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LIVER Publication-derived

Hepatotoxicity evaluation method through multiple-factor analysis using human pluripotent stem cell derived hepatic organoids

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

👤 Dae-Seop Shin, Jung Yoon Yang, Ha Neul Jeong, Seon Ju Mun, Hyunwoo Kim, Myung Jin Son, Myung Ae Bae ⏱ 48 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Shin et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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