization, recellularization, and recent developments are discussed in this review. Additionally, the criteria for assessment and major obstacles in using a decellularized scaffold are covered in detail.

• National Natural Science Foundation of China

• Angiogenesis
• Growth factors
• Stem cells
• Tissue engineering
• Vascular diseases

• Animals
• Mesenchymal Stem Cells
• Mice
• Mice, Inbred NOD
• Mice, SCID
• Neovascularization, Physiologic
• Proteomics

• angiogenesis
• heparinization
• kidney scaffolds
• vascular endothelial growth factor

• Animals
• Anticoagulants/pharmacology
• Coculture Techniques
• Drug Liberation
• Heparin/pharmacology
• Human Umbilical Vein Endothelial Cells/drug effects
• Humans
• Kidney/cytology
• Kidney/drug effects
• Neovascularization, Physiologic/drug effects
• Rats, Sprague-Dawley
• Tensile Strength
• Tissue Scaffolds/chemistry
• Vascular Endothelial Growth Factor A/metabolism
• Rats

• Decellularization
• Liver
• Liver regeneration
• Liver scaffold
• Liver transplantation
• Tissue engineering

• extracellular matrix
• liver decellularization
• regenerative potential
• scanning probe nanotomography

• Biocompatibility
• Cell-ECM interactions
• Decellularized scaffold
• Growth factors
• Immunogenicity
• Xenogeneic implant

• Cell sheet technology
• Decellularization
• Liver bioengineering

Sterilization is the process of killing all microorganisms, while disinfection is the process of killing or removing all kinds of pathogenic microorganisms except bacterial spores. Biomaterials involved in cell experiments, animal experiments, and clinical applications need to be in the aseptic state, but their physical and chemical properties as well as biological activities can be affected by sterilization or disinfection. Decellularized matrix (dECM) is the low immunogenicity material obtained by removing cells from tissues, which retains many inherent components in tissues such as proteins and proteoglycans. But there are few studies concerning the effects of sterilization or disinfection on dECM, and the systematic introduction of sterilization or disinfection for dECM is even less. Therefore, this review systematically introduces and analyzes the mechanism, advantages, disadvantages, and applications of various sterilization and disinfection methods, discusses the factors influencing the selection of sterilization and disinfection methods, summarizes the sterilization and disinfection methods for various common dECM, and finally proposes a graphical route for selecting an appropriate sterilization or disinfection method for dECM and a technical route for validating the selected method, so as to provide the reference and basis for choosing more appropriate sterilization or disinfection methods of various dECM.

•

Asepsis is the prerequisite for the experiment and application of biomaterials.

• Alcohol
• Antibiotic
• Decellularized matrix
• Disinfection
• Ethylene oxide
• Hydrogen peroxide
• Irradiation
• Peracetic acid
• Sterilization
• Supercritical carbon dioxide

Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.

• H&E
• convolutional neural networks
• decellularization
• liver
• semantic segmentation
• tissue engineering

• Image Processing, Computer-Assisted
• Liver/diagnostic imaging
• Neural Networks, Computer
• Semantics

Instantaneous blood coagulation after bioengineered liver transplantation is a major issue, and the key process in its prevention is the construction of the endothelial vascular bed on biomimetic scaffolds. However, the specific molecules involved in the regulation of the vascular bed formation remain unclear. Syndecan-4 is a type I transmembrane glycoprotein commonly expressed in the human body; its receptor has been reported as critical for optimal cell adhesion and initiation of intracellular signaling, indicating its promising application in vascular bed formation. In the current study, bioinformatics analysis and in vitro experiments were performed to evaluate whether syndecan-4 promoted endothelial cell migration and functional activation. Exogenous syndecan-4-overexpressing endothelial cells were perfused into the decellularized liver scaffold, which was assessed by Masson’s trichrome staining. Western blotting and qRT-PCR were used to evaluate the effects of syndecan-4 on the thrombospondin 1 (THBS1) stability. We found that syndecan-4 promoted the adhesion of vascular endothelial cells and facilitated cell migration and angiogenesis. Furthermore, syndecan-4 overexpression resulted in a well-aligned endothelium on the decellularized liver scaffolds. Mechanistically, syndecan-4 destabilized THBS1 at the protein level. Therefore, our data revealed that syndecan-4 promoted the biological activity of endothelial cells on the bionic liver vascular bed through THBS1. These findings provide scientific evidences for solving transient blood coagulation after bionic liver transplantation.

• Syndecan-4
• thrombospondin-1
• tissue engineered liver
• vascular bed formation

• Decellularization
• HepG2 cells
• Porcine liver
• Sodium deoxycholate

• Animals
• Extracellular Matrix
• Humans
• Liver
• Perfusion
• Swine
• Tissue Engineering/methods
• Tissue Scaffolds

• Alginate hydrogel
• Dentin matrix
• Dentin regeneration
• Injectable scaffold
• Pulp capping

• Collagen IV
• Extracellular matrix
• Integrin
• Laminin
• Neurite outgrowth
• Optic nerve

• Acute liver failure
• Coagulation
• Intracranial hypertension
• Liver transplantation

The incidence of liver disease is increasing significantly worldwide and, as a result, there is a pressing need to develop new technologies and applications for end-stage liver diseases. For many of them, orthotopic liver transplantation is the only viable therapeutic option. Stem cells that are capable of differentiating into all liver cell types and could closely mimic human liver disease are extremely valuable for disease modeling, tissue regeneration and repair, and for drug metabolism studies to develop novel therapeutic treatments. Despite the extensive research efforts, positive results from rodent models have not translated meaningfully into realistic preclinical models and therapies. The common marmoset Callithrix jacchus has emerged as a viable non-human primate model to study various human diseases because of its distinct features and close physiologic, genetic and metabolic similarities to humans. C. jacchus embryonic stem cells (cjESC) and recently generated cjESC-derived hepatocyte-like cells (cjESC-HLCs) could fill the gaps in disease modeling, liver regeneration and metabolic studies. They are extremely useful for cell therapy to regenerate and repair damaged liver tissues in vivo as they could efficiently engraft into the liver parenchyma. For in vitro studies, they would be advantageous for drug design and metabolism in developing novel drugs and cell-based therapies. Specifically, they express both phase I and II metabolic enzymes that share similar substrate specificities, inhibition and induction characteristics, and drug metabolism as their human counterparts. In addition, cjESCs and cjESC-HLCs are advantageous for investigations on emerging research areas, including blastocyst complementation to generate entire livers, and bioengineering of discarded livers to regenerate whole livers for transplantation.

• Callithrix jacchus
• bioengineering
• cell therapy
• cytochrome P450
• drug metabolism
• embryonic stem cell
• hepatocyte
• liver
• liver disease
• marmoset
• regeneration
• transplantation

• Cell transplantation
• Liver transplantation
• Organ transplantation
• Scaffold
• Tissue engineering
• Xenotransplantation

• extracellular matrices
• immune response
• recellularization
• scaffolds
• stem cells
• transplantation

Allogeneic liver transplantation is still deemed the gold standard solution for end-stage organ failure; however, donor organ shortages have led to extended waiting lists for organ transplants. In order to overcome the lack of donors, the development of new therapeutic options is mandatory. In the last several years, organ bioengineering has been extensively explored to provide transplantable tissues or whole organs with the final goal of creating a three-dimensional growth microenvironment mimicking the native structure. It has been frequently reported that an extracellular matrix-based scaffold offers a structural support and important biological molecules that could help cellular proliferation during the recellularization process. The aim of the present review is to underline the recent developments in cell-on-scaffold technology for liver bioengineering, taking into account: (1) biological and synthetic scaffolds; (2) animal and human tissue decellularization; (3) scaffold recellularization; (4) 3D bioprinting; and (5) organoid technology. Future possible clinical applications in regenerative medicine for liver tissue engineering and for drug testing were underlined and dissected.

• extracellular matrix
• liver
• liver bioengineering
• regenerative medicine
• scaffold

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here, we show that decellularized whole porcine livers revascularized with human umbilical endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleen has been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). On heterotopic implantation of the rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for 3 days, followed by significant immune responses directed against the human endothelial cells. A 10-day steroid-based immunosuppression protocol and a splenectomy at time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help overcome the chronic shortage of transplantable human livers.

• decellularization
• liver
• scaffold
• sodium lauryl ether sulfate

The burden of liver diseases continues to grow worldwide, and liver transplantation is the only option for patients with end-stage liver disease. This procedure is limited by critical issues, including the low availability of donor organs; thus, novel therapeutic strategies are greatly needed. Recently, bioengineering approaches using decellularized liver scaffolds have been proposed as a novel strategy to overcome these challenges. The aim of this systematic literature review was to identify the major advances in the field of bioengineering using decellularized liver scaffolds and to identify obstacles and challenges for clinical application. The main findings of the articles and each contribution for technique optimization were highlighted, including the protocols of perfusion and decellularization, duration, demonstration of quality control—scaffold acellularity, matrix composition, and preservation of growth factors—and tissue functionality after recellularization. In previous years, many advances have been made as this technique has evolved from studies in animal models to human livers. As the field develops and this promising technique has become much more feasible, many challenges remain, including the selection of appropriate cell types for recellularization, route of cell administration, cell-seeding protocol, and scalability that must be standardized prior to clinical application.

• Clinical application
• Decellularized cartilage
• Extracellular matrix
• Recellularization

Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold.

Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO₂ (5%) cell incubator at 37 °C.

After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium.

Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.

•

Human iPSC-derived hepatocytes were engrafted in a rat decellularized liver scaffold.

• Artificial liver
• Decellularized liver
• Hepatocyte
• Human iPSC
• Recellularization

• Biocompatibility
• Decellularization
• Liver
• Tissue engineering
• Triton X-100

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.

• ECM
• decellularization
• hepatocytes
• liver
• solubilization

• Bioartificial dialysis devices
• Cell and organ engineering
• Dialysis
• End-stage kidney disease
• Renal transplantation

• Mouse embryonic stem cells
• Neural engineering
• Tissue engineering

• Animals
• Astrocytes/drug effects
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/physiology
• Extracellular Matrix/chemistry
• Hyaluronic Acid/chemistry
• Hydrogels/chemistry
• Interneurons/drug effects
• Mice
• Nerve Regeneration/drug effects
• Rats
• Spinal Cord Injuries/surgery
• Tissue Engineering/methods