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REPRODUCTIVE Publication-derived

Reproductive Organoids for Disease Modelling

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: 14 days 7 phases Cell source: Human fallopian tube epithelial cells (normal)

Abstract

This protocol describes the establishment and long-term maintenance of 3D organoid cultures derived from human fallopian tube epithelium. Single bipotent stem cells give rise to organoids that faithfully recapitulate the native mucosal fold architecture and can be stably expanded for over one year in culture. The organoids contain both ciliated and secretory cells and respond physiologically to Wnt, Notch, and hormonal signaling.

Cell source
Human fallopian tube epithelial cells (normal)
Application
Disease modeling; Developmental study; Stem cell biology

Protocol overview

27 steps across 7 phases

Phase 1 · Epithelial progenitor isolation from fallopian tube samples Day 1-2
  1. a Tissue collection and transport
  2. b Tissue preparation and dissociation
  3. c Cell collection and resuspension
  4. d Primary culture in 2D
Phase 2 · Organoid initiation and 3D culture establishment Day 5-14
  1. a Cell detachment and preparation for 3D seeding
  2. b Matrigel polymerization
  3. c Expansion medium overlay
  4. d Initial organoid formation and assessment
  5. e Media exchange
Phase 3 · Long-term organoid maintenance and passaging Week 2 onwards
  1. a Assessment of passaging interval
  2. b Matrigel dissolution
  3. c Mechanical fragmentation
  4. d Organoid replating
  5. e Post-passage culture establishment
  6. f Long-term stability assessment
Phase 4 · Monoclonal organoid generation from single cells (optional) Variable (3-6 weeks per clone)
  1. a FACS isolation of EpCAM+ cells
  2. b Single-cell seeding in Matrigel
  3. c Monoclonal organoid expansion
Phase 5 · Notch pathway modulation (optional - for differentiation studies) Variable
  1. a Notch inhibition with γ-secretase inhibitor DBZ
  2. b Assessment of differentiation phenotype
Phase 6 · Hormone stimulation studies (optional - for physiological response assessment) 2 weeks
  1. a Hormone treatment
  2. b Tissue collection and RNA isolation
  3. c Gene expression analysis
Phase 7 · Organoid characterization by immunofluorescence and microscopy Variable
  1. a Organoid fixation and processing
  2. b Immunofluorescent labeling
  3. c Confocal microscopy imaging
  4. d Transmission electron microscopy (optional)

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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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