• combinatorial detergents
• decellularization
• extracellular matrix
• glycosaminoglycans
• growth factors

• Animals
• Swine
• Tissue Scaffolds/chemistry
• Liver/cytology
• Decellularized Extracellular Matrix/chemistry
• Glycosaminoglycans/metabolism
• Extracellular Matrix/chemistry
• Tissue Engineering/methods
• Detergents
• Transplantation, Heterologous/methods
• Sodium Dodecyl Sulfate
• Deoxycholic Acid
• Humans

• SCTIMST
• 6241 TDFProject
• 09/523(0099)/2020-EMR-I CSIR

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

• SERB Start-up research
• SERB POWER grant

• decellularization
• extracellular matrix
• liver
• scaffold
• tissue engineering

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

• Biliary duct
• Decellularization
• Hepatic vein
• Portal vein
• Tissue engineering

• Decellularized extracellular matrix
• Gelatin
• Sodium alginate
• Three-dimensional bioprinting
• Tumor model

• Perfusion
• decellularization
• recellularization
• vascularized composite allotransplantation

• Decellularized Scaffolds
• Foreskin
• Mesenchymal stem cell

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

Organoid technology holds great promise for regenerative medicine. Recent studies show feasibility for bile duct tissue repair in humans by successfully transplanting cholangiocyte organoids in liver grafts during perfusion. Large-scale expansion of cholangiocytes is essential for extending these regenerative medicine applications. Human cholangiocyte organoids have a high and stable proliferation capacity, making them an attractive source of cholangiocytes. Commercially available basement membrane extract (BME) is used to expand the organoids. BME allows the cells to self-organize into 3D structures and stimulates cell proliferation. However, the use of BME is limiting the clinical applications of the organoids. There is a need for alternative tissue-specific and clinically relevant culture substrates capable of supporting organoid proliferation. Hydrogels prepared from decellularized and solubilized native livers are an attractive alternative for BME. These hydrogels can be used for the culture and expansion of cholangiocyte organoids in a clinically relevant manner. Moreover, the liver-derived hydrogels retain tissue-specific aspects of the extracellular microenvironment. They are composed of a complex mixture of bioactive and biodegradable extracellular matrix (ECM) components and can support the growth of various hepatobiliary cells. In this review, we provide an overview of the clinical potential of native liver ECM-based hydrogels for applications with human cholangiocyte organoids. We discuss the current limitations of BME for the clinical applications of organoids and how native ECM hydrogels can potentially overcome these problems in an effort to unlock the full regenerative clinical potential of the organoids.

• Descellularization
• Gravity Perfusion System
• extracellular matrix

Three-dimensional (3D) culture via micropattern arrays to generate cellular spheroids seems a promising in vitro biomimetic system for liver tissue engineering applications, such as drug screening. Recently, organ-derived decellularized extracellular matrix emerges as arguably the most biomimetic bioink. Herein, decellularized liver matrix (DLM)-derived micropattern array chips were developed to fabricate size-controllable and arrangement-orderly HepG2 spheroids for drug screening. The porcine DLM was obtained by the removal of cellular components and then ground into powder, followed by enzymolysis. DLM as a coating substrate was compared with collagen type I (Col I) and Matrigel in terms of biological performance for enhancing cell adhesion, proliferation, and functions. Subsequently, we used poly(dimethylsiloxane) (PDMS) to adsorb DLM as the bioink to fabricate micropattern array chips. The optimal shape and size of micropattern were determined by evaluating the morphology, viability, and functions of HepG2 3D cellular aggregates. In addition, drug-susceptibility testing (paclitaxel, doxorubicin HCl, and disulfiram) was performed on this novel platform. The DLM provided the tissue-specific microenvironment that provided suitable supports for HepG2 cells, compared to Col I and Matrigel. A circular micropattern with a diameter of 100 μm was the optimal processing parameter to rapidly fabricate large-scale, size-controllable, and arrangement-orderly HepG2 cellular aggregates with 3D spheroid’s shape and high cell viability. Drug screening testing showed that the effect of a drug could be directly demonstrated on-chip by confocal microscopy measuring the viability of spheroids. We provide a novel platform for the large-scale generation of HepG2 spheroids with uniform size and arrangement, thus bringing convenience, reducing error, and increasing reproducibility for a rapid drug discovery by fluorescence quantitative analysis. This methodology may be possible to apply in advancing personalized medicine and drug discovery.

• Wnt/β-catenin pathway
• decellularization
• iPSC
• liver regeneration
• recellularization
• regenerative medicine

• Albumins/metabolism
• Animals
• Cell Differentiation
• Hepatocytes/cytology
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/ultrastructure
• Liver/cytology
• Male
• Rats
• Tissue Scaffolds/chemistry
• Up-Regulation
• Urea/metabolism
• Wnt Signaling Pathway
• alpha-Fetoproteins/metabolism
• beta Catenin/metabolism

• Decellularization
• Extracellular matrix
• Liver engineering
• Organ engineering

• 3D matrices
• 3D printing and bioprinting
• biomechanical cues
• decellularization
• hydrogel
• micro-bioreactor
• microenvironment remodeling
• nanofiber-based scaffolds
• nanomedicine
• tissue engineering

• Animals
• Biomechanical Phenomena
• Bioprinting/methods
• Bioreactors
• Cell Culture Techniques
• Culture Techniques
• Extracellular Matrix/metabolism
• Female
• Humans
• Hydrogels/chemistry
• Male
• Nanofibers
• Nanoparticles
• Ovary/physiology
• Printing, Three-Dimensional
• Regeneration
• Signal Transduction
• Testis/physiology
• Tissue Engineering/trends
• Tissue Scaffolds/chemistry

• 2019-2021 Carraresi Foundation
• 1176436 MiND FoodS Hub

Liver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration.

To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel.

Perfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20.

The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro.

Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

• Organoid
• Primary hepatocytes
• Stem cells
• Liver therapies
• Extracellular matrix
• Drug screening

• Decellularization
• HepG2 cells
• Porcine liver
• Sodium deoxycholate

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

• adipose tissue engineering
• bone tissue engineering
• decellularization
• plant tissues
• tendon tissue engineering

• end-stage liver diseases
• liver bioreactors
• liver regeneration
• liver tissue bioengineering
• regenerative medicine

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.

Liver disease contributes significantly to global disease burden and is associated with rising incidence and escalating costs. It is likely that innovative approaches, arising from the emerging field of liver regenerative medicine, will counter these trends.

Liver regenerative medicine is a rapidly expanding field based on a rich history of basic investigations into the nature of liver structure, physiology, development, regeneration, and function. With a bioengineering perspective, we discuss all major subfields within liver regenerative medicine, focusing on the history, seminal publications, recent progress within these fields, and commercialization efforts. The areas reviewed include fundamental aspects of liver transplantation, liver regeneration, primary hepatocyte cell culture, bioartificial liver, hepatocyte transplantation and liver cell therapies, mouse liver repopulation, adult liver stem cell/progenitor cells, pluripotent stem cells, hepatic microdevices, and decellularized liver grafts.

These studies highlight the creative directions of liver regenerative medicine, the collective efforts of scientists, engineers, and doctors, and the bright outlook for a wide range of approaches and applications which will impact patients with liver disease.

• Adult liver stem cell/progenitor cells
• Bioartificial liver
• Decellularized liver grafts
• Hepatocyte transplantation
• Hepatoxicity and engineered devices
• Liver cell therapies
• Liver regeneration
• Liver transplantation
• Mouse liver repopulation
• Pluripotent stem cells
• Primary hepatocyte cell culture

The accumulated evidence points to the microenvironment as the primary mediator of cellular fate determination. Comprised of parenchymal cells, stromal cells, structural extracellular matrix proteins, and signaling molecules, the microenvironment is a complex and synergistic edifice that varies tissue to tissue. Furthermore, it has become increasingly clear that the microenvironment plays crucial roles in the establishment and progression of diseases such as cardiovascular disease, neurodegeneration, cancer, and ageing. Here we review the historical perspectives on the microenvironment, and how it has directed current explorations in tissue engineering. By thoroughly understanding the role of the microenvironment, we can begin to correctly manipulate it to prevent and cure diseases through regenerative medicine techniques.

The development of novel culture systems that mimic the in vivo microenvironment may be beneficial for inducing the differentiation of stem cells and promoting liver function. In the present study, spheroid cultures and decellularized liver scaffolds (DLSs) were utilized to obtain differentiated hepatocyte-like cells. Mouse bone marrow (BM)-derived mesenchymal stem cells (MSCs) self-aggregated into spheroids under low-attachment conditions and implanted into the DLSs via a negative pressure suction device. The Albp-ZsGreen adenoviral vector was utilized for real-time monitoring of hepatocyte-like cell differentiation. To detect the differentiation stages of the MSCs, immunostaining of hepatocyte markers and functional analysis was performed. Compared with traditional 2D monolayer induction, mouse BM-MSCs spheroids and DLSs in 3D culture generated greater yields of mature, differentiated hepatocytes. In conclusion, this 3D culture system may provide a strategy for generating hepatocyte-like cells for portable liver micro-organs, and aid clinical hepatocyte transplantation and liver tissue engineering.

• DP2 DK102258 NIDDK NIH HHS
• R01 HL130488 NHLBI NIH HHS
• UH2 DK107343 NIDDK NIH HHS
• UH3 DK107343 NIDDK NIH HHS

Genetic constructs with promoters fused to reporter genes for simultaneous monitoring of cellular events have been the focus of attention in recent years. Adenoviral vectors, which have distinctive characteristics, have been used to monitor the differentiation of stem cells in vitro. In the present study, a modified adenoviral vector was constructed, containing a mouse, rat, and human general albumin promoter sequence fused to a ZsGreen reporter gene, and evaluated its efficiency in different cell types. Two hepatocyte cell lines (Hepa1-6 and HepG2), rat primary hepatocytes, rat bone marrow mesenchymal stem cells (BM-MSCs) and rat BM-MSCs-derived hepatocyte-like cells were transduced with this vector, and the transfection efficiency and functional capabilities of the promoter were evaluated by fluorescent microscopy. The results demonstrated efficient expression of ZsGreen in Hepa1-6 cells, HepG2 cells, rat primary hepatocytes, and rat BM-MSCs-derived hepatocyte-like cells, but not in rat BM-MSCs. In conclusion, the current study demonstrates a simple, high-efficiency, general tool for real-time monitoring of the differentiation status of hepatocytes from stem cells in mice, rats, and humans. This tool may be useful for evaluating different protocols to generate functional hepatocytes from stem cells in multiple species.

• adenoviral vector
• albumin promoter
• gene reporter
• hepatocyte differentiation
• stems cells

• Animals
• Extracellular Matrix/physiology
• Extracellular Matrix Proteins/metabolism
• Female
• Male
• Swine
• Tissue Engineering/methods
• Tissue Scaffolds
• Urethra/cytology
• Urethra/physiology

• Alginate
• Chitosan
• Microcapsule
• Tissue Engineering
• Vascularization

• R01 EB012108 NIBIB NIH HHS

• animal model
• disector
• liver surgery
• morphometry
• nucleator
• pig
• stereology
• swine

In the present study, we aimed to determine whether the combination of aggregate culture and decellularized liver scaffolds (DLSs) promoted the hepatic differentiation of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into high yields of mature hepatocytes in vitro. Four culturing methods for differentiation [single cell (2D), spheroids (3D), 2D + DLS and 3D + DLS] were studied. To determine the differentiation stages of the MSCs, RT-qPCR of the hepatocyte genes, immunostaining of hepatocyte markers, and functional analyses were all performed. Compared with the other groups, hepatocyte-like cells which differentiated from BM-MSC spheroids on extracellular matrix (ECM) exhibited more intensive staining of stored glycogen, an elevated level of urea biosynthesis and albumin secretion as well as the higher expression of hepatocyte-specific genes. Our results indicated that DLSs combined with spheroidal aggregate culture may be used as an effective method to facilitate the hepatic maturation of BM-MSCs and may have future applications in stem cell-based liver regenerative medicine.