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LIVER Publication-derived

Liver Models for Disease Modelling

Full SOP with Academic or a plan

Source Huch et al. · Hubrecht Institute, Utrecht / Wellcome Sanger Institute, Cambridge · 10.1016/j.cell.2014.11.050

Authors: Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MMA, Clevers H Duration: 180 days 7 phases Cell source: Human Adult Liver (EpCAM+ Ductal Cells)

Abstract

Das erste humane Leber-Organoid-Protokoll. EpCAM+ Gallengangsepithelzellen aus adulter humaner Leber werden in Matrigel mit Wnt3a/R-Spondin/EGF/HGF/A83-01-Medium kultiviert und expandieren klonal über Monate mit stabiler Genomintegrität. Krankheitsmodelle von A1AT-Mangel und Alagille-Syndrom wurden erfolgreich rekonstituiert. Goldstandard-Referenz für alle humanen Leber-ASC-Organoid-Protokolle.

Cell source
Human Adult Liver (EpCAM+ Ductal Cells)
Application
Disease Modeling

Protocol overview

47 steps across 7 phases

Phase 1 · Human Liver Cell Isolation and Initial Culture Setup Day 0-3
  1. a Liver Tissue Preparation and Collagenase Digestion
  2. b Cell Collection and Straining
  3. c Secondary Accutase Digestion
  4. d Cell Counting and Viability Assessment
  5. e Matrigel or BME Preparation and Cell Seeding
  6. f First Three Days of Culture Medium Supplementation
Phase 2 · Expansion Medium Culture and Serial Passaging Day 3-180+
  1. a Expansion Medium Preparation
  2. b Medium Changes During Organoid Growth
  3. c First Organoid Passage (Day 10-14)
  4. d Serial Passaging (Every 7-10 Days)
  5. e Freezing Organoid Stocks
  6. f Thawing Frozen Organoid Stocks
Phase 3 · EpCAM+ Cell Sorting (Optional) Day 0 (from fresh tissue)
  1. a EpCAM Antibody Staining
  2. b Cell Sorting by Flow Cytometry
  3. c Post-Sort Seeding and Recovery
Phase 4 · Hepatocyte Differentiation Day 0-13 (relative to start of differentiation protocol)
  1. a BMP7 Pre-treatment (Days −7 to 0)
  2. b Culture Split and Re-seeding in EM + BMP7
  3. c Differentiation Medium Preparation
  4. d Medium Switch to Differentiation Medium (Day 0)
  5. e Differentiation Medium Changes (Days 1-13)
  6. f Observation of Morphological Changes
Phase 5 · In Vitro Functional Assessment of Differentiated Hepatocytes Day 11-13 (after 11-13 days in differentiation medium)
  1. a Sample Preparation and Medium Collection
  2. b Albumin Secretion Measurement
  3. c Glycogen Accumulation (PAS Staining)
  4. d LDL Uptake Assay
  5. e CYP3A4 Activity Measurement
  6. f Midazolam Metabolism (Phase I and II Detoxification)
  7. g Bile Acid Secretion Measurement
  8. h Ammonia Elimination Assay
Phase 6 · Transplantation into Immunocompromised Mice Day 0 (transplant day; preceded by 44 days of animal pretreatment)
  1. a Mouse Pretreatment with Retrorsine (Days −44 and −30)
  2. b Acute Liver Injury Induction (Day −1)
  3. c Immunosuppression Protocol (Days −1 to study endpoint)
  4. d Organoid Cell Harvest and Preparation (Day 0)
  5. e Mouse Anesthesia and Surgical Preparation (Day 0)
  6. f Intrasplenic Transplantation (Day 0)
  7. g Post-Operative Care and Recovery (Days 1-14)
  8. h NK Cell Depletion (Weekly, Days 7-60+)
  9. i Serial Blood Sampling and Biomarker Monitoring (Days 0, 7, 14, 28, 60, 120)
  10. j End-Point Analysis and Tissue Harvest (Days 60-120)
Phase 7 · Genomic Stability Analysis Day 0 (at harvest; can be performed at any passage)
  1. a Karyotyping Preparation
  2. b Karyotype Analysis
  3. c DNA Extraction and Whole-Genome Sequencing (WGS) Library Preparation
  4. d Whole-Genome Sequencing
  5. e Sequence Read Mapping and Variant Calling
  6. f Somatic SNV Filtering
  7. g Copy Number Variant (CNV) Analysis
  8. h Data Deposition and Accessibility

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Attribution

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

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