Bioengineered humanized livers as better three-dimensional drug testing model system

Figure 1 Schematic study plan. Representation of bioengineering technology to generate humanized livers using acellularization and human HPCs repopulation strategy for the development of three-dimensional platform for drug testing.

Figure 2 Xenogenic liver before and after perfusion acellularization. Macroscopic appearance (optical) of acellularized liver showing removal of hematopoietic, liver parenchyma and non-parenchyma cells while retaining the vascular tree. Histological comparison of native and acellularized liver. The expression and distribution of liver extracellular matrix proteins such as collagen type 1 was seen in the parenchymal space as well as around the blood vessels. Fibronectin staining demonstrated conserved meshwork in sinusoidal spaces and biliary ducts post-acellularization. Laminin staining showed intact vasculature throughout the liver scaffold (magnification: 10 ×).

Figure 3 Characterization of acellularized liver scaffold. A: Ultra-structural characterization of acellularized xenogenic liver showing acellularity in the scaffold with preserved three-dimensional microanatomy of the portal tract surrounded by lobular structures. The parenchymal spaces were found enriched with connective tissue fibres arranged with honeycomb-like structures which are an exceptionally preserved anatomy of connective tissues which structures the hepatocyte-free spaces; B: Methylene blue dye infusion showing intact vasculature post-acellularization; Residual DNA content in liver perfusate (C) and in liver tissues (D) before (black bar) and after acellularization (red bar) (^bP < 0.0001); E: Mixed tensile strength; F: Mixed suture retention strength; G: Mixed comprehensive strength plots showing higher degree of retention of mechanical properties post-acellularization.

Figure 4 Characterization of humanized liver. A: Optical image of humanized liver after 7 d of repopulation with human HPCs into a customized bioreactor; B: SEM image of humanized liver at day 7 showing cellular proliferation and natural arrangements; C: H and E staining of thin micro-section of humanized liver (Magnification: 10 ×); Albumin (D) and G6PC (E) immunofluorescence staining (green) representing the functional activity of repopulated human liver cells (Magnification: 20 ×); F: TUNEL staining of repopulated cells at day 7 (RL/d7) showed significantly high expression as compared with the negative control. SEM: Scanning electron microscopy.

Figure 5 Functional assessment of humanized liver. A and B: Quantification of double stranded DNA (dsDNA) in (A) liver perfusate showing reduction in quantity at day 7 whereas (B) in humanized liver post-repopulation showed reciprocal relationship which was significantly high (^bP < 0.001) at day 7 as compared to day 1 and was almost similar to the fresh liver (FL); C-E: The rate of (C) albumin secretion (D) lactate dehydrogenase release and (E) urea synthesis in humanized livers at different time points showing improved response as compared to the conventional 2D-cultures.

Figure 6 Drug metabolism studies using 3D humanized liver system. A: Schematic representation showing the timeline of humanized liver development and drug metabolism study; B: Bar graphs showing the rate of cellular metabolism of six cytochrome P-450 probe substrates in humanized liver as compared to 2D-culture system (^bP < 0.01, ^dP < 0.001).