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LIVER Publication-derived

Off-the-Shelf Engineered Liver Tissue (ELT) Generation from iPSC-Derived Organoids

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

👤 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 ⏱ 90 days 📋 8 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Raggi et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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