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LIVER Publication-derived

Fatty Liver Disease Organoids (2024)

Full SOP with Academic or a plan

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

Authors: Mohammad Majd Hammour, Guanqiao Chen, Yuxuan Xin, Georg Damm, Massoud Vosough, Benedikt Kaufmann, Andreas Klaus Nussler, Romina Haydeé Aspera-Werz Duration: 35 days 10 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Hammour et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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