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LIVER Publication-derived

High-Fidelity DILI Screening

Source Shinozawa et al. · Cincinnati Children's Hospital / Tokyo Medical and Dental University · 10.1053/j.gastro.2020.10.002

👤 Shinozawa T, Kimura M, Cai Y, Saiki N, Yoneyama Y, Ouchi R, Koike H, Maezawa M, Zhang RR, Dunn A, Takebe T ⏱ 27 days 📋 6 phases 🧫 Human iPSC → Polar Hepatocyte Organoids

Abstract

Shinozawa und Takebe entwickelten eine 384-Well-DILI-Screening-Plattform auf Basis polarer iPSC-Hepatocytenorganoids mit simultaner Viabilitäts-, Cholestase- und Mitochondrientoxizitätsmessung. Validiert mit 238 Referenzarzneimitteln: 88% Sensitivität, 89% Spezifität — übertrifft PHH in Cholestase-Detektion. Bosentan-induzierte Cholestase wurde CYP2C9-genotypabhängig erkannt. Dies ist das direkteste Referenzprotokoll für OrganCore's initialen DILI-Marktansatz.

Cell source
Human iPSC → Polar Hepatocyte Organoids
Application
DILI Prediction

Protocol overview

26 steps across 6 phases

Pluripotent Stem Cell Maintenance Ongoing
  1. 1 Plate PSCs on Laminin-coated Dishes
Foregut Induction Days 1-6
  1. 1 Dissociate and Seed PSCs
  2. 2 Day 1: Activate Endoderm Induction
  3. 3 Day 2: Modify Medium Composition
  4. 4 Day 3: Increase Serum Concentration
  5. 5 Days 4–6: Foregut Specification
  6. 6 Harvest and Cryopreserve Foregut Cells
Human Liver Organoid (HLO) Generation Days 1-18 (from thawed foregut cells)
  1. 1 Thaw and Prepare Foregut Cells
  2. 2 Organoid Formation (Days 1–4)
  3. 3 Liver Specification (Days 5–8)
  4. 4 Harvest Organoids from Matrigel
  5. 5 Hepatocyte Maturation (Days 9–18)
HLO Functional Characterization (Optional) Days 15-27
  1. 1 Quantify Albumin Secretion
  2. 2 Measure Bile Acid Production
  3. 3 Assess Bile Transport Activity with Fluorescent Probe
  4. 4 Immunofluorescent Staining for Structural Validation
High-Throughput Drug Screening (384-Well Format) Days 15-21 (from thawed foregut cells) plus screening days
  1. 1 Prepare HLOs for Floating Culture
  2. 2 Prepare Test Compound Plates
  3. 3 Seed Organoids into Drug-Containing 384-Well Plates
  4. 4 Add Fluorescent Bile Acid Probe
  5. 5 Acquire Multichannel Fluorescence Images
  6. 6 Quantify Cell Viability
Image Analysis and Data Processing Post-imaging
  1. 1 Process Fluorescence Images
  2. 2 Analyze Cell Viability (CellTiter-Glo) Data
  3. 3 Normalize to Vehicle Control
  4. 4 Statistical Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Shinozawa et al.. 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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