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LIVER Publication-derived

Autologous Organoid-T Cell Co-Culture Platform for Modeling of Immune-Mediated Drug-Induced Liver Injury

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

👤 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 ⏱ 72 days 📋 15 phases 🧫 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

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

Full SOP

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Attribution

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

This wording is awaiting legal review.

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