The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.
An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFβ signaling. This was also supported by the presence and release of higher concentration of endogenous TGFβ1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFβ1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFβ1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFβ-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.
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.SignificanceUnequivocal 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.
Hepatitis B virus (HBV) study is hampered by lacking of idea cell model which support effective HBV infection and meanwhile recapitulate hepatocyte biology function in vivo. In this study, we developed decellularized human liver scaffolds for cell culture and further applied for HBV infection. As a result, primary human hepatocytes (PHHs) engrafted into liver scaffolds and maintained differentiation with stable albumin secretion and liver‐specific gene expression. Comparing to mono‐layer cell culture, scaffold‐based three‐dimensional (3D) culture system significantly augment HBV DNA (including cccDNA), RNA level as well as HBsAg secretion. Moreover, HepG2‐NTCP cells cultured on 3D system exhibited higher infection efficiency and longer infection period in vitro. In addition, HBV DNA level was suppressed when anti‐HBV medicine Entecavir (ETV) introduced into HepG2‐NTCP 3D system. Herein, we evaluated the potential of decellularized human liver scaffold‐based in 3D cell culture and disclosed that scaffold‐based 3D culture system can facilitate HBV infection in vitro. This 3D culture system could be further applied in HBV‐related study. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1744–1753, 2019.
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