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LIVER Publication-derived

Robust, Long-Term Culture of Endoderm-Derived Hepatic Organoids for Disease Modeling

Source Akbari et al., 2019 · Izmir Biomedicine and Genome Center / Dokuz Eylul University · 10.1016/j.stemcr.2019.08.007

👤 Soheil Akbari, Gülben Gürhani Sevinç, Nevin Ersoy, Onur Basak, Kubra Kaplan, Kenan Sevinç, Erkin Ozel, Berke Sengun, Eray Enustun, Burcu Ozcimen, Alper Bagriyanik, Nur Arslan, Tamer Tevfik Önder, Esra Erdal ⏱ 24 days 📋 6 phases 🧫 Human iPSC, Patient-Derived iPSC (Citrullinemia type 1)

Abstract

This protocol describes the generation and long-term expansion of endoderm-derived hepatic organoids (eHEPOs) from human iPSCs. eHEPOs are produced within 2 weeks using EpCAM-positive endodermal cells as an intermediate and can be expanded for over 16 months without loss of differentiation capacity. The system enables generation of functional hepatocytes and is amenable to genetic manipulation, as demonstrated by disease modeling of citrullinemia type 1 with rescue of the disease phenotype.

Cell source
Human iPSC, Patient-Derived iPSC (Citrullinemia type 1)
Application
Disease modeling, Drug screening, Personalized medicine

Protocol overview

27 steps across 6 phases

Endodermal Induction from iPSCs Days 1–5
  1. 1 iPSC Culture Preparation
  2. 2 Replace Culture Medium with Endoderm Induction Medium
  3. 3 Culture for Endoderm Differentiation
  4. 4 Assess Endoderm Differentiation
Sorting and Seeding of EpCAM+ Cells for Organoid Formation Days 5–6
  1. 1 Harvest and Dissociate Endodermal Cells
  2. 2 Stain and Sort EpCAM+ Cells
  3. 3 Prepare Matrigel-Containing Culture Plates
Expansion Medium (EM) Culture and Organoid Growth Days 6–16 (initial phase; can extend >16 months)
  1. 1 Prepare Expansion Medium (EM)
  2. 2 Add First Dose of EM with Supplementary Factors
  3. 3 Continue Culture with Standard EM (Days 4+)
  4. 4 Observe Organoid Formation
  5. 5 Passage Organoids
  6. 6 Maintain Long-Term Culture
Hepatocyte Differentiation (Differentiation Medium, DM) Days 10–24 (14 days post-organoid formation)
  1. 1 Pre-Differentiation BMP7 Supplementation
  2. 2 Prepare Differentiation Medium (DM)
  3. 3 Switch to Differentiation Medium
  4. 4 Assess Hepatocyte Maturation
Functional Assessment and Disease-Specific Studies Post-differentiation (Days 24+)
  1. 1 Albumin Secretion Assay
  2. 2 CYP3A4 Activity Assay
  3. 3 Low-Density Lipoprotein (LDL) Uptake
  4. 4 Glycogen Storage Assessment
  5. 5 Ammonia and Urea Measurement (Disease Model)
  6. 6 Gene Expression Analysis
In Vivo Transplantation (Optional) Post-differentiation
  1. 1 Prepare eHEPO Cells and GFP Vector Transduction
  2. 2 Induce Acute Liver Damage in NSG Mice
  3. 3 Transplant eHEPO Cells Intrasplenically
  4. 4 Assess Engraftment and Functionality

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Akbari et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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