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LIVER Publication-derived

Three-Dimensional Liver-derived Extracellular Matrix Hydrogel (LEMgel) for Liver Organoid Culture

Source Saheli et al. · Royan Institute for Stem Cell Biology and Technology and Shahid Beheshti University of Medical Sciences, Tehran, Iran · 10.1002/jcb.26622

👤 Mona Saheli, Mohammadmajid Sepantafar, Behshad Pournasr, Zahra Farzaneh, Massoud Vosough, Abbas Piryaei, Hossein Baharvand ⏱ 10 days 📋 4 phases 🧫 Human hepatocarcinoma cells (Huh7) with human bone marrow-derived mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs)

Abstract

This protocol describes the generation and characterization of a three-dimensional liver-derived extracellular matrix hydrogel (LEMgel) from decellularized sheep liver, and its use to culture self-organized liver organoids using Huh7 hepatocarcinoma cells co-cultured with human MSCs and HUVECs. The LEMgel preserves critical ECM components including glycosaminoglycans, collagen, laminin, and fibronectin, resulting in organoids with enhanced hepatic-specific gene expression and functional activity compared to traditional 2D and collagen-based 3D cultures.

Cell source
Human hepatocarcinoma cells (Huh7) with human bone marrow-derived mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs)
Application
Tissue engineering and organoid development for drug screening and toxicology studies

Protocol overview

30 steps across 4 phases

Preparation of Liver-Derived Extracellular Matrix (LEM) Day 0–7
  1. 1 Slice and freeze liver tissue
  2. 2 Mechanical agitation in deionized water
  3. 3 SDS decellularization
  4. 4 Triton X-100 treatment
  5. 5 Final rinse with deionized water
  6. 6 Store decellularized liver ECM
Preparation of Liver-Derived Extracellular Matrix Hydrogel (LEMgel) Day 7–8
  1. 1 Lyophilization of LEM
  2. 2 Mill lyophilized LEM to powder
  3. 3 Pepsin digestion
  4. 4 UV sterilization of pre-gel solution
  5. 5 pH neutralization for gelation
  6. 6 Incubation for gel formation
Cell Culture Preparation and Organoid Generation Day 0–10
  1. 1 HUVEC isolation from umbilical cord
  2. 2 Culture Huh7 hepatocarcinoma cells
  3. 3 Culture bone marrow-derived MSCs
  4. 4 Culture HUVECs
  5. 5 Mix cell types for organoid generation
  6. 6 Mix cells with LEMgel
  7. 7 Plate cell-gel mixture in non-adherent dish
  8. 8 Initial incubation for gel solidification
  9. 9 Add culture medium
  10. 10 Maintain organoid culture
Characterization and Functional Assessment (Day 10) Day 10
  1. 1 Live/Dead cell viability assay
  2. 2 Immunofluorescence staining for hepatic markers
  3. 3 Periodic acid-Schiff (PAS) staining for glycogen
  4. 4 Indocyanine green (ICG) uptake assay
  5. 5 Quantitative reverse transcriptase-PCR (qRT-PCR) for hepatic genes
  6. 6 Albumin and alpha-1 antitrypsin (AAT) secretion assay
  7. 7 Urea production assay
  8. 8 CYP3A4 enzyme activity assay (PROD assay)

Full SOP

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Attribution

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

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