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LIVER Publication-derived

Fatty Liver Disease Organoids with Gene Editing

Full SOP with Academic or a plan

Source Hendriks et al., 2023 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands · 10.1038/s41587-023-01680-4

Authors: Delilah Hendriks, Jos F. Brouwers, Karien Hamer, Maarten H. Geurts, Léa Luciana, Simone Massalini, Carmen López-Iglesias, Peter J. Peters, Maria J. Rodríguez-Colman, Susana Chuva de Sousa Lopes, Benedetta Artegiani, Hans Clevers Duration: 30 days 9 phases Cell source: Human fetal hepatocyte organoids

Abstract

This protocol describes the generation and culture of human fetal hepatocyte organoids engineered to model hepatic steatosis through multiple mechanisms: free fatty acid loading (diet-related), genetic variants (PNPLA3 I148M), and monogenic lipid disorders (APOB and MTTP knockouts). The organoids are used for drug screening to identify steatosis-reducing compounds and for CRISPR-based genetic screening (FatTracer platform) to identify novel steatosis modulators, exemplified by the discovery of FADS2 as a critical regulator.

Cell source
Human fetal hepatocyte organoids
Application
Disease modeling, Drug screening, Target discovery

Protocol overview

45 steps across 9 phases

Phase 1 · Organoid Establishment and Culture Days 1-10
  1. a Hepatocyte isolation
  2. b Organoid seeding in BME suspension
  3. c HEP medium preparation and culture initiation
  4. d Organoid passaging
Phase 2 · FFA-Induced Steatosis Model Generation Days 3-10
  1. a FFA stock preparation
  2. b Organoid replating for FFA exposure
  3. c FFA dose-response treatment
Phase 3 · CRISPR-Based Gene Editing and Knockout Generation Days 5-25
  1. a sgRNA design and plasmid cloning
  2. b Cas9 and selection plasmid preparation
  3. c Organoid dissociation for electroporation
  4. d Electroporation
  5. e Post-electroporation recovery and replating
  6. f Antibiotic selection (if using hygromycin-piggyBac system)
  7. g Single organoid picking and clonal line establishment
  8. h Genotyping of picked clonal lines
Phase 4 · Drug Screening in Steatotic Organoid Models Days 10-17
  1. a Pre-steatosis induction in FFA model (WT organoids)
  2. b Drug preparation and concentration standardization
  3. c Drug treatment of steatotic organoids
  4. d Organoid harvesting for lipid analysis
Phase 5 · Lipid Analysis and Quantification Days 17-19
  1. a Formaldehyde fixation of organoids
  2. b Nile Red lipid staining
  3. c Confocal microscopy imaging
  4. d Lipid scoring and quantification analysis
Phase 6 · FatTracer CRISPR Loss-of-Function Screening Days 1-20
  1. a FatTracer library and sgRNA design
  2. b FatTracer organoid transfection
  3. c Selection and clonal outgrowth
  4. d Phenotypic assessment and scoring
  5. e Single organoid picking and genotyping
  6. f Nile Red staining of FatTracer clonal lines
Phase 7 · FADS2 Validation and Functional Analysis Days 15-30
  1. a FADS2 knockout generation and validation
  2. b FFA sensitivity testing in FADS2 knockout organoids
  3. c FADS2 overexpression construct generation
  4. d FADS2 overexpression transfection and selection
  5. e FADS2 expression quantification in overexpression lines
  6. f FADS2 overexpression steatosis rescue
Phase 8 · Lipidomics and Mechanistic Analysis Days 15-25
  1. a Lipidomics sample preparation
  2. b Lipid extraction
  3. c Neutral lipid mass spectrometry analysis
  4. d Lipidomics data analysis
Phase 9 · RNA-Sequencing and Transcriptomic Analysis Days 7-15
  1. a Drug-treated organoid sample preparation for RNA-seq
  2. b RNA extraction and quality assessment
  3. c RNA library preparation and sequencing
  4. d RNA-seq data processing and alignment
  5. e Differential gene expression analysis
  6. f Gene set enrichment analysis (GSEA)

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Attribution

This SOP was written by Organthis based on the published method in Hendriks et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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