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LIVER Publication-derived

Validating Well-Functioning Hepatic Organoids for Toxicity Evaluation

Source Choi et al., 2024 · National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety · 10.3390/toxics12050371

👤 Seo Yoon Choi, Tae Hee Kim, Min Jeong Kim, Seon Ju Mun, Tae Sung Kim, Ki Kyung Jung, Il Ung Oh, Jae Ho Oh, Myung Jin Son, Jin Hee Lee ⏱ 10 days 📋 19 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation and functional validation of human iPSC-derived hepatic organoids for drug-induced liver injury (DILI) assessment. The organoids recapitulate key hepatic functions including glycogen storage, albumin and bile acid secretion, and cytochrome P450 activity, enabling reliable toxicity testing of pharmaceutical compounds.

Cell source
Human iPSC
Application
Toxicity evaluation and drug screening

Protocol overview

72 steps across 19 phases

iPSC Derivation and Initial Culture Setup Prior to protocol start
  1. 1 Obtain iPSC source material
Organoid Generation and Embedding Days 0–5
  1. 1 Prepare cells in mature hepatocyte state
  2. 2 Embed detached cells in Matrigel with hepatic medium
  3. 3 Maintain organoids in hepatic medium
Mechanical Passage and Maintenance Every 7 days
  1. 1 Prepare organoids for mechanical passage
  2. 2 Mechanically cut organoids into small pieces
  3. 3 Resuspend cells in fresh Matrigel
Cryopreservation Protocol As needed
  1. 1 Prepare organoids for freezing
  2. 2 Freeze and thaw organoids
  3. 3 Post-thaw recovery
Hepatic Organoid Differentiation Days 0–10 (from start of differentiation)
  1. 1 Initiate differentiation with expansion medium
  2. 2 Switch to differentiation medium
Functional Characterization — Gene Expression Day 8 of differentiation
  1. 1 Isolate total RNA from organoids
  2. 2 Reverse-transcribe RNA to cDNA
  3. 3 Perform quantitative real-time PCR
Immunofluorescence Assay for Protein Localization Day 8 of differentiation
  1. 1 Seed organoids on chamber slides
  2. 2 Fix organoids with paraformaldehyde
  3. 3 Permeabilize and block organoids
  4. 4 Apply primary antibody
  5. 5 Apply secondary antibody and acquire images
Tissue Clearing and Immunostaining Day 8 of differentiation
  1. 1 Prepare and fix tissue samples
  2. 2 Clear tissue using Binaree kit
  3. 3 Permeabilize tissues
  4. 4 Apply primary and secondary antibodies
  5. 5 Stain nuclei and mount samples
Functional Assays — Glycogen Storage Day 8 of differentiation
  1. 1 Fix and prepare organoids for PAS staining
  2. 2 Slice tissue sections
  3. 3 Perform Periodic Acid-Schiff (PAS) staining
  4. 4 Acquire images
Functional Assays — Indocyanine Green Uptake and Release Day 8 of differentiation
  1. 1 Prepare organoids for ICG assay
  2. 2 Incubate organoids with indocyanine green
  3. 3 Image ICG uptake
  4. 4 Assess ICG efflux
  5. 5 Image ICG release
Functional Assays — Bile Canaliculi Polarization Day 8 of differentiation
  1. 1 Prepare organoids for functional polarization assay
  2. 2 Wash and prepare for imaging
  3. 3 Acquire confocal images
Functional Assays — Albumin Secretion Day 6–10 of differentiation (measure secretion rates)
  1. 1 Collect culture supernatant
  2. 2 Measure albumin using ELISA
  3. 3 Measure absorbance and normalize
Functional Assays — Alpha-1 Antitrypsin Secretion Days 6–10 of differentiation (measure secretion rates)
  1. 1 Collect culture supernatant
  2. 2 Measure AAT using ELISA
  3. 3 Measure absorbance and normalize
Functional Assays — Bile Acid Production Day 8 of differentiation
  1. 1 Lyse organoids and prepare lysate
  2. 2 Clarify lysate by centrifugation
  3. 3 Quantify bile acids
  4. 4 Measure and normalize absorbance
Functional Assays — CYP3A4 Enzyme Activity Day 8 of differentiation
  1. 1 Prepare organoids and induce CYP activity
  2. 2 Prepare organoids for enzyme assay
  3. 3 Treat organoids with luminogenic CYP substrate
  4. 4 Measure luciferase signal
  5. 5 Normalize to cell number
Toxicity Testing — Drug Treatment and Viability Assessment Days 0–5 of toxicity test
  1. 1 Differentiate organoids for toxicity test
  2. 2 Seed organoids in 96-well plate
  3. 3 Prepare drug solutions
  4. 4 Treat organoids with test substances
  5. 5 Collect supernatant for albumin analysis
  6. 6 Assess cell viability
  7. 7 Measure absorbance and calculate viability
Toxicity Assessment — TC50 Determination After viability data collection
  1. 1 Generate dose-response curves
  2. 2 Fit curve using log(inhibitor) vs. normalized response equation
  3. 3 Determine TC50 value
Gene Expression Analysis in Drug-Treated Organoids After 5-day drug treatment
  1. 1 Isolate RNA from drug-treated organoids
  2. 2 Prepare RNA library
  3. 3 Quantify and qualify libraries
  4. 4 Perform RNA sequencing
  5. 5 Perform quality control and alignment
  6. 6 Identify differentially expressed genes
  7. 7 Perform gene ontology and pathway analysis
Cell Counting and Normalization As needed for data normalization
  1. 1 Dissociate organoids to single cells
  2. 2 Count viable cells
  3. 3 Calculate viable cell concentration

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Choi et al., 2024. 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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