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LIVER Publication-derived

ICO (Huch/Clevers)

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

👤 Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MMA, Clevers H ⏱ 180 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

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

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