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LIVER Publication-derived

MASLD-Induced Liver Micro-Organoids Protocol

Source Hammour et al., 2024 · Eberhard Karls University, Department of Traumatology, Siegfried Weller Institute, BG Clinic Tübingen · 10.20944/preprints202412.2546.v1

👤 Mohammad Majd Hammour, Guanqiao Chen, Yuxuan Xin, Georg Damm, Massoud Vosough, Benedikt Kaufmann, Andreas Klaus Nussler, Romina Haydeé Aspera-Werz ⏱ 35 days 📋 10 phases 🧫 Human iPSC-derived HepaRG cells, LX-2 stellate cells, Human HUVEC

Abstract

This protocol describes the establishment of a 3D human liver micro-organoid model incorporating HepaRG hepatocytes, LX-2 stellate cells, and HUVECs to model MASLD and investigate the impact on vitamin D metabolism. MASLD is induced by palmitic and oleic acid treatment, resulting in lipid accumulation, altered CYP expression, and impaired vitamin D synthesis.

Cell source
Human iPSC-derived HepaRG cells, LX-2 stellate cells, Human HUVEC
Application
Disease modeling

Protocol overview

23 steps across 10 phases

Cell Culture Preparation Days 1-14
  1. 1 HepaRG Cell Culture Setup
  2. 2 LX-2 Stellate Cell Culture
  3. 3 HUVEC Culture Preparation
Agarose Microwell Plate Preparation Day 14
  1. 4 Agarose Microwell Plate Preparation
Liver Micro-Organoid Formation and Differentiation Days 14-28
  1. 5 Cell Suspension Preparation and Seeding
  2. 6 Initial Organoid Culture (Days 0-2)
  3. 7 HepaRG Differentiation Induction (Days 2-14)
MASLD Induction Days 28-35
  1. 8 Free Fatty Acid Preparation
  2. 9 MASLD Induction Treatment (Days 0-7)
Mitochondrial Activity and Cell Viability Assessment Day 35
  1. 10 Resazurin Conversion Assay
  2. 11 Live-Dead Staining
Lactate Dehydrogenase and DNA Quantification Day 35
  1. 12 Lactate Dehydrogenase (LDH) Measurement
  2. 13 DNA Isolation and Quantification
Lipid Accumulation Analysis Day 35
  1. 14 Bodipy Staining and Fluorescence Microscopy
Gene Expression Analysis by RT-PCR Day 35
  1. 15 RNA Isolation
  2. 16 cDNA Synthesis
  3. 17 PCR Amplification
  4. 18 Densitometric Analysis
Dot Blot Analysis for Secreted Proteins Day 35
  1. 19 Protein Spotting onto Nitrocellulose Membrane
  2. 20 Antibody Incubation
  3. 21 Visualization and Quantification
Vitamin D Metabolism Analysis Day 35
  1. 22 Vitamin D Incubation
  2. 23 ELISA for 25(OH)D3

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Hammour et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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