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LIVER Publication-derived

Liver Models for Drug Screening

Full SOP with Academic or a plan

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

Authors: Shinozawa T, Kimura M, Cai Y, Saiki N, Yoneyama Y, Ouchi R, Koike H, Maezawa M, Zhang RR, Dunn A, Takebe T Duration: 27 days 6 phases Cell source: 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

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

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Attribution

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

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