Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

Abstract

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.

Decellularized human liver scaffold‐based three‐dimensional culture system facilitate hepatitis B virus infection

“…16 Briefly, human liver was cut into 125 mm 3 cubes and stored at −80 C. Prior to decellularization, scaffolds were thawed at 37 C followed by repeated agitation with Milli-Q and Reagents Mixture (RM). 16 Briefly, human liver was cut into 125 mm 3 cubes and stored at −80 C. Prior to decellularization, scaffolds were thawed at 37 C followed by repeated agitation with Milli-Q and Reagents Mixture (RM).…”

“…The decellularized human liver scaffolds (hDLS) which not fit for liver transplantation were collected and decellularized as previously described. 16 Briefly, human liver was cut into 125 mm 3 cubes and stored at −80 C. Prior to decellularization, scaffolds were thawed at 37 C followed by repeated agitation with Milli-Q and Reagents Mixture (RM). For the decellularized human liver cirrhotic scaffolds (hDLCS), human cirrhotic liver was collected from patients diagnosed as end stage liver disease (ESLD) and underwent liver transplantation in Children's Hospital of ChongQing Medical University.…”

3D organ models—Revolution in pharmacological research?

“…Organ structures can also be reconstituted by recellularizing scaffolds derived from excised animal organs [31] or non-transplantable human organs [32] or by 3D bioprinting. Detergents are used to remove all cells leaving a dECM composed of collagen fibers and proteinaceous factors facilitating cell growth, differentiation and function after repopulation with human cells [33].…”

Liver tissue engineering: From implantable tissue to whole organ engineering

“…A logical approach to identify the ideal composition of the bio‐ink is to analyze the composition and distribution of ECM proteins in decellularized tissue scaffolds 33, 34, 35. The ability to image, map, and reproduce complex 3D structures composed of biologically relevant ECM proteins would represent a major technical advancement.…”