Fast formation and maturation enhancement of human liver organoids using a liver-organoid-on-a-chip

Abstract

Background: Spatial and functional hepatic zonation, established by the heterogeneous tissue along the portal-central axis of the liver, is important for ensuring optimal liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver function using hepatocytes cultured in the developed systems. However, mimicking the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. Methods: In this study, we established a microphysiological system that operated under continuous flush medium flow, thereby allowing the supply of nutrients and oxygen to liver organoids and the removal of waste and release of cytokines therefrom, similar to the function of blood vessels. Results: The application of microphysiological system to organoid culture was advantageous for reducing the differentiation time and enhancing the functional maturity of human liver organoid. Conclusion: Hence, our microphysiological culture system might open the possibility of the miniaturized liver model system into a single device to enable more rational in vitro assays of liver response.

Keywords: 3D cell culture; hepatic models; liver maturation; liver microenvironment; microphysiological system (MPS).

FIGURE 1

Schematic diagram of the microphysiological system for culturing human liver organoids. (A) Photographs showing examples of the experimental set-up for the MPS. (B) Illustration of the operating principle of hLO formation using the MPS device. (C) The mixture of mechanically dissociated cells was seeded in the MPS and cultured in the expansion medium for 4 days. Then, the cells were treated with BMP7 for an additional 2 days. The medium was changed to the differentiation medium for 5 days. MP,: microphysiological system; GF, growth factors; hLO, human liver organoid; BMP7, bone morphogenetic protein 7.

FIGURE 2

Comparison of the morphologies of human liver organoids cultured in Matrigel droplets or the microphysiological system. (A) Morphologies of the organoids formed on Matrigel droplets at the indicated time points. Bar, 500 μm. (B) Morphologies of the organoids formed on the microfluidic-based organoid culture device at the indicated time points. Bar, 200 μm. EM, expansion medium; BMP7, bone morphogenetic protein 7; DM, differentiation medium.

FIGURE 3

Characterisation of liver tissue-derived organoids maintained in an expansion medium. (A) mRNA expression of key liver progenitor/bipotent markers (EpCAM, SOX9, CD133, and KRT19), as analyzed by qRT-PCR on the last day of expansion. The gene expression levels of the tissue-derived organoids in static or flow cultures were compared with those of human liver tissue. Error bars show the means ± S.D. of biological triplicates. (B, C) Immunofluorescence staining for progenitor/bipotent markers (EpCAM, KRT19, LGR5, and SOX9) in static (B) and flow (C) cultures. The scale bars in (B) and (C) represent 50 μm. EpCAM, epithelial cell adhesion molecule; SOX9, SRY-box transcription factor 9; CD133, prominin 1; KRT19, type 1 cytoskeletal 19 (or keratin 19); LGR5, leucine-rich repeat-containing G-protein coupled receptor 5.

FIGURE 4

Characterization of the differentiation of tissue-derived organoids to hepatocytes. (A) mRNA expression of progenitor and hepatocyte markers (EpCAM, SOX9, CD133, KRT19, albumin, and CYP3A4) analyzed by qRT-PCR on the last day of differentiation. The expression levels of these genes in the tissue-derived organoids in the static or flow cultures were compared with those of human liver tissue. The error bars show the means ± S.D. of biological triplicates. Whole-mount immunofluorescence staining of MRP2, albumin, ZO-1, and HNF4α following differentiation under either static (B) or flow conditions (C), followed by tissue clearing. The scale bars in (B, C) represent 100 μm. EpCAM, epithelial cell adhesion molecule; SOX9, SRY-box transcription factor 9; CD133, prominin 1; KRT19, type 1 cytoskeletal 19 (or keratin 19); CYP3A4, cytochrome P450 family 3 subfamily A member 4; MRP2, multidrug resistance-associated protein 2; ZO-1, zona occludens protein 1; HNF4α, hepatocyte nuclear factor 4-alpha.

FIGURE 5

Functional characterization of hepatocyte-like cells differentiated from tissue-derived organoids. (A) Albumin concentrations in the supernatant were measured by ELISA (ng/ml/10³ cells) on the last day of differentiation. The data are expressed as the mean ± S.D. from triplicate experiments. (B) Representative histograms of the A1AT expression after hepatic differentiation of organoids under the static (grey) or flow (pink). (C) Relative basal expression of CYP1A2, 2A6, 2E1, and 3A7 in hLOs shown as genes among samples. The unpaired Student’s t-test was used to determine the statistical significance of the data (****P < 0.001). (D) Heat map of various bile acid classes. EM, expansion medium; DM, differentiation medium.