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LIVER Publication-derived

Drug-Induced Liver Injury Organoids

Full SOP with Academic or a plan

Source Soussi et al., 2025 · Cincinnati Children's Hospital Medical Center · 10.1002/advs.202508584

Authors: Fadoua El Abdellaoui Soussi, Michael Brusilovsky, Emma Buck, W Clark Bacon, Sina Dadgar, Aaron Fullerton, Victoria Marsh Durban, Riccardo Barrile, Michael A. Helmrath, Takanori Takebe, Adrian Roth, Magdalena Kasendra Duration: 72 days 15 phases Cell source: Patient-Derived iPSC (HLA-B*57:01)

Abstract

This protocol establishes a scalable, matrix-free human liver organoid (HLO) microarray platform that enables controlled co-culture of iPSC-derived HLOs with autologous CD8+ T cells in a genetically defined, HLA-matched context. The system supports antigen-specific T cell activation and reproduces immune-mediated hepatotoxicity phenotypes, particularly for HLA-B*57:01-restricted flucloxacillin-induced liver injury.

Cell source
Patient-Derived iPSC (HLA-B*57:01)
Application
Disease modeling; Drug screening; Immune-mediated toxicity assessment

Protocol overview

55 steps across 15 phases

Phase 1 · iPSC Culture and Maintenance Ongoing prior to differentiation
  1. a iPSC Expansion on Laminin-coated Dishes
Phase 2 · Posterior Foregut Cell Induction (Days 0-6) Days 0-6
  1. a Seed iPSCs and Initiate Foregut Differentiation
  2. b Day 1-2 Media Change
  3. c Day 2-3 Media Change
  4. d Days 4-6 FGF4/CHIR99021 Culture
Phase 3 · HLO Formation in Gri3D Microarrays (Days 0-4) Days 0-4
  1. a Dissociate and Prepare Foregut Cells for Seeding
  2. b Seed Cells into Gri3D Microcavities
  3. c Add Initial Culture Medium
  4. d Organoid Formation Phase (Days 0-4)
Phase 4 · HLO Specification Phase (Days 4-8) Days 4-8
  1. a Liver Specification Medium Culture
Phase 5 · HLO Maturation Phase (Days 8-23) Days 8-23
  1. a Initiate Liver Maturation
  2. b Daily Media Changes During Maturation
  3. c Maturation Verification (Day 23)
Phase 6 · HLA Typing and PBMC Processing Prior to T cell priming
  1. a Isolate Genomic DNA from PBMCs
  2. b High-Resolution HLA Typing by NGS
  3. c PBMC Isolation by Density Gradient
Phase 7 · Dendritic Cell Differentiation 4 days
  1. a Isolate Monocytes from PBMCs
  2. b Differentiate Monocytes into Immature DCs (Days 0-4)
Phase 8 · Naïve CD8+ T Cell Isolation and Initial Priming Days 0-12
  1. a Isolate Naïve CD8+ T Cells from PBMCs
  2. b Mature and Activate Dendritic Cells
  3. c Load Dendritic Cells with Flucloxacillin or Mock Medium
  4. d Initial T Cell Priming (Days 0-3)
  5. e T Cell Expansion Phase (Days 3-12)
  6. f Second Priming (Re-stimulation) (Days 12-14)
Phase 9 · HLO Pre-treatment with Flucloxacillin 3 days prior to T cell co-culture
  1. a Prepare Flucloxacillin Stock
  2. b Treat Mature HLOs with Flucloxacillin (72 h)
Phase 10 · HLO-T Cell Co-Culture (Immune Challenge) 3 days
  1. a Label CD8+ T Cells with CFSE
  2. b Prepare Co-culture Medium
  3. c Initiate HLO-T Cell Co-Culture at Defined E:T Ratios
  4. d Maintain Co-Culture for 72 hours with Media Changes
  5. e Optional T Cell Receptor (TCR) Co-stimulation
Phase 11 · Assessment of Hepatotoxicity (Single-Drug Studies) 7 days
  1. a Prepare Compound Stock Solutions
  2. b Dose HLO Microarrays with Test Compounds
  3. c Assess Cell Viability by CellTiter-Glo 3D Assay (Day 7)
  4. d Assess Dead Cell Counts by DRAQ7 Staining
  5. e Measure Albumin Secretion by ELISA (Day 7)
  6. f Determine IC50 and Margin of Safety (MOS)
Phase 12 · Immunological Readouts in Co-Cultures 72 hours of co-culture
  1. a Quantify Cell Death in Co-Cultures (DRAQ7 Staining)
  2. b Measure Cytokeratin-18 (CK18) Release as Hepatocyte Death Marker
  3. c Measure TNF-α Secretion
  4. d Measure Granzyme B Secretion
  5. e Assess Albumin Secretion in Co-Culture
Phase 13 · Flow Cytometry Analysis of T Cell Activation Following second priming (day 14)
  1. a Prepare T Cells for Flow Cytometry
  2. b Stain T Cells with Activation Marker Antibodies
  3. c Acquire Data on Flow Cytometer
  4. d Calculate Stimulation Index
Phase 14 · Immunofluorescence Staining and Imaging Following fixation
  1. a Fix HLOs in Paraformaldehyde
  2. b Permeabilize and Block HLOs
  3. c Incubate with Primary Antibodies
  4. d Incubate with Secondary Antibodies and DAPI
  5. e Image Organoids Using High-Content Imaging
Phase 15 · Gene Expression Analysis (RT-qPCR) Following RNA extraction
  1. a Extract and Purify RNA from Organoids
  2. b Synthesize cDNA
  3. c Perform qPCR
  4. d Analyze Gene Expression Data

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Attribution

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

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