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LIVER Publication-derived

Liver Organoids for Drug Screening (2025)

Full SOP with Academic or a plan

Source Shin et al., 2025 · Korea Research Institute of Chemical Technology · 10.1038/s41598-025-95071-1

Authors: Dae-Seop Shin, Jung Yoon Yang, Ha Neul Jeong, Seon Ju Mun, Hyunwoo Kim, Myung Jin Son, Myung Ae Bae Duration: 48 days 10 phases Cell source: Human pluripotent stem cells (hPSCs)

Abstract

This protocol describes a method for generating hepatic organoids (HOs) from human pluripotent stem cells and cocultured with hepatic stellate cells (HSCs) and THP-1 macrophages in Matrigel domes to model the cellular microenvironment of human liver. The system evaluates hepatotoxicity through measurement of oxidative stress markers (ROS, GSSG, catalase), proinflammatory cytokines (IL-1β, IL-6, IL-10), and hepatic function markers (ALT, AST, albumin) using radar graph analysis and polygon area quantification for prediction of drug-induced liver injury in early-stage drug development.

Cell source
Human pluripotent stem cells (hPSCs)
Application
Drug-induced liver injury (DILI) prediction and hepatotoxicity evaluation

Protocol overview

39 steps across 10 phases

Phase 1 · Hepatic organoid (HO) culture and preparation Days 1-7
  1. a Prepare and solidify organoid-Matrigel domes
  2. b Submerge domes in hepatic differentiation medium
  3. c Replace medium every 3 days
  4. d Wash organoids to remove Matrigel
  5. e Dissociate and passage organoids
Phase 2 · Preparation of nonparenchymal cells (THP-1 macrophages and HSCs) Days 1-7 (parallel to HO culture)
  1. a Culture THP-1 human macrophages
  2. b Differentiate THP-1 cells to macrophage-like cells
  3. c Recovery of differentiated THP-1 macrophages
  4. d Maintain HSC stellate cells
Phase 3 · Coculture of HOs with nonparenchymal cells Day 7 onwards (after HO passaging)
  1. a Prepare HO single-cell suspension
  2. b Prepare cell mixture for coculture
  3. c Suspend cells in Matrigel and form domes
  4. d Add hepatic medium to coculture domes
Phase 4 · Drug treatment and sample collection Days 3-5 post-seeding
  1. a Prepare test compounds
  2. b First drug addition (9-hour timepoint)
  3. c Collect medium for oxidative stress analysis (9-hour timepoint)
  4. d Second drug addition (48-hour total treatment)
  5. e Final medium collection (48-hour timepoint)
Phase 5 · Cell viability and mitochondrial function assessment Day 2 post-seeding (for viability); Day 3 post-seeding (for mitochondrial function)
  1. a Cell viability assay (CellTiter-Glo 3D)
  2. b Mitochondrial function assessment (Mitochondrial ToxGlo™ Assay)
Phase 6 · Measurement of oxidative stress markers Performed at 9-hour timepoint
  1. a ROS level measurement (DCFDA assay)
  2. b Catalase activity assay
  3. c GSH/GSSG ratio measurement
Phase 7 · Measurement of cytokine levels Performed at 48-hour timepoint
  1. a IL-1β quantification (ELISA)
  2. b IL-6 quantification (ELISA)
  3. c IL-10 quantification (ELISA)
Phase 8 · Measurement of hepatic function markers Performed at 48-hour timepoint
  1. a ALT activity measurement
  2. b AST activity measurement
  3. c Albumin level measurement
Phase 9 · RT-qPCR analysis of gene expression As needed for validation (performed on mono- and cocultured HOs)
  1. a RNA extraction
  2. b RNA quantification
  3. c RT-qPCR setup and execution
  4. d mRNA level analysis
Phase 10 · Data analysis and hepatotoxicity prediction After all measurements are complete
  1. a Normalize factor levels relative to untreated control
  2. b Generate radar graphs
  3. c Calculate relative polygon areas
  4. d Generate comparative bar graphs
  5. e Statistical analysis
  6. f Classify hepatotoxicity severity

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Attribution

This SOP was written by Organthis based on the published method in Shin et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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