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LIVER Publication-derived

Antiretroviral Therapy-Induced Liver Injury Organoids

Full SOP with Academic or a plan

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

Authors: Setjie Welcome Maepa, Hlumani Ndlovu Duration: 30 days 10 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Maepa et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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