Decellularized Liver Scaffolds Research Articles

Proteomic analysis of decellularized mice liver and kidney extracellular matrices

BackgroundThe extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM.ResultsIn this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected.ConclusionThe identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell–matrix interactions and their contribution to a tissue-specific microenvironment.

Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

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.

Generation and metabolomic characterization of functional ductal organoids with biliary tree networks in decellularized liver scaffolds.

Developing functional ductal organoids (FDOs) is essential for liver regenerative medicine. We aimed to construct FDOs with biliary tree networks in rat decellularized liver scaffolds (DLSs) with primary cholangiocytes isolated from mouse bile ducts. The developed FDOs were dynamically characterized by functional assays and metabolomics for bioprocess clarification. FDOs were reconstructed in DLSs retaining native structure and bioactive factors with mouse primary cholangiocytes expressing enriched biomarkers. Morphological assessment showed that biliary tree-like structures gradually formed from day 3 to day 14. The cholangiocytes in FDOs maintained high viability and expressed 11 specific biomarkers. Basal-apical polarity was observed at day 14 with immunostaining for E-cadherin and acetylated α-tubulin. The rhodamine 123 transport assay and active collection of cholyl-lysyl-fluorescein exhibited the specific functions of bile secretion and transportation at day 14 compared to those in monolayer and hydrogel culture systems. The metabolomics analysis with 1075 peak pairs showed that serotonin, as a key molecule of the tryptophan metabolism pathway linked to biliary tree reconstruction, was specifically expressed in FDOs during the whole period of culture. Such FDOs with biliary tree networks and serotonin expression may be applied for disease modeling and drug screening, which paves the way for future clinical therapeutic applications.

Orthotopic Transplantation of Functional Bioengineered Livers in Rats.

Functional bioengineered livers (FBLs) are promising alternatives to orthotopic liver transplantation. However, orthotopic transplantation of FBLs has not yet been reported. This study aimed to perform the orthotopic transplantation of FBLs in rats subjected to complete hepatectomy. FBLs were developed using rat whole decellularized liver scaffolds (DLSs) with human umbilical vein endothelial cells implanted via the portal vein, and human bone marrow mesenchymal stem cells (hBMSCs) and mouse hepatocyte cell line implanted via the bile duct. FBLs were evaluated in terms of endothelial barrier function, biosynthesis, and metabolism and orthotopically transplanted into rats to determine the survival benefit. The FBLs with well-organized vascular structures exhibited endothelial barrier function, with reduced blood cell leakage. The implanted hBMSCs and hepatocyte cell line were well aligned in the parenchyma of the FBLs. The high levels of urea, albumin, and glycogen in the FBLs indicated biosynthesis and metabolism. Orthotopic transplantation of FBLs achieved a survival time of 81.38 ± 4.263 min in rats (n = 8) subjected to complete hepatectomy, whereas control animals (n = 4) died within 30 min (p < 0.001). After transplantation, CD90-positive hBMSCs and the albumin-positive hepatocyte cell line were scattered throughout the parenchyma, and blood cells were limited within the vascular lumen of the FBLs. In contrast, the parenchyma and vessels were filled with blood cells in the control grafts. Thus, orthotopic transplantation of whole DLS-based FBLs can effectively prolong the survival of rats subjected to complete hepatectomy. In summary, this work was the first to perform the orthotopic transplantation of FBLs, with limited survival benefits, which still has important value for the advancement of bioengineered livers.

Repopulation of decellularized organ scaffolds with human pluripotent stem cell-derived pancreatic progenitor cells

Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.

Key Issues and Novel Strategies of Stem Cell Intervention and Decellularized Scaffold Liver Tissue Engineer for End-Stage Liver Disease

End-stage liver disease (ESLD) is ultimation of all kinds of chronic liver diseases. It is highly morbidity and mortality disease and currently no effective treatment is available. Liver transplantation (LTx) is the only way for the treatment. However, the organ source limitation interferes with clinical application of LTx. For the goal of crossing liver source shortage barrier, researches attempt to explore techniques or methods like stem cells and tissue engineering, and recent years sounds developed rapidly and bright prospects, for example, artificial liver, tissue-engineered liver and xenotransplantation etc., in particular adult stem cells like bone marrow-derived mesenchymal stem cells (<SUB>BM</SUB>MSCs) and decellularized liver scaffold for large scale liver tissue engineer seems more eye-catching, and such decellularization / recellularization technology used has become the first strategy to build complex organs. We have previously discussed some points in the <Chinese Journal of Digestive Surgery > about optimizing bioscaffold materials that probably will be benificial for tissue engineer. In the present review, we would like to discuss some new developments based on research status and provide a little new thoughts, hopefully those can help people in this field in order to treat ESLD in the near future.

Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation

Decellularized extracellular matrix scaffold, widely utilized for organ engineering, often undergoes matrix decomposition after transplantation and produces byproducts that cause inflammation, leading to clinical failure. Here we propose a strategy using nano-graphene oxide to modify the biophysical properties of decellularized liver scaffolds. Notably, we demonstrate that scaffolds crosslinked with nano-graphene oxide show high resistance to enzymatic degradation via direct inhibition of matrix metalloproteinase activity and increased mechanical rigidity. We find that M2-like macrophage polarization is promoted within the crosslinked scaffolds, which reduces graft-elicited inflammation. Moreover, we show that low activities of matrix metalloproteinases, attributed to both nano-graphene oxide and tissue inhibitors of metalloproteinases expressed by M2c, can protect the crosslinked scaffolds against in vivo degradation. Lastly, we demonstrate that bioengineered livers fabricated with the crosslinked scaffolds remain functional, thereby effectively regenerating damaged livers after transplantation into liver failure mouse models. Overall, nano-graphene oxide crosslinking prolongs allograft survival and ultimately improves therapeutic effects of bioengineered livers, which offer an alternative for donor organs.

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life‐saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large‐animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte‐ and cholangiocyte‐like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large‐animal model.

Dynamic alterations of metabolites revealed the vascularization progression of bioengineered liver.

Vascularization is a critical but challenging process in developing functional bioengineered livers with the decellularized liver scaffolds (DLSs) and the process is accompanied by cell-specific metabolic alterations. To elucidate the dynamic alterations of metabolites during vascularization, rat DLSs were vascularized with human umbilical vein endothelial cells and liquid chromatography mass spectrometry-based metabolomics was performed on culture supernatants collected at 0, 1, 3, 7, 14, and 21 days. Overall, 1698 peak pairs or metabolites were detected in the culture supernatants, with 309 metabolites being positively identified. The orthogonal partial least-squares discriminant analysis and functional enrichment analysis revealed three phases that could be clearly discriminated, including Phase D1 (cell proliferation and migration), Phase D3D7 (vascular lumen formation), and Phase D14D21 (functional endothelial barrier formation). Seventy-two common differentially abundant metabolites of known identity were detected in these three phases when compared with Day 0. Of these metabolites, a high level of β-Alanine indicated a better degree of vascularization and 14 days of in vitro dynamic culture is required to develop a functionalized vascular structure. These results enriched our understanding of the metabolic mechanism of DLS vascularization and indicated that β-Alanine could function as a potential predictor of the patency of vascularized bioengineered livers.

OS085 - Complete vascular tree reconstruction in rat decellularized liver scaffolds using differential recellularization pressures

OS085 - Complete vascular tree reconstruction in rat decellularized liver scaffolds using differential recellularization pressures

Hepatocyte Differentiation from iPSCs or MSCs in Decellularized Liver Scaffold: Cell–ECM Adhesion, Spatial Distribution, and Hepatocyte Maturation Profile

ABSTRACT Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin–eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell–ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

Re-Endothelialization of Decellularized Liver Scaffolds: A Step for Bioengineered Liver Transplantation.

Bioengineered livers (BELs) are an attractive therapeutic alternative to address the donor organ shortage for liver transplantation. The goal of BELs technology aims at replacement or regeneration of the native human liver. A variety of approaches have been proposed for tissue engineering of transplantable livers; the current review will highlight the decellularization-recellularization approach to BELs. For example, vascular patency and appropriate cell distribution and expansion are critical components in the production of successful BELs. Proper solutions to these components of BELs have challenged its development. Several strategies, such as heparin immobilization, heparin-gelatin, REDV peptide, and anti-CD31 aptamer have been developed to extend the vascular patency of revascularized bioengineered livers (rBELs). Other novel methods have been developed to enhance cell seeding of parenchymal cells and to increase graft functionality during both bench and in vivo perfusion. These enhanced methods have been associated with up to 15 days of survival in large animal (porcine) models of heterotopic transplantation but have not yet permitted extended survival after implantation of BELs in the orthotopic position. This review will highlight both the remaining challenges and the potential for clinical application of functional bioengineered grafts.

Decellularized Whole-Organ Pre-vascularization: A Novel Approach for Organogenesis.

Introduction: Whole-organ decellularization is an attractive approach for three-dimensional (3D) organ engineering. However, progress with this approach is hindered by intra-vascular blood coagulation that occurs after in vivo implantation of the re-cellularized scaffold, resulting in a short-term graft survival. In this study, we explored an alternative approach for 3D organ engineering through an axial pre-vascularization approach and examined its suitability for pancreatic islet transplantation. Methods: Whole livers from male Lewis rats were decellularized through sequential arterial perfusion of detergents. The decellularized liver scaffold was implanted into Lewis rats, and an arteriovenous bundle was passed through the scaffold. At the time of implantation, fresh bone marrow preparation (BM; n = 3), adipose-derived stem cells (ADSCs; n = 4), or HBSS (n = 4) was injected into the scaffold through the portal vein. After 5 weeks, around 2,600 islet equivalents (IEQs) were injected through the portal vein of the scaffold. The recipient rats were rendered diabetic by the injection of 65 mg/kg STZ intravenously 1 week before islet transplantation and were followed up after transplantation by measuring the blood glucose and body weight for 30 days. Intravenous glucose tolerance test was performed in the cured animals, and samples were collected for immunohistochemical (IHC) analyses. Micro-computed tomography (CT) images were obtained from one rat in each group for representation. Results: Two rats in the BM group and one in the ADSC group showed normalization of blood glucose levels, while one rat from each group showed partial correction of blood glucose levels. In contrast, no rats were cured in the HBSS group. Micro-CT showed evidence of sprouting from the arteriovenous bundle inside the scaffold. IHC analyses showed insulin-positive cells in all three groups. The number of von-Willebrand factor-positive cells in the islet region was higher in the BM and ADSC groups than in the HBSS group. The number of 5-bromo-2′-deoxyuridine-positive cells was significantly lower in the BM group than in the other two groups. Conclusions: Despite the limited numbers, the study showed the promising potential of the pre-vascularized whole-organ scaffold as a novel approach for islet transplantation. Both BM- and ADSCs-seeded scaffolds were superior to the acellular scaffold.

Ectopic expansion and vascularization of engineered hepatic tissue based on heparinized acellular liver matrix and mesenchymal stromal cell spheroids

Engineered liver organogenesis is not yet a viable therapeutic option, but ectopic liver histogenesis may be possible. Accumulating evidence has suggested that cell-cell interactions and cell-matrix interactions play an important role in determining the properties of engineered hepatic tissue in vitro and in vivo. In the current study, we utilized heparinized decellularized liver scaffolds and bone marrow mesenchymal stromal cell spheroids to fabricate engineered hepatic tissue, which was subsequently implanted into the omentum of Sprague-Dawley rats with or without liver injury. The survival, liver-specific functions, differentiation level and regenerative potential of the implanted hepatocyte-like cells in this ectopic liver system were evaluated, together with the vascularization status and therapeutic potential of the engineered hepatic tissue. We demonstrated that these hepatic grafts could survive and possess hepatocyte specific function in this ectopic liver system but could also efficiently anastomose with host vascular networks. Furthermore, we found that hepatocyte-like cells within grafts expanded more than 9-fold over the course of 4 weeks in immunocompetent rats with injured livers. Immunostaining revealed that these hepatocyte-like cells could self-organize into cord-like structures in vivo. In addition, these hepatic grafts exhibited therapeutic potential in liver injury induced by CCl4. To our knowledge, this is the first report demonstrating the generation of long-term vascularized hepatic parenchyma at ectopic sites based on decellularized liver scaffolds and stem cells. These results provide an economic and feasible method for engineering hepatic tissue from construction to transplantation. This methodology may be applicable in clinical medicine, especially metabolic liver diseases. Statement of significanceIn this manuscript, we presented an optimized method for the hepatic engineered tissue (HET) from construction to transplantation. The core of this method is utilizing the combination of heparinized decellularized liver scaffolds and stem cell spheroids, which could provide necessary cell-cell and cell-extracellular matrix interactions for HET in vitro and in vivo. We proved that these hepatic grafts could possess hepatocyte specific function and exhibit strong proliferative activity in ectopic liver system, but also able to anastomose with the host vascular networks efficiently and be compatible with the host immune system. This methodology may be possible one day to apply in clinical medicine, especially metabolic liver diseases.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Biocompatibility of a decellularized liver scaffold in studies in vitro

The development of multicomponent threedimensional structures based on decellularized tissue is a perspective alternative for organ transplantation in end-stage liver disease. The technology of rat liver decellularization is presented which consist in sequential perfusion of organ through the portal vein and use of 0.1 % sodium dodecyl sulfate as an active solution. The absence of the cytotoxic effect of decellularized scaffolds on allogeneic splenocytes and multipotent mesenchymal stromal cells was established. The obtained liver scaffolds are biocompatible in cells cultures and correspond criteria for cell carriers.

Identification of tissue sections from decellularized liver scaffolds for repopulation experiments

BackgroundBiological organ engineering is a novel experimental approach to generate functional liver grafts by decellularization and repopulation. Currently, healthy organs of small or large animals and human organs with preexisting liver diseases are used to optimize decellularization and repopulation.However, the effects of morphological changes on allo- and xenogeneic cell-scaffold interactions during repopulation procedure, e.g., using scaffold-sections, are unknown. We present a sequential morphological workflow to identify murine liver scaffold-sections with well-preserved microarchitecture. MethodsNative livers (CONT, n = 9) and livers with experimentally induced pathologies (hepatics steatosis: STEA, n = 7; hepatic fibrosis induced by bile duct ligation: BDL, n = 9; nodular regenerative hyperplasia induced by 90% partial hepatectomy: PH, n = 8) were decellularized using SDS and Triton X-100 to generate cell-free scaffolds. Scaffold-sections were assessed using a sequential morphological workflow consisting of macroscopic, microscopic and morphological evaluation: (1) The scaffold was evaluated by a macroscopic decellularization score. (2) Regions without visible tissue remnants were localized for sampling and histological processing. Subsequent microscopical examination served to identify tissue samples without cell remnants. (3) Only cell-free tissue sections were subjected to detailed liver-specific morphological assessment using a histological and immunohistochemical decellularization score. ResultsDecellularization was feasible in 33 livers, which were subjected to the sequential morphological workflow. In 11 of 33 scaffolds we achieved a good macroscopic decellularization result (CONT: 3 scaffolds; STEA: 3 scaffolds; BDL: 3 scaffolds; PH: 2 scaffolds). The microscopic assessment resulted in the selection of 88 cell-free tissue sections (CONT: 15 sections; STEA: 38 sections; BDL: 30 sections; PH: 5 sections). In 27 of those sections we obtained a good histological decellularization result (CONT: 3 sections; STEA: 6 sections; BDL: 17 sections; PH: 1 section). All experimental groups contained sections with a good immunohistochemical decellularization result (CONT: 6 sections; STEA: 5 sections; BDL: 4 sections; PH: 1 section). DiscussionDecellularization was possible in all experimental groups, irrespectively of the underlying morphological alteration. Furthermore, our proposed sequential morphological workflow was suitable to detect tissue sections with well-preserved hepatic microarchitecture, as needed for further repopulation experiments.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

An InVitro Whole-Organ Liver Engineering for Testing of Genetic Therapies.

SummaryExplosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics.The in vitro whole-organ “Bioreactor grown Artificial Liver Model” (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection.Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system.We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.