In vitro construction of liver organoids with biomimetic lobule structure by a multicellular 3D bioprinting strategy

Abstract

Liver disease is one of the serious threats to human life and health. Three-dimensional (3D) liver models, which simulate the structure and function of natural liver tissue in vitro, have become a common demand in medical, scientific and pharmaceutical fields nowadays. However, the complex cellular composition and multi-scale spatial arrangement of liver tissue make it extremely challenging to construct liver models in vitro. According to HepaRG preference and printing strategy, the formulation of bioink system with opposite charge is optimized. The sodium alginate-based bioink 1 and dipeptide-based bioink 2 are used to ensure structural integrity and provide flexible designability, respectively. The HepaRG/HUVECs/LX-2-laden liver organoids with biomimetic lobule structure are fabricated by a multicellular 3D droplet-based bioprinting strategy, to mimic the cell heterogeneity, spatial structure and extracellular matrix (ECM) features. The liver organoids can maintain structural integrity and multicellular distribution within the printed lobule-like structure after 7 days of culture. Compared with the 2D monolayer culture, the constructed 3D organoids show high cell viability, ALB secretion and urea synthesis levels. This study provides a droplet-based and layer-by-layer 3D bioprinting strategy for in vitro construction of liver organoids with biomimetic lobule structure, giving meaningful insights in the fields of new drugs, disease modelling, and tissue regeneration.

FIGURE 1

Studies on the bioink formulation, determination of the preference of HepaRG cells on the dipeptide bioinks with different sequences. (A) Molecular structure and AFM images of the Fmoc‐dipeptides. (B) Cytocompatibility of dipeptide materials on HepaRG cells after 24 h of incubation. The asterisks in figure indicate the level of significant difference (*p < 0.05, **p < 0.01). (C) Zeta potentials of Fmoc‐dipeptide solutions at a concentration of 0.5 mM. (D) Mechanical strength of 15 mM Fmoc‐YK ^d hydrogel and (E) the mechanical strength enhancement after immersion in DMEM. (F) Zeta potentials of bioinks 1, 2 and the mixture of bioink 1/bioink 2 at a volume ratio of 1:1. Bioink 1 was composed of 0.5% sodium alginate (SA), 0.125% hyaluronic acid (HA) and 0.5 mg/mL Arg‐Gly‐Asp (RGD). Bioink 2 was consisted of 10 mM Fmoc‐YK ^d and 50 mM calcium chloride. The bioink samples were diluted for 20‐fold prior to the zeta potential determination. (G) Amplitude and (H) frequency sweeps on the hydrogel prepared by mixing bioinks 1 and 2 at a volume ratio of 1:1.

FIGURE 2

(A) Schematic diagram of in vitro construction of liver organoids with biomimetic lobular structure by a multicellular 3D bioprinting strategy. (B) Programming of 3D droplet‐based bioprinting for the in vitro liver model fabrication. (C) Multicellular composition of the constructed liver organoid: HUVECs‐RFP, LX‐2‐BFP, and HepaRG‐GFP. (D) Photos of the printing process. (E) Optical image of 3D constructed liver organoid with hydrogel scaffold. (F) Confocal image of the hexagonal central region in the constructed lobule‐like structure after 1 day of culture.

FIGURE 3

(A) Confocal images of the constructed liver organoids by multicellular 3D printing with HepaRG, LX‐2 and HUVECs cells, after 7 days of culture. (B) Cell proliferation determined by the absorbance at 450 nm. The asterisks in figure indicate the level of significant difference (*p < 0.05, **p < 0.01). (C) Live‐dead stain of 2D culture and the constructed liver organoids on days 1 and 7 during culture.

FIGURE 4

Hepatic function evaluations of 2D and 3D models. (A) Immunostaining of ALB (green) and nuclei (bule, DAPI) of cells on Day 7 during culture period. Determination of the total (B) ALB and (C) urea secretions in the supernatant at days 1, 4 and 7 during culture. The asterisks in figure indicate the level of significant difference (*p < 0.05, **p < 0.01).