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REPRODUCTIVE Publication-derived

Human Fallopian Tube Organoid Establishment and Long-Term Culture

Source Kessler et al., 2015 · Max Planck Institute for Infection Biology, Berlin · 10.1038/ncomms9989

👤 Mirjana Kessler, Karen Hoffmann, Volker Brinkmann, Oliver Thieck, Susan Jackisch, Benjamin Toelle, Hilmar Berger, Hans-Joachim Mollenkopf, Mandy Mangler, Jalid Sehouli, Christina Fotopoulou, Thomas F. Meyer ⏱ 21 days 📋 6 phases 🧫 Human fallopian tube epithelial cells

Abstract

This protocol describes the establishment and maintenance of long-term, 3D organoid cultures from human fallopian tube epithelium. Single epithelial stem cells give rise to polarized, differentiated organoids containing both ciliated and secretory cells, requiring active Wnt and Notch paracrine signalling for stemness maintenance and proper differentiation.

Cell source
Human fallopian tube epithelial cells
Application
Developmental study; Disease modeling (high-grade serous ovarian cancer)

Protocol overview

27 steps across 6 phases

Epithelial Progenitor Isolation from Fallopian Tube Tissue Day 0–5
  1. 1 Tissue collection and transport
  2. 2 Tissue cleaning and preparation
  3. 3 Enzymatic dissociation of epithelial cells
  4. 4 Cell scraping and collection
  5. 5 Cell resuspension and 2D culture initiation
Transition to 3D Organoid Culture Day 5–14
  1. 1 Detachment and preparation of epithelial cells
  2. 2 Embedding cells in Matrigel
  3. 3 Overlay with expansion medium
  4. 4 Organoid formation and monitoring
  5. 5 Observation of mature organoid architecture
Long-Term Organoid Maintenance and Passaging Day 14 onwards (every 14–21 days)
  1. 1 Assessment of passaging requirement
  2. 2 Matrigel dissolution
  3. 3 Mechanical fragmentation of organoids
  4. 4 Centrifugation and pellet recovery
  5. 5 Re-embedding in fresh Matrigel
  6. 6 Resume expansion medium culture
FACS Isolation of EpCAM+ Stem Cells and Single-Cell Cloning Day 0–3 (optional, for monoclonal organoid derivation)
  1. 1 Preparation of epithelial cells for FACS
  2. 2 Antibody labelling of EpCAM+ cells
  3. 3 FACS sorting with viability gating
  4. 4 Single-cell embedding in Matrigel
  5. 5 Single-cell organoid culture
Notch Pathway Inhibition Experiments (Optional) Day 7 (7 days post-passaging)
  1. 1 Preparation of DBZ stock solution
  2. 2 DBZ treatment of organoids
  3. 3 Microscopic observation of phenotypic changes
Hormone Stimulation Experiments (Optional) Day 0–14 (2-week treatment)
  1. 1 Preparation of hormone stock solutions
  2. 2 Addition of hormones to organoid cultures
  3. 3 Sample collection for gene expression analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kessler et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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