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LIVER Publication-derived

Generation of Multicellular 3D Liver Organoids From Induced Pluripotent Stem Cells as a Tool for Modelling Liver Diseases

Source Maepa et al., 2024 · University of Cape Town · 10.21769/bioprotoc.5042

👤 Setjie W. Maepa, Mohlopheni J. Marakalala, Hlumani Ndlovu ⏱ 30 days 📋 13 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of multicellular 3D liver organoids (HLO) composed of hepatocytes, stellate cells, and Kupffer cells from induced pluripotent stem cells. The organoids recapitulate hepatic structure and function and can be used to model liver diseases including steatohepatitis, drug-induced liver injury, and inflammatory responses to drugs and pathogens.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

89 steps across 13 phases

Plate coating with extracellular matrix Day 0
  1. 1 Prepare and dilute Vitronectin solution
  2. 2 Coat plates with Vitronectin
  3. 3 Thaw and prepare Geltrex
  4. 4 Store Geltrex aliquots
hiPSC resuscitation and maintenance Days 0-7 (before differentiation)
  1. 1 Resuscitate cryopreserved hiPSCs
  2. 2 Pellet and resuspend cells
  3. 3 Ensure even cell distribution and incubate
  4. 4 Check cell attachment and daily media changes
  5. 5 Passage cells using EDTA dissociation
  6. 6 Collect dissociated cells and dilute
  7. 7 Alternative passaging using Accutase
  8. 8 Complete Accutase dissociation and prepare for seeding
  9. 9 Count viable cells and prepare for seeding
  10. 10 Seed cells with ROCK inhibitor
  11. 11 Day 0: Replace media without ROCK inhibitor
Definitive endoderm (DE) induction Days 1-3
  1. 1 Assess cell density and prepare for DE induction
  2. 2 Day 1: Add Activin A and BMP-4
  3. 3 Day 2: Replace with KSR-containing media
  4. 4 Day 3: Increase KSR concentration
  5. 5 Day 4: Confirm DE differentiation
Foregut spheroid (FGS) induction Days 4-6
  1. 1 Prepare days 4-6 media
  2. 2 Days 4-6: Daily media changes
  3. 3 Confirm 3D structure formation
HLO formation (enzymatic dissociation method) Days 7-10
  1. 1 Collect floating spheroids and dissociate with Accutase
  2. 2 Centrifuge and prepare cells for Geltrex embedding
  3. 3 Mix cells with Geltrex
  4. 4 Plate cells in Geltrex drops
  5. 5 Allow Geltrex to solidify
  6. 6 Add day 7 media and begin culture
  7. 7 Day 9: Replace with fresh HLO formation media
HLO formation (alternative: mechanical dissociation and cryopreservation) Days 6-10 (optional alternative pathway)
  1. 1 Mechanically dissociate FGS cells
  2. 2 Centrifuge and prepare cryopreservation medium
  3. 3 Slow-freeze and store cryotubes
  4. 4 Thaw and resuscitate FGS cells
  5. 5 Prepare cells for Geltrex embedding
  6. 6 Plate cells in Geltrex and solidify
  7. 7 Add formation medium 2 every two days for four days
Hepatocyte maturation Days 11-25
  1. 1 Day 11: Switch to hepatocyte culture medium (HCM)
  2. 2 Days 11-17: Every 3-day media changes
  3. 3 Day 18: Dissociate Geltrex and passage organoids
  4. 4 Centrifuge and resuspend organoids in Geltrex
  5. 5 Replate organoids in new Geltrex drops
  6. 6 Day 21 onwards: Continue HCM maintenance
  7. 7 Day 21: Confirm organoid health and maturity
Total RNA isolation and RT-qPCR analysis Days 21+ (for maturity assessment)
  1. 1 Isolate organoids from Geltrex
  2. 2 Wash organoids to remove Geltrex
  3. 3 Extract total RNA using TRIzol
  4. 4 Quantify extracted RNA
  5. 5 Prepare first-strand cDNA synthesis mixture
  6. 6 Prepare cDNA synthesis master mix
  7. 7 Combine primer mix and template RNA
  8. 8 Anneal primers and synthesize cDNA
  9. 9 Inactivate reverse transcriptase
  10. 10 Perform qRT-PCR in triplicates
  11. 11 Calculate relative mRNA gene expression
Flow cytometry analysis of organoid cellular composition Days 21+ (after maturation)
  1. 1 Optional: Coat tubes and tips with BSA
  2. 2 Collect organoids from culture wells
  3. 3 First centrifugation and supernatant removal
  4. 4 Second wash with DPBS
  5. 5 Dissociate organoids into single cells
  6. 6 Remove TryLE by washing
  7. 7 Incubate cells with fluorophore-labeled antibodies
  8. 8 Wash cells and prepare for flow cytometry
  9. 9 Transfer to flow cytometry tubes
  10. 10 Acquire samples on flow cytometer
Modeling drug-induced liver injury (ARV and TB drug hepatotoxicity) Day 23 (or maturity)
  1. 1 Prepare organoids for drug treatment
  2. 2 Aspirate supernatant and resuspend in drug mixture
  3. 3 Treat organoids with drugs for 24 hours
  4. 4 Image organoids and collect supernatants
  5. 5 Continue with cell viability and metabolic function assays
Modeling inflammatory liver injury (Schistosome eggs antigen treatment) Day 23-30 (7-day treatment)
  1. 1 Prepare organoids for SEA treatment
  2. 2 Stimulate organoids with Schistosome eggs antigen (SEA)
  3. 3 Monitor organoid morphology
  4. 4 Collect supernatants for cytokine analysis
Cell viability determination using CellTiter-GLO assay Day 23+ (after drug or SEA treatment)
  1. 1 Treat organoids with positive and negative controls
  2. 2 Remove supernatant and add CellTiter-GLO reagent
  3. 3 Induce cell lysis by vigorous mixing
  4. 4 Allow signal stabilization
  5. 5 Read luminescence on plate luminometer
  6. 6 Calculate percent viability
Cytochrome P450 3A4 enzyme activity assay Day 23+ (after drug treatment)
  1. 1 Treat organoids with P450 inducers or inhibitors
  2. 2 Prepare organoids for assay
  3. 3 Add P450-Glo CYP3A4 assay substrate
  4. 4 Incubate to allow P450 metabolism
  5. 5 Prepare for luciferase detection
  6. 6 Add luciferase detection reagent
  7. 7 Incubate for signal stabilization
  8. 8 Read luminescence on plate luminometer
  9. 9 Analyze CYP3A4 enzyme activity

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Maepa et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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