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, 7 (1), 5534

Rapid Production of Human Liver Scaffolds for Functional Tissue Engineering by High Shear Stress Oscillation-Decellularization


Rapid Production of Human Liver Scaffolds for Functional Tissue Engineering by High Shear Stress Oscillation-Decellularization

Giuseppe Mazza et al. Sci Rep.


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.

Conflict of interest statement

David Hughes and Mohsen Shaeri (employees of CN-Bio Innovations) declare that there are no conflicts of interest. The other authors declare no competing financial interests.


Figure 1
Figure 1
Decellularization of human liver cubes. (a) Macroscopic appearance and histological analysis after decellularization, confirming elimination of nuclear (blue; H&E) and cellular material (yellow; SR) and preservation of collagen (red; SR) and elastin (blue/black; EVG). (b) Macroscopic appearance and histological images of fresh human liver. (c) DNA quantification showing significant elimination of DNA in the decellularized cubes. (d) Comparison of the expression and distribution of several ECM proteins, namely collagen I, collagen III, collagen IV, fibronectin and Laminin, evaluated by immunohistochemistry showing consistency between decellularized cubes (top panel) and fresh samples (bottom panel). Data are expressed as mean ± s.d. ***p < 0.0001. Scale bars, 1 mm macroscopic images (a,b) or 200 μm top panel; a, b or 50 μm bottom panel; a, b or 100 μm (d). Biological replicates (n = 16) are performed for all samples.
Figure 2
Figure 2
Confirmation of preservation of the micro-anatomy, biochemical and biomechanical properties of the ALTCs. SEM imaging of; (a–c), fresh liver samples, and (d–f), decellularized liver cubes showing the preservation of a portal tract (asterisks), collagen fibrils and hepatocyte pockets (octothorpe). Second harmonic generation analysis of fibrillar collagens structure (green) of (g), fresh liver samples with the presence of cells (red), (h), fresh liver samples with the subtraction of cells and (i), decellularized liver cubes. (j,k) Confocal auto-fluorescence microscopy showing preservation of the vascular trees in both samples. (l,m) Pseudo-colour images based on the first six components, highlighting the overall morphology of the tissue samples. (n,o) Raman spectra from different points on the cubes, showing similar spectra pattern to collagen. (p), Biochemical peaks of collagen proteins demonstrating no variability between the 2 decellularized cubes. (q) AFM comparison of tissue stiffness between fresh and decellularized liver cubes. Data are expressed as mean ± s.d. *, p < 0.05. Scale bars, 100 μm (a,d), or 10 μm (b,c,e,f), or 100 μm (g–i). Biological replicates (n = 8) are performed for (a–i), or (n = 3) for q.
Figure 3
Figure 3
Neo-angiogenesis and re-endothelisation of ALTCs. CAM assay of (A), decellularized liver cube, (B), sponge soaked in PBS, and (C), sponge loaded with VEGF. (D) Quantification of observed vessels at 0 and 7 days, showing a significant difference between the ALTCs after 7 days when compared to the negative control (PBS). Recellularization of ALTCs with HUVECs after 7 days, characterized by positive staining with (E–G), H&E, (H–J), CD31 and (K–M), FVIII, confirming the migration and attachment of the endothelial cells to the lumen of the vessels and their functionality. Data are expressed as mean ± sem. *p < 0.05, **P < 0.005. Scale bars, 50 μm (E,G,H,J,K,M), or 200 μm (F,I,L). Biological replicates (n > 5) are performed for all samples.
Figure 4
Figure 4
Biocompatibility of ALTCs with various cell lines. Reseeded HepG2 cells were positive for (a,d), H&E, (b,e), AFP and (c,f), EPCAM staining at both 7 (left panel) and 14 days (right panel). At 7 days the cells appear to have only attached to the outer surface of the ALTCs, however, at 14 days, they were able to migrate and occupy the ALTC’s parenchymal space. (g) SEM analysis confirming cellular attachment of the HepG2s cells to the ALTCs at 14 days.Quantitative comparison of (h), albumin and (i), UGT1A1 mRNA expressions of HepG2 grown on 2D plastic (black bar) and those reseeded on ALTCs (blue bar), show contrasting patterns after 7 and 14 days. Similarly, reseeded LX2 cells stained positive for (j,n), H&E, (k,o), PDGFB-β and (l,p), TFG-β at both 7 and 14 days. In contrast to HepG2, LX2 cells were able to migrate and occupy the ALTCs sinusoidal space at 7 days and were more abundant after 14 days. (p), SEM analysis confirming cellular attachment of the LX2 cells to the ALTCs at 14 days. Quantitative comparison of (q), COL1A1 and (r), LOX mRNA expressions of LX2 cells grown on 2D plastic (black bar) and those reseeded n ALTCs blue bar), confirming significantly different patterns. Data are expressed as mean ± s.e.m *p < 0.05, **p < 0.005, ***p < 0.0001. Scale bars, 50 μm (a–f, j–o), or 10 μm (g,p). Biological replicates (n > 4) are performed for all samples.
Figure 5
Figure 5
Biocompatibility of ALTCs with primary hepatocytes in a dynamic perfusion culture system.: Human primary hepatocytes cultures were established in ATLC using the LiverChip dynamic system for up to 10 days. (a) Schematic representation of ALTCs with cells in the perfusion system. H&E staining showing the attachment of primary hepatocytes in the, (b), core and, (c), outer surface of the ALTC after 10 days in culture. Hepatocytes supernatants were collected every 48 hours for measurement of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), Albumin, and factor IX. Data are expressed as mean ± s.e.m. and analysed via one-way ANOVA with Dunnet’s multiple comparisons test, *p < 0.05, **p < 0.005, ***p < 0.0001. Scale bars, 50 μm. Biological replicates (n = 6) are performed for all samples.

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