Harvesting decellularized liver extracellular matrix from rodents for 3D scaffold fabrication.

Decellularization is a process to harvest the decellularized extra cellular matrix (dECM) that helps develop 3D scaffolds which mimic the native tissue composition. The decellularized tissues retain the structural and functional properties of the extracellular matrix (ECM) by an efficient decellularization process that retains tissue-specific biochemical and biophysical cues for tissue regeneration. In this study, we report an injection-based decellularization method, without perfusion setup. This study also compares the efficiency of the proposed protocol in the two animal models viz rat and mice. This method harvests rat and mice liver dECM using ethylenediamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS) within 08 h and 02 h respectively and preserved significant amount of ECM proteins. We reported that the harvested mice decellularized extracellular matrix (mdECM) and rat decellularized extracellular matrix (rdECM) had significant reduction in their DNA content (∼97%) and retained structural architecture resembling their native tissue counterparts. The total protein content retained in mdECM was ∼39% while that retained in rdECM was ∼65%. It was also found that the sGAG (sulphated glycosaminoglycan) content showed a no List of Figures.

Strategies for decellularization, re-cellularIzation and crosslinking in liver bioengineering.

Liver transplantation is the only definitive treatment for end-stage liver disease and its availability is restricted by organ donor shortages. The development of liver bioengineering provides the probability to create a functional alternative to reduce the gap in organ demand and supply. Decellularized liver scaffolds have been widely applied in bioengineering because they can mimic the native liver microenvironment and retain extracellular matrix (ECM) components. Multiple approaches including chemical, physical and biological methods have been developed for liver decellularization in current studies, but a full set of unified criteria has not yet been established. Each method has its advantages and drawbacks that influence the microstructure and ligand landscape of decellularized liver scaffolds. Optimizing a decellularization method to eliminate cell material while retaining as much of the ECM intact as possible is therefore important for biological scaffold applications. Furthermore, crosslinking strategies can improve the biological performance of scaffolds, including reinforcing biomechanics, delaying degradation in vivo and reducing immune rejection, which can better promote the integration of re-cellularized scaffolds with host tissue and influence the reconstruction process. In this review, we aim to present the different liver decellularization techniques, the crosslinking methods to improve scaffold characteristics with crosslinking and the preparation of soluble ECM.

A review on the application of chitosan-based polymers in liver tissue engineering.

Chitosan-based polymers have enormous structural tendencies to build bioactive materials with novel characteristics, functions, and various applications, mainly in liver tissue engineering (LTE). The specific physicochemical, biological, mechanical, and biodegradation properties give the effective ways to blend these biopolymers with synthetic and natural polymers to fabricate scaffolds matrixes, sponges, and complexes. A variety of natural and synthetic biomaterials, including chitosan (CS), alginate (Alg), collagen (CN), gelatin (GL), hyaluronic acid (HA), hydroxyapatite (HAp), polyethylene glycol (PEG), polycaprolactone (PCL), poly(lactic-co-glycolic) acid (PGLA), polylactic acid (PLA), and silk fibroin gained considerable attention due to their structure-properties relationship. The incorporation of CS within the polymer matrix results in increased mechanical strength and also imparts biological behavior to the designed PU formulations. The significant and growing interest in the LTE sector, this review aims to be a detailed exploration of CS-based polymers biomaterials for LTE. A brief explanation of the sources and extraction, properties, structure, and scope of CS is described in the introduction. After that, a full overview of the liver, its anatomy, issues, hepatocyte transplantation, LTE, and CS LTE applications are discussed.

3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions.

The cell spheroid technology, which greatly enhances cell-cell interactions, has gained significant attention in the development of in vitro liver models. However, existing cell spheroid technologies still have limitations in improving hepatocyte-extracellular matrix (ECM) interaction, which have a significant impact on hepatic function. In this study, we have developed a novel bioprinting technology for decellularized ECM (dECM)-incorporated hepatocyte spheroids that could enhance both cell-cell and -ECM interactions simultaneously. To provide a biomimetic environment, a porcine liver dECM-based cell bio-ink was developed, and a spheroid printing process using this bio-ink was established. As a result, we precisely printed the dECM-incorporated hepatocyte spheroids with a diameter of approximately 160-220 µm using primary mouse hepatocyte (PMHs). The dECM materials were uniformly distributed within the bio-printed spheroids, and even after more than 2 weeks of culture, the spheroids maintained their spherical shape and high viability. The incorporation of dECM also significantly improved the hepatic function of hepatocyte spheroids. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed approximately 4.3- and 2.5-fold increased levels of albumin and urea secretion, respectively, and a 2.0-fold increase in CYP enzyme activity. These characteristics were also reflected in the hepatic gene expression levels of ALB, HNF4A, CPS1, and others. Furthermore, the dECM-incorporated hepatocyte spheroids exhibited up to a 1.8-fold enhanced drug responsiveness to representative hepatotoxic drugs such as acetaminophen, celecoxib, and amiodarone. Based on these results, it can be concluded that the dECM-incorporated spheroid printing technology has great potential for the development of highly functional in vitro liver tissue models for drug toxicity assessment.

Strategic Replication of the Hepatic Zonation In Vitro Employing a Biomimetic Approach.

The varied functions of the liver are dependent on the metabolic heterogeneity exhibited by the hepatocytes within the liver lobule spanning the porto-central axis. This complex phenomenon plays an important role in maintaining the physiological homeostasis of the liver. Standard in vitro culture models fail to mimic this spatial heterogeneity of hepatocytes, assuming a homogeneous population of cells, which leads to inaccurate translation of results. Here, we demonstrate the development of an in vitro model of hepatic zonation by mimicking the microarchitecture of the liver using a 3D printed mini bioreactor and decellularized liver matrix to provide the native microenvironmental cues. There was a differential expression of hypoxic and metabolic markers across the developed mini bioreactor, showing the establishment of gradients of oxygen, Wnt/ß-catenin pathway, and other metabolic pathways. The model also showed the establishment of zone-dependent toxicity on treatment with acetaminophen. The developed model would thus be a promising avenue in the field of tissue engineering for understanding the liver physiology and pathophysiology and for drug screening to evaluate the potential of new pharmaceutical interventions.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Liver dECM-Gelatin Composite Bioink for Precise 3D Printing of Highly Functional Liver Tissues.

In recent studies, liver decellularized extracellular matrix (dECM)-based bioinks have gained significant attention for their excellent compatibility with hepatocytes. However, their low printability limits the fabrication of highly functional liver tissue. In this study, a new liver dECM-gelatin composite bioink (dECM gBioink) was developed to overcome this limitation. The dECM gBioink was prepared by incorporating a viscous gelatin mixture into the liver dECM material. The novel dECM gBioink showed 2.44 and 10.71 times higher bioprinting resolution and compressive modulus, respectively, than a traditional dECM bioink. In addition, the new bioink enabled stable stacking with 20 or more layers, whereas a structure printed with the traditional dECM bioink collapsed. Moreover, the proposed dECM gBioink exhibited excellent hepatocyte and endothelial cell compatibility. At last, the liver lobule mimetic structure was successfully fabricated with a precisely patterned endothelial cell cord-like pattern and primary hepatocytes using the dECM gBioink. The fabricated lobule structure exhibited excellent hepatic functionalities and dose-dependent responses to hepatotoxic drugs. These results demonstrated that the gelatin mixture can significantly improve the printability and mechanical properties of the liver dECM materials while maintaining good cytocompatibility. This novel liver dECM gBioink with enhanced 3D printability and resolution can be used as an advanced tool for engineering highly functional liver tissues.

Reproducible Preparation of Primary Rat Hepatocyte Sheets Using a Thermoresponsive Culture Dish.

Hepatocyte transplantation has been utilized as a therapy for congenital metabolic liver diseases such as hemophilia and for liver function support in acute liver failure. Hepatocyte sheet technology using a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted dish is expected to provide an efficient cell transplantation method because the resulting hepatocyte sheet possesses extracellular matrix (ECM) on the basal surface, which enhances attachment to the target sites. However, the cultured hepatocytes consume large amounts of oxygen, leading to the loss of a few hepatocytes within the confluent culture sheet owing to a lack of oxygen. To circumvent this problem, this work demonstrates the shortening of diffusion distance, that is, the medium depth, to accelerate oxygen supply from the gas phase/medium interface to the cultured hepatocytes, allowing them to form a monolayer hepatocyte sheet. Incubation of hepatocytes with medium at a depth of 1.3 mm facilitates confluent culture of hepatocytes for 72 h, whereas viable hepatocytes decreased at 2.6 mm depth. Hepatocyte sheets are formed on a 0.5 µg/cm2 fibronectin-physisorbed PIPAAm-grafted dish during 72 h incubation at 37°C. Detachment of the cultured hepatocyte sheet from the PIPAAm-grafted dish where the surface becomes hydrophilic at 20°C is accomplished by scraping the periphery of the sheet using a cell scraper. Furthermore, the apical side of the hepatocyte sheet can be physically grabbed using a gelatin-coated membrane, and the sheet with ECM on the basal surface can be readily transferred to the target site after melting the coated gelatin at 37°C. Both methods are beneficial for creating tissue models by layering with another type of cell sheets, and for quick transplantation, such as into the subcutaneous space and orthotopic transplantation on the surface of the liver.

Mammalian-specific decellularized matrices derived bioink for bioengineering of liver tissue analogues: A review.

The absolute shortage of compatible liver donors and the growing number of potential recipients have led scientists to explore alternative approaches to providing tissue/ organ substitutes from bioengineered sources. Bioartificial regeneration of a fully functional tissue/organ replacement is highly dependent on the right combination of engineering tools, biological principles, and materiobiology horizons. Over the past two decades, remarkable achievements have been made in hepatic tissue engineering by converging various advanced interdisciplinary research approaches. Three-dimensional (3D) bioprinting has arisen as a promising state-of-the-art tool with strong potential to fabricate volumetric liver tissue/organ equivalents using viscosity- and degradation-controlled printable bioinks composed of hydrous microenvironments, and formulations containing living cells and associated supplements. Source of origin, biophysiochemical, or thermomechanical properties and crosslinking reaction kinetics are prerequisites for ideal bioink formulation and realizing the bioprinting process. In this review, we delve into the forecast of the potential future utility of bioprinting technology and the promise of tissue/organ- specific decellularized biomaterials as bioink substrates. Afterward, we outline various methods of decellularization, and the most relevant studies applying decellularized bioinks toward the bioengineering of in vitro liver models. Finally, the challenges and future prospects of decellularized material-based bioprinting in the direction of clinical regenerative medicine are presented to motivate further developments.

A Unification of Nanotopography and Extracellular Matrix in Electrospun Scaffolds for Bioengineered Hepatic Models.

Donor liver shortage is a crucial global public health problem as whole-organ transplantation is the only definitive cure for liver disease. Liver tissue engineering aims to reproduce or restore function through in vitro tissue constructs, which may lead to alternative treatments for active and chronic liver disease. The formulation of a multifunctional scaffold that has the potential to mimic the complex extracellular matrix (ECM) and their influence on cellular behavior, are essential for culturing cells on a construct. The separate employment of topographic or biological cues on a scaffold has both shown influences on hepatocyte survival and growth. In this study, we investigate both of these synergistic effects and developed a new procedure to directly blend whole-organ vascular perfusion-decellularized rat liver ECM (dECM) into electrospun fibers with tailored surface nanotopography. Water contact angle, tensile test, and degradation studies were conducted to analyze scaffold hydrophilicity, mechanical properties, and stability. The results show that our novel hybrid scaffolds have enhanced hydrophilicity, and the nanotopography retained its original form after hydrolytic degradation for 14 days. Human hepatocytes (HepG2) were seeded to analyze the scaffold biocompatibility. Cell viability and DNA quantification imply steady cell proliferation over the culture period, with the highest albumin secretion observed on the hybrid scaffold. Scanning electron microscopy shows that cell morphology was distinctly different on hybrid scaffolds compared to control groups, where HepG2 began to form a monolayer toward the end of the culture period; meanwhile, typical hepatic markers and ECM genes were also influenced, such as an increasing trend of albumin appearing on the hybrid scaffolds. Taken together, our findings provide a reproducible approach and utilization of animal tissue-derived ECM and emphasize the synergism of topographical stimuli and biochemical cues on electrospun scaffolds in liver tissue engineering.

Advances in Recellularization of Decellularized Liver Grafts with Different Liver (Stem) Cells: Towards Clinical Applications.

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.

3D-printed PLA/Gel hybrid in liver tissue engineering: Effects of architecture on biological functions.

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific three-dimensional (3D) structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, poly-lactic acid (PLA) polymer was used as the input of the fused deposition modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-sectional designs were printed. One percent and 2% of gelatin coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. Scanning electron microscopy pictures were used to analyze the structural properties of PLA-Gel hybrid scaffolds, energy dispersive spectroscopy to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ± 0.02-fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.

Polyethylene glycol crosslinked decellularized single liver lobe scaffolds with vascular endothelial growth factor promotes angiogenesis in vivo.

BACKGROUND: Improving the mechanical properties and angiogenesis of acellular scaffolds before transplantation is an important challenge facing the development of acellular liver grafts. The present study aimed to evaluate the cytotoxicity and angiogenesis of polyethylene glycol (PEG) crosslinked decellularized single liver lobe scaffolds (DLSs), and establish its suitability as a graft for long-term liver tissue engineering. METHODS: Using mercaptoacrylate produced by the Michael addition reaction, DLSs were first modified using N-succinimidyl S-acetylthioacetate (SATA), followed by cross-linking with PEG as well as vascular endothelial growth factor (VEGF). The optimal concentration of agents and time of the individual steps were identified in this procedure through biomechanical testing and morphological analysis. Subsequently, human umbilical vein endothelial cells (HUVECs) were seeded on the PEG crosslinked scaffolds to detect the proliferation and viability of cells. The scaffolds were then transplanted into the subcutaneous tissue of Sprague-Dawley rats to evaluate angiogenesis. In addition, the average number of blood vessels was evaluated in the grafts with or without PEG at days 7, 14, and 21 after implantation. RESULTS: The PEG crosslinked DLS maintained their three-dimensional structure and were more translucent after decellularization than native DLS, which presented a denser and more porous network structure. The results for Young's modulus proved that the mechanical properties of 0.5 PEG crosslinked DLS were the best and close to that of native livers. The PEG-VEGF-DLS could better promote cell proliferation and differentiation of HUVECs compared with the groups without PEG cross-linking. Importantly, the average density of blood vessels was higher in the PEG-VEGF-DLS than that in other groups at days 7, 14, and 21 after implantation in vivo. CONCLUSIONS: The PEG crosslinked DLS with VEGF could improve the biomechanical properties of native DLS, and most importantly, their lack of cytotoxicity provides a new route to promote the proliferation of cells in vitro and angiogenesis in vivo in liver tissue engineering.

Recent Advances in Liver Tissue Engineering as an Alternative and Complementary Approach for Liver Transplantation.

Acute and chronic liver diseases cause significant morbidity and mortality worldwide, affecting millions of people. Liver transplantation is the primary intervention method, replacing a non-functional liver with a functional one. However, the field of liver transplantation faces challenges such as donor shortage, postoperative complications, immune rejection, and ethical problems. Consequently, there is an urgent need for alternative therapies that can complement traditional transplantation or serve as an alternative method. In this review, we explore the potential of liver tissue engineering as a supplementary approach to liver transplantation, offering benefits to patients with severe liver dysfunctions.

Efficient hepatic differentiation of hydrogel microsphere-encapsulated human pluripotent stem cells for engineering prevascularized liver tissue.

Liver tissue engineering is promising as an alternative strategy to treat liver failure. However, generating functional hepatocytes from stem cells is conventionally restricted by the immature status of differentiated cells. Besides, embedding hepatocytes in bulk scaffold is limited by a lack of vascularity and low cell-packing density. Here, we fabricate collagen type I (COL1) microspheres for efficient hepatic differentiation of pluripotent stem cells and subsequent assembly of prevascularized liver tissue (PLT). Using a microfluidic platform, we demonstrate that hydrogel COL1 microspheres (mCOL1) encapsulating human embryonic stem cells (hESCs) can be reproducibly generated and efficiently differentiated into hepatocyte-like cells (HLCs) microspheres for the first time. Compared with other culture configurations such as encapsulation of hESC in a bulk COL1 hydrogel and 2D monolayer culture, mCOL1 with high uniformity produce HLC microspheres of improved maturity based on comprehensive analyses of cell morphology, transcriptome profile, hepatic marker expression and hepatic functions. In addition, these HLC microspheres can be applied as building blocks to self-assemble with endothelial cells to construct a dense PLT. The PLT resembles native liver tissue with high cell-packing density, shows successful engraftment in mice liver following implantation, and exhibits improved hepatic functionin vivo. Overall, it is believed that this multiscale technology will advance the fabrication of stem cell-based liver tissue for regenerative medicine, drug screening, andin vitroliver modeling.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Evolution of Electrospinning in Liver Tissue Engineering.

The major goal of liver tissue engineering is to reproduce the phenotype and functions of liver cells, especially primary hepatocytes ex vivo. Several strategies have been explored in the recent past for culturing the liver cells in the most apt environment using biological scaffolds supporting hepatocyte growth and differentiation. Nanofibrous scaffolds have been widely used in the field of tissue engineering for their increased surface-to-volume ratio and increased porosity, and their close resemblance with the native tissue extracellular matrix (ECM) environment. Electrospinning is one of the most preferred techniques to produce nanofiber scaffolds. In the current review, we have discussed the various technical aspects of electrospinning that have been employed for scaffold development for different types of liver cells. We have highlighted the use of synthetic and natural electrospun polymers along with liver ECM in the fabrication of these scaffolds. We have also described novel strategies that include modifications, such as galactosylation, matrix protein incorporation, etc., in the electrospun scaffolds that have evolved to support the long-term growth and viability of the primary hepatocytes.

Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering.

Constructing an engineered hepatic lobule-mimetic model is challenging owing to complicated lobular architecture and crucial hepatic functionality. Our previous study has demonstrated the feasibility of using silk fibroin (SF) scaffolds as functional templates for engineering hepatic lobule-like constructs. But the unsatisfactory chemical and physical performances of the SF-only scaffold and the inherent defect in the functional activity of the carcinoma-derived seeding cells remain to be addressed to satisfy the downstream application demand. In this study, SF-collagen I (SFC) composite scaffolds with improved physical and chemical properties were fabricated, and their utilization for bioengineering a more hepatic lobule-like construct was explored using the immortalized human hepatocyte-derived liver progenitor-like cells (iHepLPCs) and endothelial cells incorporated in the dynamic culture system. The SFC scaffolds prepared through the directional lyophilization process showed radially aligned porous structures with increased swelling ratio and porosity, ameliorative mechanical stiffness that resembled the normal liver matrix more closely, and improved biocompatibility. The iHepLPCs displayed a hepatic plate-like distribution and differentiated into matured hepatocytes with improved hepatic function in vitro and in vivo. Moreover, hepatocyte-endothelial cell interphase arrangement was generated in the co-culture compartment with improved polarity, bile capillary formation, and enhanced liver functions compared with the monocultures. Thus, a more biomimetic hepatic lobule-like model was established and could provide a valuable and robust platform for various applications, including bioartificial liver and drug screening.

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model.

Drug-induced liver injury (DILI) is a leading cause of attrition in drug development or withdrawal; current animal experiments and traditional 2D cell culture systems fail to precisely predict the liver toxicity of drug candidates. Hence, there is an urgent need for an alternative in vitro model that can mimic the liver microenvironments and accurately detect human-specific drug hepatotoxicity. Here, for the first time we propose the fabrication of an albumin methacryloyl cryogel platform inspired by the liver's microarchitecture via emulating the mechanical properties and extracellular matrix (ECM) cues of liver. Engineered crosslinkable albumin methacryloyl is used as a protein-based building block for fabrication of albumin cryogel in vitro models that can have potential applications in 3D cell culture and drug screening. In this work, protein modification, cryogelation, and liver ECM coating were employed to engineer highly porous three-dimensional cryogels with high interconnectivity, liver-like stiffness, and liver ECM as artificial liver constructs. The resulting albumin-based cryogel in vitro model provided improved cell-cell and cell-material interactions and consequently displayed excellent liver functional gene expression, being conducive to detection of fialuridine (FIAU) hepatotoxicity.

Galactose Tethered Decellularized Liver Matrix: Toward a Biomimetic and Biofunctional Matrix for Liver Tissue Engineering.

The major challenge in liver tissue engineering is the replication of the microenvironment and microarchitecture of the liver tissue at the nanoscale. Decellularized liver matrix (DLM) provides an ideal material for scaffold preparation, as it retains the relevant structural and biochemical composition. However, the loss of bioactive factors during decellularization needs to be taken into account when using DLM and should be supplemented accordingly for an expected outcome. This study reports on the modification of DLM by the addition of galactose residues using a two-step thiol-ene-mediated photoclick chemistry for the coupling of galactose moieties to the DLM. Modification with galactose enhanced the function of hepatocytes and provides many advantages over currently used DLM and DLM-based materials. The galactose modified DLM enhanced the initial HepG2 cell adhesion to the substrate with changes in dynamics over time such as spheroid formation and further migration on the matrix. Our observation is that the galactose ligand decoration can also enhance the liver-specific metabolism of HepG2 compared to unmodified DLM. Galactosylated DLM also showed a better establishment of cellular polarity which also contributes to the function of HepG2 cells. Together our results demonstrate the advantages of adding galactose residues to currently available biomaterials, which makes this approach an attractive method for ECM-based liver tissue engineering.