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LIVER Publication-derived

Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis

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

👤 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 ⏱ 30 days 📋 9 phases 🧫 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

Organoid Establishment and Culture Days 1-10
  1. 1 Hepatocyte isolation
  2. 2 Organoid seeding in BME suspension
  3. 3 HEP medium preparation and culture initiation
  4. 4 Organoid passaging
FFA-Induced Steatosis Model Generation Days 3-10
  1. 5 FFA stock preparation
  2. 6 Organoid replating for FFA exposure
  3. 7 FFA dose-response treatment
CRISPR-Based Gene Editing and Knockout Generation Days 5-25
  1. 8 sgRNA design and plasmid cloning
  2. 9 Cas9 and selection plasmid preparation
  3. 10 Organoid dissociation for electroporation
  4. 11 Electroporation
  5. 12 Post-electroporation recovery and replating
  6. 13 Antibiotic selection (if using hygromycin-piggyBac system)
  7. 14 Single organoid picking and clonal line establishment
  8. 15 Genotyping of picked clonal lines
Drug Screening in Steatotic Organoid Models Days 10-17
  1. 16 Pre-steatosis induction in FFA model (WT organoids)
  2. 17 Drug preparation and concentration standardization
  3. 18 Drug treatment of steatotic organoids
  4. 19 Organoid harvesting for lipid analysis
Lipid Analysis and Quantification Days 17-19
  1. 20 Formaldehyde fixation of organoids
  2. 21 Nile Red lipid staining
  3. 22 Confocal microscopy imaging
  4. 23 Lipid scoring and quantification analysis
FatTracer CRISPR Loss-of-Function Screening Days 1-20
  1. 24 FatTracer library and sgRNA design
  2. 25 FatTracer organoid transfection
  3. 26 Selection and clonal outgrowth
  4. 27 Phenotypic assessment and scoring
  5. 28 Single organoid picking and genotyping
  6. 29 Nile Red staining of FatTracer clonal lines
FADS2 Validation and Functional Analysis Days 15-30
  1. 30 FADS2 knockout generation and validation
  2. 31 FFA sensitivity testing in FADS2 knockout organoids
  3. 32 FADS2 overexpression construct generation
  4. 33 FADS2 overexpression transfection and selection
  5. 34 FADS2 expression quantification in overexpression lines
  6. 35 FADS2 overexpression steatosis rescue
Lipidomics and Mechanistic Analysis Days 15-25
  1. 36 Lipidomics sample preparation
  2. 37 Lipid extraction
  3. 38 Neutral lipid mass spectrometry analysis
  4. 39 Lipidomics data analysis
RNA-Sequencing and Transcriptomic Analysis Days 7-15
  1. 40 Drug-treated organoid sample preparation for RNA-seq
  2. 41 RNA extraction and quality assessment
  3. 42 RNA library preparation and sequencing
  4. 43 RNA-seq data processing and alignment
  5. 44 Differential gene expression analysis
  6. 45 Gene set enrichment analysis (GSEA)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Hendriks et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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