Development of Human Liver Extracellular Matrix Hydrogel for Three Dimensional Cell Culture and Cell Transplantation

Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications

Exogenous Liposomal ceramide-c6 ammeliorates lipidomic profile, energy homeostasis and anti-oxidant systems in NASH

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation

Increasing the accuracy of proteomic typing by decellularisation of amyloid tissue biopsies

Diagnosis and treatment of systemic amyloidosis depend on accurate identification of the specific amyloid fibril protein forming the tissue deposits. Confirmation of monoclonal immunoglobulin light chain amyloidosis (AL), requiring cytotoxic chemotherapy, and avoidance of such treatment in non-AL amyloidosis, are particularly important. Proteomic analysis characterises amyloid proteins directly. It complements immunohistochemical staining of amyloid to identify fibril proteins and gene sequencing to identify mutations in the fibril precursors. However, proteomics sometimes detects more than one potentially amyloidogenic protein, especially immunoglobulins and transthyretin which are abundant plasma proteins. Ambiguous results are most challenging in the elderly as both AL and transthyretin (ATTR) amyloidosis are usually present in this group. We have lately described a procedure for tissue decellularisation which retains the structure, integrity and composition of amyloid but removes proteins that are not integrated within the deposits. Here we show that use of this procedure before proteomic analysis eliminates ambiguity and improves diagnostic accuracy. SIGNIFICANCE: Unequivocal identification of the protein causing amyloidosis disease is crucial for correct diagnosis and treatment. As a proof of principle, we selected a number of cardiac and fat tissue biopsies from patients with various types of amyloidosis and show that a classical procedure of decellularisation enhances the specificity of the identification of the culprit protein reducing ambiguity and the risk of misdiagnosis

Whole Human liver decellularisation-recellularisation for future liver transplantation and extracorporeal device application

Background and Aims: An estimated 29 million people in the European Union (EU) suffer from a chronic liver condition, with liver transplantation still remaining the only treatment for end-stage hepatic disease. Currently, there are approximately 6700 people awaiting liver transplantation in the EU. Considering that 15- 25% of donated organs are discarded, whole human liver regeneration represents a novel approach to overcome current organ shortages. One possible approach is the use of native extracellular matrix (ECM) as a suitable environment for cells to restore tissue function. Therefore, the aim of this project was to demonstrate, for the first time, the recellularisation of a decellularised whole human liver for future transplantation and extracorporeal device applications. Method: A human liver explant, diagnosed with Crigler–Najjar syndrome, was decellularised using a well- established method, previously characterized for cellular material elimination and preservation of ECM proteins and micro-architecture. Temperature, pH, oxygen and pressure sensors were incorporated into the Harvard Apparatus’ ORCA system, as well as compressed air, O 2 and CO 2 reservoirs. Whole human liver scaffolds (840g) was recellularised by IVC infusion with 2x10 9 HepG2. The liver was maintained in 6 L of complete media with a flow-rate of 400ml/min. The media was changed by replacing 3L of existing media with fresh complete media after 48 hours. The experiment was stopped after 72 hours and the liver was fixed in 4% formaldehyde. The liver was sectioned into 21 parts to investigate repopulation by H&E stating. Albumin secretion was measured using an ELISA kit at 0, 24 and 72 hours. Results: Histological analysis using H&E staining showed that cells have infiltrated all liver segments, excluding segment one. HepG2 cells were seen microscopically to have been migrating from the central vein towards the portal triad, including penetrating into the parenchymal space. Oxygen consumption during the course of three days decreased from 20% to 10%. Additionally, pH was reduced by 0.4. Finally, albumin present in the media increased from 0 ng/ml on day 0, to 200 ng/ml on day 1, to 1500 ng/ml on day 3. Conclusion: This is the first report describing the recellularisation of whole human liver ECM scaffolds with a human hepatocyte cell line. This is a key advance in the development of a bioengineered human liver for future liver transplantation and extracorporeal device applications

Tissue-Specific Human Extracellular Matrix Scaffolds Promote Pancreatic Tumour Progression and Chemotherapy Resistance

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture

Tissue-Specific Human Extracellular Matrix Scaffolds Promote Pancreatic Tumour Progression and Chemotherapy Resistance.

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture