The advent of tissue engineering and biomedical techniques has significantly advanced the development of three-dimensional (3D) cell culture systems, particularly tumor organoids. These self-assembled 3D cell clusters closely replicate the histopathological, genetic, and phenotypic characteristics of primary tissues, making them invaluable tools in cancer research and drug screening.

This review addresses the challenges in developing in vitro models that accurately reflect tumor heterogeneity and explores the application of tumor organoids in cancer research, with a specific focus on the screening of natural products for antitumor therapies.

This review synthesizes information from major databases, including Chemical Abstracts, Medicinal and Aromatic Plants Abstracts, ScienceDirect, Google Scholar, Scopus, PubMed and Springer Link. Publications were selected without date restrictions, using terms such as 'organoid', 'natural product', 'pharmacological', 'extract', 'nanomaterial' and 'traditional uses'. Articles related to agriculture, ecology, synthetic work or published in languages other than English were excluded.

The review identifies key challenges related to the efficiency and variability of organoid generation and discusses ongoing efforts to enhance their predictive capabilities in drug screening and personalized medicine. Recent studies utilizing patient-derived organoid models for natural compound screening are highlighted, demonstrating the potential of these models in developing new classes of anticancer agents. The integration of natural products with patient-derived organoid models presents a promising approach for discovering novel anticancer compounds and elucidating their mechanisms of action.

Culture platforms that closely mimic the spatial architecture, cellular diversity, and extracellular matrix composition of native tissues can serve as invaluable tools for a range of scientific discovery and biomedical applications. Organoids have emerged as a promising alternative to both traditional 2D cell culture and animal models, offering a physiologically relevant 3D culture system for studying human cell biology. Organoids provide a manipulable platform to investigate organ development and function as well as to model patient-specific phenotypes. This mini review examines various methods used for culturing organoids to model normal and disease conditions in gastrointestinal tissues. We focus on how the matrix composition and media formulations can impact cell signaling, altering the baseline cellular phenotypes as well as response to perturbations. We discuss future directions for optimizing organoid culture conditions to improve basic and translational potential.

As an emerging technology, organoids are promising new tools for basic and translational research in disease. Currently, the culture of organoids relies mainly on a type of unknown composition scaffold, namely Matrigel, which may pose problems in studying the effect of mechanical properties on organoids. Hydrogels, a new material with adjustable mechanical properties, can adapt to current studies. In this review, we summarized the synthesis of recent advance in developing definite hydrogel scaffolds for organoid culture and identified the critical parameters for regulating mechanical properties. In addition, classified by different mechanical properties like stiffness and viscoelasticity, we concluded the effect of mechanical properties on the development of organoids and tumor organoids. We hope this review enhances the understanding of the development of organoids by hydrogels and provides more practical approaches to investigating them.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

Hepatic organoid-based modelling, through the elucidation of a range of in vivo biological processes and the recreation of the intricate liver microenvironment, is yielding groundbreaking insights into the pathophysiology and personalized medicine approaches for liver diseases.

This study was designed to analyse the global scientific output of hepatic organoid research and assess current achievements and future trends through bibliometric analysis.

Articles were retrieved from the Web of Science Core Collection, and CiteSpace 6.3.R1 was employed to analyse the literature, including outputs, journals, and countries, among others.

Between 2010 and 2024, a total of 991 articles pertaining to hepatic organoid research were published. The journal Hepatology published the greatest number of papers, and journals with an impact factor greater than 10 constituted 60% of the top 10 journals. The United States and Utrecht University were identified as the most prolific country and institution, respectively. Clevers H emerged as the most prolific author, whereas Huch M had the highest number of cocitations, suggesting that both are ideal candidates for academic collaboration. Research on hepatic organoids has exhibited a progressive shift in focus, evolving from initial investigations into model building, differentiation research in stem cells, bile ducts, and progenitor cells, to a broader spectrum encompassing lipid metabolism, single-cell RNA sequencing, and therapeutic applications. The phrases exhibiting citation bursts from 2022 to 2024 include "drug resistance", "disease model", and "patient-derived tumor organoids".

Research on hepatic organoids has increased over the past decade and is expected to continue to grow. Key research areas include applications for liver diseases and drug development. Future trends likely to gain focus include patient-derived tumour organoids, disease modelling, and personalized medicine.

The biochemical complexity of a material determines the biological response of cells triggered by a cell-material interaction. The degree in which this complexity influences basic cell-material interactions such as cell adhesion, spreading, and mechanotransduction is not entirely clear. To this end, we compared three different hydrogel systems, ranging from completely natural to synthetic, in their ability to induce mechanotransduction in kidney epithelial cells (HK-2). A natural hydrogel system was developed based on a decellularized kidney extracellular matrix (dECM). Supramolecular ureido-pyrimidinone (UPy)-glycinamide molecules, with self-associative behavior, were used for a hybrid and complete synthetic system. A hybrid system was engineered by co-assembling this monovalent UPy molecule with a hyaluronic acid, functionalized with ∼7 UPy-groups (UPy-HA), into a transient network. A similar approach was used for the synthetic hydrogel system, in which the multivalent UPy-HA was replaced with a bivalent UPy-PEG molecule with bioinert properties. Both hybrid and synthetic hydrogel systems were more mechanically tunable compared to the dECM hydrogel. The higher bulk stiffness in combination with the introduction of collagen type I mimicking UPy-additives allowed these materials to induce more nuclear yes-associated protein translocation in HK-2 cells compared to the biochemically complex dECM hydrogel. This demonstrated that minimal biochemical complexity is sufficient for inducing mechanotransduction.

Peripheral nerve tissue engineering is a field that uses cells, growth factors and biological scaffold material to provide a nutritional and physical support in the repair of nerve injuries. The specific properties of injectable human amniotic membrane-derived hydrogel including growth factors as well as anti-inflammatory and neuroprotective agents make it an ideal tool for nerve tissue repair, and metformin may also aid in nerve regeneration. The aim of this study was to investigate the effects of hydrogel derived from amniotic membrane (AM) along with metformin (MET) administration in the repair of sciatic nerve injury in male rats. We randomly divided 60 male rats into five groups. A control and four sciatic nerve compression groups including model; hydrogel; metformin and mix which received hydrogel and metformin. The recovery rate was assessed by Sciatic Functional Index (SFI), Static Sciatic Index (SSI) and von-frey test. Conduction velocity of the sciatic nerve was measured by Electrophysiological studies, and histological evaluations were performed 14 days after injury. SFI, SSI, latency time, remyelination rate and the expression of NF-200 and S-100β improved in hydrogel group. Response to mechanical stimulus, myelin density, axonal regeneration, and myelin sheath reconstruction improved in the mix group. The gastrocnemius muscle index was significantly reduced in the experimental groups while collagen fibers increased in these groups. These findings suggest that injection of hydrogel derived from decellularized amniotic membrane into the epineurium can be promoted reconstruction of peripheral nerve injury and improved functional nerve recovery. Also, metformin administration can reinforce the therapeutic effect of the hydrogel.

In vitro drug screening endeavors to replicate cellular states closely resembling those encountered in vivo, thereby maximizing the fidelity of drug effects and responses within the body. Decellularized extracellular matrix (dECM)-based materials offer a more authentic milieu for crafting disease models, faithfully emulating the extracellular components and structural complexities encountered by cells in vivo. This review discusses recent advancements in leveraging dECM-based materials as biomaterials for crafting cell models tailored for drug screening. Initially, we delineate the biological functionalities of diverse ECM components, shedding light on their potential influences on disease model construction. Further, we elucidate the decellularization techniques and methodologies for fabricating cell models utilizing dECM substrates. Then, the article delves into the research strides made in employing dECM-based models for drug screening across a spectrum of ailments, including tumors, as well as heart, liver, lung, and bone diseases. Finally, the review summarizes the bottlenecks, hurdles, and promising research trajectories associated with the dECM materials for drug screening, alongside their prospective applications in personalized medicine. Together, by encapsulating the contemporary research landscape surrounding dECM materials in cell model construction and drug screening, this review underscores the vast potential of dECM materials in drug assessment and personalized therapy.

The incorporation of silver nanoparticles (AgNPs) into alginate-gelatin (Alg-Gel) hydrogels can enhance the properties of these materials for bone regeneration applications, due to the antimicrobial properties of AgNPs and non-cytotoxic concentrations, osteoinductive properties, and regulation of stem cell proliferation and differentiation. Here, the hydrogel formulation included 2% (w/v) sodium alginate, 4 µg/mL AgNPs, and 2.5% (w/v) gelatin. AgNPs were synthesized using a 2% (w/v) aqueous extract of roasted green tea with silver nitrate. The aqueous extract of roasted green tea for AgNP synthesis was characterized using HPLC and UHPLC-ESI-QTOF-MS/MS, and antioxidant capacity was measured in Trolox equivalents (TE) from 4 to 20 nmol/well concentrations. Stem cells from human exfoliated deciduous tooth cells were used for differentiation assays including positive (SHEDs/hydrogel with AgNPs) and negative controls (hydrogel without AgNPs). FTIR was used for hydrogel chemical characterization. Statistical analysis (p < 0.05, ANOVA) confirmed significant findings. Roasted green tea extract contained caffeine (most abundant), (-)-Gallocatechin, gallic acid, and various catechins. XRD analysis revealed FCC structure, TEM showed quasispheroidal AgNPs (19.85 ± 3 nm), and UV-Vis indicated a plasmon surface of 418 nm. This integration of nanotechnology and biomaterials shows promise for addressing bone tissue loss in clinical and surgical settings.

Over the past decade, organoids have emerged as a prevalent and promising research tool, mirroring the physiological architecture of the human body. However, as the field advances, the traditional use of animal or tumor-derived extracellular matrix (ECM) as scaffolds has become increasingly inadequate. This shift has led to a focus on developing synthetic scaffolds, particularly hydrogels, that more accurately mimic three-dimensional (3D) tissue structures and dynamics in vitro. The ECM-cell interaction is crucial for organoid growth, necessitating hydrogels that meet organoid-specific requirements through modifiable physical and compositional properties. Advanced composite hydrogels have been engineered to more effectively replicate in vivo conditions, offering a more accurate representation of human organs compared to traditional matrices. This review explores the evolution and current uses of decellularized ECM scaffolds, emphasizing the application of decellularized ECM hydrogels in organoid culture. It also explores the fabrication of composite hydrogels and the prospects for their future use in organoid systems.

Stem cell-derived β-cells (SC-BCs) represent a potential source for curing diabetes. To date, in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation, indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here, we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards β-cells in vitro. To this aim, a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most β-cell markers, but did not improve β-cell identity. In conclusion, the study describes the production of a PanMa hydrogel, which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs.

Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

The incidence of liver diseases is high worldwide. Many factors can cause liver fibrosis, which in turn can lead to liver cirrhosis and even liver cancer. Due to the shortage of donor organs, immunosuppression, and other factors, only a few patients are able to undergo liver transplantation. Therefore, how to construct a bioartificial liver that can be transplanted has become a global research hotspot. With the rapid development of three-dimensional (3D) bioprinting in the field of tissue engineering and regenerative medicine, researchers have tried to use various 3D bioprinting technologies to construct bioartificial livers in vitro. In terms of the choice of bioinks, liver decellularized extracellular matrix (dECM) has many advantages over other materials for cell-laden hydrogel in 3D bioprinting. This review mainly summarizes the acquisition of liver dECM and its application in liver 3D bioprinting as a bioink with respect to availability, printability, and biocompatibility in many aspects and puts forward the current challenges and prospects.

The importance of decellularized extracellular matrix (dECM) as a natural biomaterial in tissue engineering and regenerative medicine is rapidly growing. The core objective of the decellularization process is to eliminate cellular components while maximizing the preservation of the ECM's primary structure and components. Establishing a rapid, effective, and minimally destructive decellularization technique is essential for obtaining high-quality dECM to construct regenerative organs. This study focused on human umbilical cord tissue, designing different reagent combinations for decellularization protocols while maintaining a consistent processing time. The impact of these protocols on the decellularization efficiency of human umbilical cord tissue was evaluated. The results suggested that the composite decellularization strategy utilizing trypsin/EDTA + Triton X-100 + sodium deoxycholate was the optimal approach in this study for preparing decellularized human umbilical cord dECM. After 5 h of decellularization treatment, most cellular components were eliminated, confirmed through dsDNA quantitative detection, hematoxylin and eosin (HE) staining, and DAPI staining. Meanwhile, Masson staining, periodic acid-silver methenamine (PASM) staining, periodic acid-Schiff (PAS) staining, and immunofluorescent tissue section staining results revealed that the decellularized scaffold retained extracellular matrix components, including collagen and glycosaminoglycans (GAGs). Compared to native umbilical cord tissue, electron microscopy results demonstrated that the microstructure of the extracellular matrix was well preserved after decellularization. Furthermore, Fourier-transform infrared spectroscopy (FTIR) findings indicated that the decellularization process successfully retained the main functional group structures of extracellular matrix (ECM) components. The quantitative analysis of collagen, elastin, and GAG content validated the advantages of this decellularization process in preserving and purifying ECM components. Additionally, it was confirmed that this decellularized matrix exhibited no cytotoxicity in vitro. This study achieved short-term decellularization preparation for umbilical cord tissue through a combined decellularization strategy.

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid-lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte-like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three-dimensional (3D) approach using Biomimesys®, a hyaluronic acid-based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28-day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low-density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC-derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a).

Organ and tissue dysfunction represents a clinically significant condition. By integrating cell biology with materials science, tissue engineering enables the reconstruction and restoration of damaged tissues or organs, offering a noninvasive repair approach. In our study, we replicated the cellular growth environment by utilizing a human umbilical cord-derived decellularized extracellular matrix (dECM) as a modifying agent for the polyethylene terephthalate (PET) polymeric fiber scaffold. This allowed us to create a dECM-coated polyester fiber-based scaffold, PET-dECM, tailored for liver tissue engineering purposes. We effectively produced a decellularized human umbilical cord-derived ECM through a combined decellularization process involving trypsin/EDTA, TritonX-100, and sodium deoxycholate. The application of the dECM coating onto the PET material was accomplished through several steps, such as ester hydrolysis, EDC/NHS-activated crosslinking, and dECM conjugation. The biological performance of the PET-dECM was validated using RG cell culture assays. Notably, the dECM coating significantly improved PET's hydrophilicity and biocompatibility, thereby aiding cell adhesion, proliferation, and functional differentiation (p < 0.05). It was further found that the hepatocyte function of HepaRG was significantly enhanced on the PET-dECM, which may be attributed to the dECM's ability to facilitate the restoration of cell polarity. The PET-dECM holds promise as an effective hepatocyte culture carrier and could potentially find application in liver tissue engineering.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 μm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Cervical cancer, primarily squamous cell carcinoma, is the most prevalent gynecologic malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment. This limitation compromises the accurate representation of tumor heterogeneity. We collected para-cancerous cervical tissues from patients diagnosed with cervical squamous cell carcinoma (CSCC) and prepared uterine cervix extracellular matrix (UCEM) hydrogels. Proteomic analysis of UCEM identified several tissue-specific signaling pathways including human papillomavirus, phosphatidylinositol 3-kinase-AKT, and extracellular matrix receptor. Secreted proteins like FLNA, MYH9, HSPA8, and EEF1A1 were present, indicating UCEM successfully maintained cervical proteins. UCEM provided a tailored microenvironment for CSCC organoids, enabling formation and growth while preserving tumorigenic potential. RNA sequencing showed UCEM-organoids exhibited greater similarity to native CSCC and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways including virus protein-cytokine, nuclear factor κB, tumor necrosis factor, and oncogenes EGR1, FPR1, and IFI6. Moreover, UCEM-organoids developed chemotherapy resistance. Our research provides insights into advanced organoid technology through native matrix hydrogels.

The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.

Non-alcoholic fatty liver disease (NAFLD) is a high-prevalence and progressive disorder. Due to lack of reliable in vitro models to recapitulate the consecutive phases, the exact pathogenesis mechanism of this disease and approved therapeutic medications have not been revealed yet. It has been proven that the interplay between multiple hepatic cell types and liver extracellular matrix (ECM) are critical in NAFLD initiation and progression. Herein, a liver microtissue (LMT) consisting of Huh-7, THP-1, and LX-2 cell lines and human umbilical vein endothelial cells (HUVEC), which could be substituted for the main hepatic cells (hepatocyte, Kupffer, stellate, and sinusoidal endothelium, respectively), encapsulated in liver derived ECM-Alginate composite, was bioengineered. When the microtissues were treated with free fatty acids (FFAs) including Oleic acid (6.6×10-4M) and Palmitic acid (3.3×10-4M), they displayed the key features of NAFLD, including similar pattern of transcripts for genes involved in lipid metabolism, inflammation, insulin-resistance, and fibrosis, as well as pro-inflammatory and pro-fibrotic cytokines' secretions and intracellular lipid accumulation. Continuing FFAs supplementation, we demonstrated that the NAFLD phenomenon was established on day 3 and progressed to the initial fibrosis stage by day 8. Furthermore, this model was stable until day 12 post FFAs withdrawal on day 3. Moreover, administration of an anti-steatotic drug candidate, Liraglutide (15 μM), on the NAFLD microtissues significantly ameliorated the NAFLD phenomenon. Overall, we bioengineered a drug-responsive, cost-benefit liver microtissues which can simulate the initiation and progression of NAFLD. It is expected that this platform could potentially be used for studying molecular pathogenesis of NAFLD and high-throughput drug screening. See also the graphical abstract(Fig. 1).

Recent breakthroughs in developing human-relevant organotypic models led to the building of highly resemblant tissue constructs that hold immense potential for transplantation, drug screening, and disease modeling. Despite the progress in fine-tuning stem cell multilineage differentiation in highly controlled spatiotemporal conditions and hosting microenvironments, 3D models still experience naive and incomplete morphogenesis. In particular, existing systems and induction protocols fail to maintain stem cell long-term potency, induce high tissue-level multicellularity, or drive the maturity of stem cell-derived 3D models to levels seen in their in vivo counterparts. In this review, we highlight the use of extracellular matrix (ECM)-derived biomaterials in providing stem cell niche-mimicking microenvironment capable of preserving stem cell long-term potency and inducing spatial and region-specific differentiation. We also examine the maturation of different 3D models, including organoids, encapsulated in ECM biomaterials and provide looking-forward perspectives on employing ECM biomaterials in building more innovative, transplantable, and functional organs.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

With the increase in weight and age of the population, the consumption of tobacco, inappropriate foods, and the reduction of sports activities in recent years, bone and joint diseases such as osteoarthritis (OA) have become more common in the world. From the past until now, various treatment strategies (e.g., microfracture treatment, Autologous Chondrocyte Implantation (ACI), and Mosaicplasty) have been investigated and studied for the prevention and treatment of this disease. However, these methods face problems such as being invasive, not fully repairing the tissue, and damaging the surrounding tissues. Tissue engineering, including cartilage tissue engineering, is one of the minimally invasive, innovative, and effective methods for the treatment and regeneration of damaged cartilage, which has attracted the attention of scientists in the fields of medicine and biomaterials engineering in the past several years. Hydrogels of different types with diverse properties have become desirable candidates for engineering and treating cartilage tissue. They can cover most of the shortcomings of other treatment methods and cause the least secondary damage to the patient. Besides using hydrogels as an ideal strategy, new drug delivery and treatment methods, such as targeted drug delivery and treatment through mechanical signaling, have been studied as interesting strategies. In this study, we review and discuss various types of hydrogels, biomaterials used for hydrogel manufacturing, cartilage-targeting drug delivery, and mechanosignaling as modern strategies for cartilage treatment.

Digestive system tumors are the leading cause of cancer-related deaths worldwide. Despite ongoing research, our understanding of their mechanisms and treatment remain inadequate. One promising tool for clinical applications is the use of gastrointestinal tract tumor organoids, which serve as an important in vitro model. Tumor organoids exhibit a genotype similar to the patient's tumor and effectively mimic various biological processes, including tissue renewal, stem cell, and ecological niche functions, and tissue response to drugs, mutations, or injury. As such, they are valuable for drug screening, developing novel drugs, assessing patient outcomes, and supporting immunotherapy. In addition, innovative materials and techniques can be used to optimize tumor organoid culture systems. Several applications of digestive system tumor organoids have been described and have shown promising results in related aspects. In this review, we discuss the current progress, limitations, and prospects of this model for digestive system tumors.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition.

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.

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

Rapid advancements in traditional bone tissue engineering have led to innovation in bone repair models and the resolution of insurmountable clinical issues like graft scarcity. The pathophysiological process of treating bone disease, however, is a multidimensional and multimodal regenerative regulatory mechanism that includes numerous immune, inflammatory, or metabolic responses related to the graft or the organism itself. Based on a 3D in vitro cell culture system that is remarkably identical to the body's bone tissue, the bone organoid is a biomimicking bone organ environment. It can accurately mimic the actual repair and regeneration condition in vivo because it shares the same physiological function, structure, morphology, and metabolic process as endogenous bone tissue. As a disruptive regenerative medicine technology, it has wide application prospects in the fields of organ development, gene editing, disease modeling, and precision therapy. Herein, the development process and physiological basis of different cell-based bone organoids are reviewed, the current status of the application of different materials, cells, and construction methods for building bone organoids is described, and the prospects and challenges for the development of bone organoids in future medical fields is discussed.

Opuntia Ficus-indica, or nopal, is traditionally used for its medicinal properties in Mexico. This study aims to decellularize and characterize nopal (Opuntia Ficus-indica) scaffolds, assess their degradation and the proliferation of hDPSC, and determine potential pro-inflammatory effects by assessing the expression of cyclooxygenase 1 and 2 (COX-1 and 2). The scaffolds were decellularized using a 0.5% sodium dodecyl sulfate (SDS) solution and confirmed by color, optical microscopy, and SEM. The degradation rates and mechanical properties of the scaffolds were determined by weight and solution absorbances using trypsin and PBS and tensile strength testing. Human dental pulp stem cells (hDPSCs) primary cells were used for scaffold-cell interaction and proliferation assays, as well as an MTT assay to determine proliferation. Proinflammatory protein expression of COX-I and -II was discovered by Western blot assay, and the cultures were induced into a pro-inflammatory state with interleukin 1-β. The nopal scaffolds exhibited a porous structure with an average pore size of 252 ± 77 μm. The decellularized scaffolds showed a 57% reduction in weight loss during hydrolytic degradation and a 70% reduction during enzymatic degradation. There was no difference in tensile strengths between native and decellularized scaffolds (12.5 ± 1 and 11.8 ± 0.5 MPa). Furthermore, hDPSCs showed a significant increase in cell viability of 95% and 106% at 168 h for native and decellularized scaffolds, respectively. The combination of the scaffold and hDPSCs did not cause an increase in the expression of COX-1 and COX-2 proteins. However, when the combination was exposed to IL-1β, there was an increase in the expression of COX-2. This study demonstrates the potential application of nopal scaffolds in tissue engineering and regenerative medicine or dentistry, owing to their structural characteristics, degradation properties, mechanical properties, ability to induce cell proliferation, and lack of enhancement of pro-inflammatory cytokines.

Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells (iPSCs) technology, solved the complications associated with human embryonic stem cells (hESC) usage. Moreover, iPSCs made significant contributions for human medicine, such as in diagnosis, therapeutic and regenerative medicine. The two-dimensional (2D) models allowed for monolayer cellular culture in vitro; however, they were surpassed by the three-dimensional (3D) cell culture system. The 3D cell culture provides higher cell-cell contact and a multi-layered cell culture, which more closely respects cellular morphology and polarity. It is more tightly able to resemble conditions in vivo and a closer approach to the architecture of human tissues, such as human organoids. Organoids are 3D cellular structures that mimic the architecture and function of native tissues. They are generated in vitro from stem cells or differentiated cells, such as epithelial or neural cells, and are used to study organ development, disease modeling, and drug discovery. Organoids have become a powerful tool for understanding the cellular and molecular mechanisms underlying human physiology, providing new insights into the pathogenesis of cancer, metabolic diseases, and brain disorders. Although organoid technology is up-and-coming, it also has some limitations that require improvements.

Over the past decade, stem cell- and tumor-derived organoids are the most promising models in developmental biology and disease modeling, respectively. The matrix is one of three main elements in the construction of an organoid and the most important module of its extracellular microenvironment. However, the source of the currently available commercial matrix, Matrigel, limits the application of organoids in clinical medicine. It is worth investigating whether the original decellularized extracellular matrix (dECM) can be exploited as the matrix of organoids and improving organoid construction are very important. In this review, tissue decellularization protocols and the characteristics of decellularization methods, the mechanical support and biological cues of extraccellular matrix (ECM), methods for construction of multifunctional dECM and responsive dECM hydrogel, and the potential applications of functional dECM are summarized. In addition, some expectations are provided for dECM as the matrix of organoids in clinical applications.

As one of the most common clinical disorders, low back pain (LBP) influences patient quality of life and causes substantial social and economic burdens. Many factors can result in LBP, the most common of which is intervertebral disc degeneration (IDD). The progression of IDD cannot be alleviated by conservative or surgical treatments, and gene therapy, growth factor therapy, and cell therapy have their own limitations. Recently, research on the use of hydrogel biomaterials for the treatment of IDD has garnered great interest, and satisfactory treatment results have been achieved. This article describes the classification of hydrogels, the methods of decellularized extracellular matrix (dECM) production and the various types of gel formation. The current research on dECM hydrogels for the treatment of IDD is described in detail in this article. First, an overview of the material sources, decellularization methods, and gel formation methods is given. The focus is on research performed over the last three years, which mainly consists of bovine and porcine NP tissues, while for decellularization methods, combinations of several approaches are primarily used. dECM hydrogels have significantly improved mechanical properties after the polymers are cross-linked. The main effects of these gels include induction of stem cell differentiation to intervertebral disc (IVD) cells, good mechanical properties to restore IVD height after polymer cross-linking, and slow release of exosomes. Finally, the challenges and problems still faced by dECM hydrogels for the treatment of IDD are summarised, and potential solutions are proposed. This paper is the first to summarise the research on dECM hydrogels for the treatment of IDD and aims to provide a theoretical reference for subsequent studies.

Tissue development, wound healing, pathogenesis, regeneration, and homeostasis rely upon coordinated and dynamic spatial and temporal remodeling of extracellular matrix (ECM) molecules. ECM reorganization and normal physiological tissue function, require the establishment and maintenance of biological, chemical, and mechanical feedback mechanisms directed by cell-matrix interactions. To replicate the physical and biological environment provided by the ECM in vivo, methods have been developed to decellularize and solubilize tissues which yield organ and tissue-specific bioactive hydrogels. While these biomaterials retain several important traits of the native ECM, the decellularizing process, and subsequent sterilization, and solubilization result in fragmented, cleaved, or partially denatured macromolecules. The final product has decreased viscosity, moduli, and yield strength, when compared to the source tissue, limiting the compatibility of isolated decellularized ECM (dECM) hydrogels with fabrication methods such as extrusion bioprinting. This review describes the physical and bioactive characteristics of dECM hydrogels and their role as biomaterials for biofabrication. In this work, critical variables when selecting the appropriate tissue source and extraction methods are identified. Common manual and automated fabrication techniques compatible with dECM hydrogels are described and compared. Fabrication and post-manufacturing challenges presented by the dECM hydrogels decreased mechanical and structural stability are discussed as well as circumvention strategies. We further highlight and provide examples of the use of dECM hydrogels in tissue engineering and their role in fabricating complex in vitro 3D microenvironments.

For the purpose of developing a 3D vehicle for the delivery of hepatocytes in cell therapy, the improved system of crosslinker and new gelling agent combinations consisting of glycerophosphate and sodium hydrogen carbonate have been employed to produce injectable, thermoresponsive hydrogels based on chitosan and silk fibroin. Adjusting the polymer-to-gelling agent ratio and utilizing a chemical crosslinker developed hydrogel scaffolds with optimal gelling time and pH. Applying sodium hydrogen carbonate neutralizes chitosan while keeping its thermoresponsive characteristics and decreases glycerophosphate from 60% to 30%. Genipin boosts the mechanical properties of hydrogel without affecting the gel time. Due to their stable microstructure and lower amine availability, genipin-containing materials have a low swelling ratio, around six compared to eight for those without genipin. Hydrogels that are crosslinked degrade about half as fast as those that are not. The slowerr degradation of Silk fibroin compared to chitosan makes it an efficient degradation inhibitor in silk-containing formulations. All of the optimized samples showed less than 5% hemolytic activity, indicating that they lacked hemolytic characteristics. The acceptable cell viability in crosslinked hydrogels ranges from 72% to 91% due to the decreasing total salt concentration, which protects cells from hyperosmolality. The pH of hydrogels and their interstitial pores kept most encapsulated cells alive and functioning for 24 h. Urea levels are higher in the encapsulation condition compared to HepG2 cultivated alone, and this may be due to cell-matrix interactions that boost liver-specific activity. Urea synthesis in genipin crosslinked hydrogels increased dramatically from day 1 (about 4 mg dl^-1) to day 3 (approximately 6 mg dl^-1), suggesting the enormous potential of these hydrogels for cell milieu preparation. All mentioned findings represent that the optimized system may be a promising candidate for liver regeneration.

Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.

Replenishing the retina with retinal pigment epithelial (RPE) cells derived from pluripotent stem cells (PSCs) has great promise for treating retinal degenerative diseases, but it is limited by poor cell survival and integration in vivo. Herein, porcine acellular sclera and uvea extracellular matrix (ECM) and their counterpart hydrogels are developed, and their effects on the biological behavior of human induced pluripotent stem cell (hiPSC)-derived RPE cells (hiPSC-RPE) and embryoid body (hiPSC-EB) differentiation are investigated. Both acellular ECM hydrogels have excellent biocompatibility and suitable biodegradability without evoking an obvious immune response. Most importantly, the decellularized uvea hydrogel-delivered cells' injection remarkably promotes the hiPSC-RPE cells' survival and integration in the subretinal space, rescues the photoreceptor cells' death and retinal gliosis, and restores vision in rats with retinal degeneration for a long duration. In addition, medium supplementation with decellularized uvea peptides promotes hiPSC-EBs onset morphogenesis and neural/retinal differentiation, forming layered retinal organoids. This study demonstrates that ECM hydrogel-delivered hiPSC-RPE cells' injection may be a useful approach for treating retinal degeneration disease, combined with an optimized retinal seeding cells' induction program, which has potential for clinical application.

Organoids are three-dimensional (3D) cell culture systems that simulate the structures and functions of organs, involving applications in disease modeling, drug screening, and cellular developmental biology. The material matrix in organoids can provide a 3D environment for stem cells to differentiate into different cell types and continuously self-renew, thereby realizing the in vitro culture of organs, which has received extensive attention in recent years. However, some challenges still exist in organoids, including low maturity, high heterogeneity, and lack of spatiotemporal regulation. Therefore, in this review, we summarized the culturing protocols and various applications of stem cell-derived organoids and proposed insightful thoughts for engineering stem cells into organoids in view of the current shortcomings, to achieve the further application and clinical translation of stem cells and engineered stem cells in organoid research.

Guided bone regeneration with osteoinductive scaffolds is a competitive edge of tissue engineering due to faster and more consistent healing. In the present study, we developed such composite beads with nanocellulose reinforced alginate hydrogel that carried β-tricalcium phosphate (β-TCP) nano-powder and liver-derived extracellular matrix (ECM) from porcine. Interestingly, it was observed that the beads' group containing ECM-βTCP-alginate-nanocellulose (ETAC) was more biocompatible with higher cellular affinity than the others comprised of βTCP-alginate-nanocellulose (TAC) and alginate-nanocellulose (AC). Cell attachment on ETAC beads was dramatically increased with time. In parallel with in vitro results, ETAC beads produced uniform cortical and cancellous bone in the femur defect model of rabbits within two months. Although the group TAC also produced noticeable bone in the defect site, the healing quality was improved and regeneration was faster after adding ECM. This conclusion was not only confirmed by micro-anatomical analysis but also demonstrated with X-ray microtomography. In addition, the characteristic moldable and injectable properties made ETAC a promising scaffold for clinical applications.

Liver organoids are primary or pluripotent stem cell-derived three-dimensional structures that recapitulate regenerative or ontogenetic processes in vitro towards biomedical applications including disease modelling and diagnostics, drug safety and efficacy prediction, and therapeutic use. The cellular composition and structural organization of liver organoids may vary depending on the goal at hand, and the key challenge in general is to direct their development in a rational and controlled fashion for gaining targeted maturity, reproducibility, and scalability. Such endeavor begins with a detailed understanding of the biological processes in space and time behind hepatogenesis, followed by precise translation of these narrative processes through a bioengineering approach. Here, we discuss advancements in liver organoid technology through the lens of 'narrative engineering' in an attempt to synergize evolving understanding around molecular and cellular landscape governing hepatogenesis with engineering-inspired approaches for organoidgenesis.

Today it is widely accepted that molecular mechanisms triggering cancer initiate with a genetic modification. However, a genetic alteration providing the aberrant clone with a growing advantage over neighboring cells is not sufficient to develop cancer. Currently, tumors are considered a heterogeneous population of cells and an extracellular matrix (ECM) that make up a characteristic microenvironment. Interactions between tumor cells and cancer microenvironment define cancer progression and therapeutic response. To investigate and clarify the role of ECM in the regulation of cancer cell behavior and response to therapy, the decellularization of ECM, a widely used technique in tissue engineering, has been recently employed to develop 3D culture model of disease. In this review, we briefly explore the different components of healthy and pathological ECM and the methods to obtain and characterize the ECM from native bioptic tissue. Finally, we highlight the most relevant applications of ECM in translational cancer research strategies: decellularized ECM, ECM-hydrogel and 3D bioprinting.

Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type.