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LIVER Publication-derived

Liver Bioprinted and Engineered Tissues for Drug Screening

Full SOP with Academic or a plan

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

Authors: Mona Saheli, Mohammadmajid Sepantafar, Behshad Pournasr, Zahra Farzaneh, Massoud Vosough, Abbas Piryaei, Hossein Baharvand Duration: 10 days 4 phases Cell source: 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

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

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Attribution

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

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