Development of a high-throughput micropatterned agarose scaffold for consistent and reproducible hPSC-derived liver organoids

Abstract

Liver organoids represent emerging human-relevant in vitro liver models that have a wide range of biomedical applications in basic medical studies and preclinical drug discovery. However, the generation of liver organoids currently relies on the conventional Matrigel dome method, which lacks precise microenvironmental control over organoid growth and results in significant heterogeneity of the formed liver organoids. Here, we demonstrate a novel high-throughput culture method to generate uniform liver organoids from human pluripotent stem cell-derived foregut stem cells in micropatterned agarose scaffold. By using this approach, more than 8000 uniformly-sized liver organoids containing liver parenchyma cells, non-parenchymal cells, and a unique stem cell niche could be efficiently and reproducibly generated in a 48-well plate with a size coefficient of variation significance smaller than that in the Matrigel dome. Additionally, the liver organoids highly expressed liver-specific markers, including ALB, HNF4α, and AFP, and displayed liver functions, such as lipid accumulation, glycogen synthesis, albumin secretion, and urea synthesis. As a proof of concept, we evaluated the acute hepatotoxicity of acetaminophen (APAP) in these organoids and observed APAP-induced liver fibrosis. Overall, we expect that the liver organoids will facilitate wide biomedical applications in hepatotoxicity analysis and liver disease modeling.

The native liver as inspiration to create superior in vitro hepatic models

“…To overcome these problems, Xu et al 200 developed a PEG-based micropatterned cell-adhesion substrate to culture individual liver organoids in a controlled way in order to promote the application of organoids for high-throughput DILI testing resulting in an in vitro model representative for the fetal liver. Jiang et al 201 developed another micropatterned organoid model using microfabricated hexagonal closely packed cavity arrays, with a single organoid in each cavity. In both micropatterned models, human hiPSC were differentiated towards liver organoids.…”

“…In these studies, exposure to APAP induced liver toxicity, and in one case, fibrosis was also induced. 201 These systems could be further improved by increasing complexity via the introduction of microfluidics enabling dynamic culturing conditions. The application of a co-culture (cfr.…”

Engineering Biomimetic Microenvironment for Organoid

“…Jiang et al designed agarose‐based microfabricated hexagonal closely packed cavity arrays (mHCPCAs) for the high‐throughput generation of homogeneous and functional liver organoids (Figure 7i). [ 69 ] By initially differentiating human‐induced hiPSCs into hFSCs and then differentiating hFSCs spheroids into liver organoids (mHCPCAs), more than 8000 uniformly‐sized liver organoids could be efficiently and reproducibly generated in a 48‐well plate (Figure 7j,k). Additionally, the liver organoids not only highly expressed liver‐specific markers including albumin (ALB), hepatocyte nuclear factor 4 alpha (HNF4α), and alpha‐fetoprotein (AFP), but also displayed liver functions, such as lipid accumulation, glycogen synthesis, ALB secretion, and urea synthesis (Figure 7l).…”

“…l) Immunofluorescence staining of liver organoids that were produced in agarose-based mHCPCAs for the biomarkers. Reproduced with permission [69]. Copyright 2023, IOP Publishing.…”

The Long Road to Develop Custom-built Livers: Current Status of 3D Liver Bioprinting

“…This approach allowed for precise control of the organoid microenvironment superior to the conventional Matrigel approach. 89 These recent advancements in optimized organoid composition improve the accuracy of these models as well as the speed at which they can be produced, allowing for high-fidelity, rapid testing crucial to pharmaceutical testing and disease modeling, with the potential for adaptation into regenerative medicine technology.…”