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REPRODUCTIVE Publication-derived

Reproductive Organoids for Disease Modelling and Long-Term Culture

Full SOP with Academic or a plan

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

Authors: 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 Duration: 21 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Kessler et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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