3D Bioprinting of an Endothelialized Liver Lobule-like Construct as a Tumor-Scale Drug Screening Platform

Abstract

3D cell culture models replicating the complexity of cell-cell interactions and biomimetic extracellular matrix (ECM) are novel approaches for studying liver cancer, including in vitro drug screening or disease mechanism investigation. Although there have been advancements in the production of 3D liver cancer models to serve as drug screening platforms, recreating the structural architecture and tumor-scale microenvironment of native liver tumors remains a challenge. Here, using the dot extrusion printing (DEP) technology reported in our previous work, we fabricated an endothelialized liver lobule-like construct by printing hepatocyte-laden methacryloyl gelatin (GelMA) hydrogel microbeads and HUVEC-laden gelatin microbeads. DEP technology enables hydrogel microbeads to be produced with precise positioning and adjustable scale, facilitating the construction of liver lobule-like structures. The vascular network was achieved by sacrificing the gelatin microbeads at 37 °C to allow HUVEC proliferation on the surface of the hepatocyte layer. Finally, we used the endothelialized liver lobule-like constructs for anti-cancer drug (Sorafenib) screening, and stronger drug resistance results were obtained when compared to either mono-cultured constructs or hepatocyte spheroids alone. The 3D liver cancer models presented here successfully recreate liver lobule-like morphology, and may have the potential to serve as a liver tumor-scale drug screening platform.

Keywords: 3D bioprinting; GelMA hydrogel; drug screening; liver lobule-like construct.

Figure 1

Fabrication of liver lobule-like constructs. (A) Schematic diagram showing the production of liver lobule-like structures using GelMA hydrogel beads generated by the DEP system. (B) Fabrication steps for liver lobule-like structures and corresponding images. Scale bar: 1 mm. (C) Images showing lobule-like structures with different layers. Scale bar: 1 mm. (D) Construct height of lobule-like structures with different layers. (E) Images showing live/dead analysis of C3A cells before and after printing. Scale bar: 1 mm. (F) Corresponding cell viabilities before and after printing.

Figure 2

(A) Morphology characterization of GelMA hydrogels with concentrations of 6%, 8%, and 10% by SEM. Scale bar: 200 µm. (B) Quantitative analysis of the average pore size in the GelMA hydrogel of 6%, 8%, 10% concentrations. (C) Compressive stress–strain curves of GelMA hydrogels at different concentrations. (D) Compression modulus of hydrogels at three different concentrations of 6%, 8%, 10%.

Figure 3

Liver lobule-like constructs fabrication and culture. (A) Bright-field and F-actin fluorescent images showing liver lobule-like constructs at days 1, 7, and 14. Scale bar: 2 mm. (B) Live/dead analysis of liver lobule-like constructs printed with different GelMA concentrations during 14 days of culturing. Scale bar: 200 µm. (C) Cell viability calculation at days 1, 7, and 14. (D) Cell diameter distributions in the liver lobule-like constructs with different GelMA concentrations after 14 days of cultivation.

Figure 4

Endothelialized liver lobule-like constructs fabrication and culture. (A) Schematic illustrations of printing endothelialized liver lobule-like constructs in four steps. (B) F-actin staining of cells to show cell morphology at day 1 and day 14. Scale bar: 200 µm.

Figure 5

Drug evaluation on co-cultured liver cancer models and mono-cultured liver models. (A) Live/dead images showing different 3D liver cancer models after incubation with different concentrations of Sorafenib. Scale bar: 200 µm. (B) Statistical analysis of cell viabilities in both constructs at different drug concentrations. * p < 0.05 and ** p < 0.01.