Skip to content
← Back to browse
LIVER Publication-derived

Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation

Source Broutier et al., 2016 · Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge · 10.1038/nprot.2016.097

👤 Laura Broutier, Amanda Andersson-Rolf, Christopher J Hindley, Sylvia F Boj, Hans Clevers, Bon-Kyoung Koo, Meritxell Huch ⏱ 21 days 📋 7 phases 🧫 Adult primary hepatic stem cells (mouse and human)

Abstract

This protocol describes methods for long-term expansion of self-renewing adult liver and pancreas organoids from primary tissue or isolated ducts. Cells are cultured in a gel-based extracellular matrix (Matrigel or BME2) with defined growth factors, maintaining genetic stability and tissue-of-origin commitment. The protocol includes procedures for organoid isolation, expansion, genetic manipulation via transfection or transduction, differentiation to hepatocytes, and analysis.

Cell source
Adult primary hepatic stem cells (mouse and human)
Application
Organoid culture establishment, genetic manipulation, and disease modeling

Protocol overview

87 steps across 7 phases

Tissue Dissociation and Duct Isolation Day 0 (4 hours for mouse/pancreas; 2 hours for human liver)
  1. 1 Tissue Collection
  2. 2 Tissue Mincing
  3. 3 Red Blood Cell Removal
  4. 4 Enzymatic Digestion
  5. 5 Duct Structure Assessment
  6. 6 Duct Collection and Washing
  7. 7 Hand-picking of Duct Structures (Option 1: No Sorting)
  8. 8 Final Duct Washing
Organoid Seeding and Initial Culture Days 0-7
  1. 9 Plate Preparation
  2. 10 Cell/Duct Resuspension
  3. 11 Seeding into Basement Matrix
  4. 12 Overlay with Isolation Medium
  5. 13 Initial Incubation
  6. 14 Medium Change (Day 3-4)
  7. 15 Organoid Formation Monitoring
Organoid Passaging and Expansion Days 7-onwards (every 5-7 days)
  1. 16 Organoid Growth Period
  2. 17 Basement Matrix Disruption
  3. 18 Initial Centrifugation
  4. 19 Organoid Disruption
  5. 20 Second Centrifugation
  6. 21 Supernatant Removal
  7. 22 Resuspension in Basement Matrix
  8. 23 Reseeding
  9. 24 Matrix Polymerization
  10. 25 Overlay with Expansion Medium
Genetic Manipulation - Transfection Days 7-14 post transfection/selection
  1. 26 Organoid Preparation for Transfection
  2. 27 Trypsinization to Single Cells
  3. 28 Stop Digestion and Wash
  4. 29 DNA and Lipofectamine Preparation
  5. 30 Complex Formation
  6. 31 Transfection
  7. 32 Spin Inoculation
  8. 33 Incubation
  9. 34 Seeding for Selection
  10. 35 Selection (FACS or Antibiotic)
Genetic Manipulation - Viral Transduction Days 0-21 (production to selection)
  1. 36 Plat-E Cell Seeding
  2. 37 Plat-E Cell Expansion
  3. 38 DNA and Lipofectamine Preparation for Plat-E
  4. 39 Complex Formation and Transfection
  5. 40 Overnight Incubation
  6. 41 Medium Refresh
  7. 42 Virus Collection and Filtration
  8. 43 Virus Ultracentrifugation
  9. 44 Virus Resuspension
  10. 45 Virus Preparation
  11. 46 Organoid Cell Dissociation for Transduction
  12. 47 Cell Preparation for Transduction
  13. 48 Retroviral Transduction
  14. 49 Post-Transduction Incubation
  15. 50 Cell Collection and Reseeding
  16. 51 Selection with Puromycin
  17. 52 Removal of Wnt3a and ROCK Inhibitor (Mouse Only)
  18. 53 Passage and Continued Culture
Hepatocyte Differentiation (Liver Organoids Only) Days 0-15 (mouse: 3 + 12 days; human: 5 + 10 days)
  1. 54 Mouse Liver Organoid Seeding for Differentiation
  2. 55 Switch to Mouse Liver Differentiation Medium
  3. 56 Dexamethasone Supplementation (Days 13-15)
  4. 57 Completion of Mouse Hepatocyte Differentiation
  5. 58 Human Liver Organoid Seeding for Differentiation
  6. 59 Switch to Human Liver Differentiation Medium
  7. 60 Completion of Human Hepatocyte Differentiation
Organoid Analysis and Characterization Days 7-onwards (variable based on analysis method)
  1. 61 DNA Isolation - Sample Collection
  2. 62 DNA Isolation - Washing
  3. 63 DNA Isolation - PBS Wash
  4. 64 DNA Isolation - Lysis
  5. 65 DNA Isolation - Recovery
  6. 66 DNA Isolation - Optional Purification
  7. 67 RNA Isolation - Medium Removal
  8. 68 RNA Isolation - Cell Lysis
  9. 69 RNA Isolation - Homogenization
  10. 70 RNA Isolation - Optional Storage
  11. 71 RNA Isolation - Purification
  12. 72 Whole-Mount Immunofluorescence - Organoid Preparation
  13. 73 Whole-Mount Staining - Organoid Transfer
  14. 74 Whole-Mount Staining - Basement Matrix Removal
  15. 75 Whole-Mount Staining - Repeat Washes
  16. 76 Whole-Mount Staining - Fixation
  17. 77 Whole-Mount Staining - PBS Washes
  18. 78 Whole-Mount Staining - Blocking
  19. 79 Whole-Mount Staining - Optional Casein Blocking
  20. 80 Whole-Mount Staining - Primary Antibody Incubation
  21. 81 Whole-Mount Staining - Primary Antibody Wash
  22. 82 Whole-Mount Staining - Secondary Antibody Incubation
  23. 83 Whole-Mount Staining - Secondary Antibody Wash
  24. 84 Whole-Mount Staining - Nuclear Staining (Optional)
  25. 85 Whole-Mount Staining - Final Wash
  26. 86 Whole-Mount Staining - Mounting
  27. 87 Whole-Mount Staining - Imaging

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Broutier et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence