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LIVER Publication-derived

Acute Liver Failure Organoids

Full SOP with Academic or a plan

Source Raggi et al., 2025 · CHU Sainte-Justine Research Center, Montreal, Canada · 10.1101/2025.10.08.680796

Authors: Claudia Raggi, Pascal Lapierre, Marie-Agnès M'Callum, Quang Toan Pham, Silvia Selleri, Radu Alexandru Paun, Nissan Baratang, Chenicka Lyn Mangahas, Basma Benabdallah, Gaël Moquin-Beaudry, Jiwoon Park, Dorothée Dal Soglio, Yves Théoret, Robert E. Schwartz, Christian Beauséjour, Elie Haddad, Massimiliano Paganelli Duration: 90 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of encapsulated liver tissue (ELT) composed of iPSC-derived liver organoids macroencapsulated in a non-degradable polyethylene glycol-vinyl sulfone (PEG-VS) hydrogel. The ELT achieves functional maturation in vitro and serves as an off-the-shelf allogeneic cell therapy for acute liver failure, providing immediate hepatic function replacement without requiring immunosuppression or vascularization.

Cell source
Human iPSC
Application
Disease modeling; acute liver failure treatment

Protocol overview

43 steps across 8 phases

Phase 1 · iPSC Generation and Maintenance Passages 1-10 and beyond
  1. a iPSC Reprogramming from Donor Cells
  2. b Single Colony Sub-cloning
  3. c Sendai Virus Removal via Temperature Shift
  4. d iPSC Culture Maintenance
Phase 2 · Differentiation into Hepatoblasts Day 0-15
  1. a Cell Dissociation and Seeding
  2. b Endoderm Induction (Days 0-2)
  3. c Continued Endoderm Specification (Days 2-5)
  4. d Hepatic Specification (Days 5-10)
  5. e Hepatoblast Maturation (Days 10-15)
Phase 3 · Differentiation into Mesenchymal Progenitor Cells (MPC) Week 0-Week 4
  1. a Single-Cell Seeding on Laminin-Coated Plates
  2. b MPC Induction (Weeks 0-2)
  3. c Passage to Plastic and Expansion (Weeks 2-6)
Phase 4 · Differentiation into Endothelial Progenitor Cells (EPC) Day 0-Day 8 (plus 1 week culture and sorting)
  1. a Single-Cell Seeding on Laminin-Coated Plates
  2. b Initial Endothelial Induction (Days 0-2)
  3. c Endothelial Specification (Days 2-6)
  4. d Passage to Endothelial Culture Medium
  5. e Fluorescence-Activated Cell Sorting (FACS) for CD31+CD144+ Cells
Phase 5 · Generation of Controlled-Size Liver Organoids Day 0-Day 5
  1. a Prepare Cell Suspension
  2. b Seed Cells into Microcavity Plates
  3. c Organoid Self-Aggregation (Days 0-5)
  4. d Harvest Organoids
Phase 6 · Encapsulation and ELT Generation Day 5 of organoid culture
  1. a Prepare Hydrogel Suspension
  2. b Cast Organoid-Hydrogel Suspension
  3. c UVA Photo-Crosslinking (First Layer)
  4. d Apply Second Thin Layer
  5. e UVA Photo-Crosslinking (Second Layer)
Phase 7 · ELT In Vitro Maturation and Functional Assessment Day 0 (post-encapsulation)–Week 12+
  1. a Culture ELT in Appropriate Medium
  2. b Monitor ELT Viability
  3. c Measure Albumin Secretion
  4. d Measure Alpha-Fetoprotein (AFP) Secretion
  5. e Assess Cytochrome P450 (CYP) Activity
  6. f Measure Urea Synthesis
  7. g Measure Ammonia Clearance
  8. h Gene Expression Analysis
  9. i Assess Cryopreservation Stability
Phase 8 · In Vivo Implantation and ALF Treatment Day 0-Day 90 (post-treatment)
  1. a Acute Liver Failure Induction
  2. b Confirm ALF Establishment (24 hours post-CCl₄)
  3. c ELT or Control Implantation
  4. d Post-Operative Monitoring (Days 0-2)
  5. e Extended Monitoring (Days 2-35)
  6. f Liver Regeneration Assessment (Day 24)
  7. g ELT Explantation (Day 35)
  8. h Post-Explantation Follow-Up (Days 35-90)

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Attribution

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

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