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LIVER Publication-derived

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Source Shrestha et al. · University of North Texas, Department of Biomedical Engineering · 10.1101/2024.03.25.586638;

👤 Sunil Shrestha, Prabha Acharya, Soo-Yeon Kang, Manav Goud Vanga, Vinod Kumar Reddy Lekkala, Jiafeng Liu, Yong Yang, Pranav Joshi, Moo-Yeal Lee ⏱ 33 days 📋 5 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of expandable (Exp-HLOs) and differentiated (Diff-HLOs) human liver organoids from iPSCs, which are bioprinted in a high-throughput pillar/perfusion plate system for dynamic culture and in situ hepatotoxicity assays. The organoids exhibit enhanced maturity and functionality under dynamic culture conditions and can be used to assess drug-induced liver injury (DILI) and liver recovery post-injury.

Cell source
Human iPSC
Application
Drug screening, Disease modeling, Hepatotoxicity testing

Protocol overview

33 steps across 5 phases

iPSC maintenance and differentiation into expandable human liver organoids (Exp-HLOs) Day -6 to Day 15
  1. 1 iPSC maintenance
  2. 2 Prepare iMatrix-511 silk-coated 6-well plates
  3. 3 Harvest and seed iPSCs
  4. 4 Definitive endoderm differentiation (Days 1-3)
  5. 5 Foregut cell differentiation (Days 4-6)
  6. 6 Harvest and encapsulate foregut cells in Matrigel
  7. 7 Matrigel gelation
  8. 8 Culture foregut cells in Exp-HLO expansion medium (Days 7-10)
  9. 9 Culture with retinoic acid (Days 11-14)
  10. 10 Differentiation into Exp-HLOs (Day 15+)
  11. 11 Enzymatic dissociation of Exp-HLOs
  12. 12 Passage or cryopreservation of Exp-HLOs
Differentiation of passaged Exp-HLOs into Diff-HLOs Day -3 to Day 20
  1. 1 Prepare Exp-HLOs in Matrigel for differentiation
  2. 2 Regenerate Exp-HLOs with BMP7 (Days -3 to 0)
  3. 3 Differentiation medium (DM) culture (Days 1-20)
  4. 4 Optional enhancement with oncostatin M (OSM)
Bioprinting and dynamic culture of Diff-HLOs in pillar/perfusion plate Day 0 to Day 20
  1. 1 Prepare dissociated Exp-HLOs in Matrigel suspension
  2. 2 Bioprint cell clumps onto 144PillarPlate
  3. 3 Matrigel gelation on pillar plate
  4. 4 Assembly of pillar plate with perfusion plate
  5. 5 Regeneration of Exp-HLOs on pillar plate (Days 1-6)
  6. 6 Switch to differentiation medium with dynamic culture (Days 7-20)
Drug testing and hepatotoxicity assessment Day 20-23
  1. 1 Prepare serial dilutions of test compounds
  2. 2 Dilute drug solutions in differentiation medium
  3. 3 Replace medium with drug-containing medium in perfusion plate
  4. 4 Drug treatment with dynamic culture
  5. 5 Measure organoid viability
  6. 6 Induce cell lysis and measure luminescence
Assessment of liver recovery post-drug-induced injury Day 23-33
  1. 1 Prepare high-dose drug treatment
  2. 2 Drug treatment at IC50 concentration
  3. 3 Replace with expansion medium for recovery
  4. 4 Dynamic culture for organoid recovery (10 days)
  5. 5 Assess recovery via gene expression and viability

Full SOP

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Attribution

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

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