Skip to content
← Back to browse
LIVER Publication-derived

Development of 3D Multicellular Human Liver Organoids from Human Induced Pluripotent Stem Cells to Model Antiretroviral Therapy Induced Liver Injury

Source Maepa et al., 2025 · University of Cape Town · 10.21769/bioprotoc.5042

👤 Setjie Welcome Maepa, Hlumani Ndlovu ⏱ 30 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of 3D multicellular human liver organoids (HLOs) from induced pluripotent stem cells (iPSCs) containing hepatocytes, Kupffer cells, and hepatic stellate cells. The HLOs are used to model antiretroviral therapy (ART) induced liver injury and to determine molecular mechanisms of drug-induced hepatotoxicity through mass spectrometry-based proteomics analysis.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

36 steps across 10 phases

iPSC Maintenance and Preparation Days -5 to 0
  1. 1 Culture undifferentiated iPSCs
  2. 2 Passage iPSCs for differentiation
  3. 3 Count and seed cells
Definitive Endoderm (DE) Induction Days 1-3
  1. 1 Initiate DE differentiation - Day 1
  2. 2 Continue DE differentiation - Day 2
  3. 3 Complete DE differentiation - Day 3
Foregut Spheroid (FGS) Formation Days 4-6
  1. 1 Switch to foregut induction medium
  2. 2 Maintain foregut spheroid culture
Hepatic Organoid Formation and Maturation Days 7-10 (embedding and RA treatment)
  1. 1 Dissociate FGS and embed in Geltrex
  2. 2 Initiate retinoic acid (RA) treatment
  3. 3 Switch to hepatocyte maturation medium
Hepatic Organoid Maturation and Characterization Days 11-30
  1. 1 Maintain HLO in maturation medium
  2. 2 Harvest HLOs for downstream analysis
  3. 3 Perform stage-specific gene marker validation
Functional Characterization - Flow Cytometry Days 20-30
  1. 1 Dissociate HLOs into single cells
  2. 2 Prepare cell suspension and stain with antibodies
  3. 3 Acquire and analyze flow cytometry data
Functional Characterization - CYP3A4 Enzyme Activity Days 20-30
  1. 1 Plate HLOs for CYP3A4 assay
  2. 2 Overlay with culture medium and pre-incubate
  3. 3 Treat HLOs with CYP3A4 substrate and measure activity
Drug Treatment and Cell Viability Assessment Days 20-30 (24-hour treatment) or Days 20-27 (7-day treatment)
  1. 1 Prepare drug treatment groups
  2. 2 Treat HLOs with drugs
  3. 3 Assess cell viability using CellTiter-GLO 3D assay
Inflammatory Cytokine Quantification Days 20-30 (post-treatment)
  1. 1 Treat HLOs with inflammatory stimuli
  2. 2 Collect culture supernatant
  3. 3 Quantify pro- and anti-inflammatory cytokines by ELISA
Proteomic Analysis by Mass Spectrometry Days 20-27 (7-day drug treatment before harvest)
  1. 1 Plate HLOs for proteomics experiment
  2. 2 Treat HLOs with drugs for 24 hours or 7 days
  3. 3 Harvest HLOs and extract proteins
  4. 4 Quantify protein concentration
  5. 5 Perform HILIC on-bead protein digest
  6. 6 Digest proteins with Trypsin and LysC
  7. 7 Perform liquid chromatography-mass spectrometry (LC-MS) analysis
  8. 8 Analyze MS/MS data using Spectronaut software
  9. 9 Perform statistical analysis and protein quantification in Perseus
  10. 10 Perform functional enrichment analysis

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Maepa 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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence