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ENDOMETRIUM Publication-derived

Endometrial Organoids for Drug Screening

Full SOP with Academic or a plan

Source Boretto et al., 2017 · KU Leuven (University of Leuven) · 10.1242/dev.148478

Authors: Matteo Boretto, Benoit Cox, Manuel Noben, Nikolai Hendriks, Amelie Fassbender, Heleen Roose, Frederic Amant, Dirk Timmerman, Carla Tomassetti, Arne Vanhie, Christel Meuleman, Marc Ferrante, Hugo Vankelecom Duration: 25 days 6 phases Cell source: Human endometrial tissue (from biopsies)

Abstract

This protocol establishes organoid cultures from mouse and human endometrium that reproduce epithelial phenotype, glandular histology, and physiological responses to estrogen and progesterone. The organoids demonstrate long-term expansion capacity (>10 passages in human; >14 passages in mouse) and recapitulate menstrual cycle-like changes in response to sequential hormone treatment.

Cell source
Human endometrial tissue (from biopsies)
Application
Disease modeling and drug screening

Protocol overview

49 steps across 6 phases

Phase 1 · Mouse endometrium tissue dissociation and preparation Day 0
  1. a Euthanize mice and isolate uterine horns
  2. b Mince uterine tissue
  3. c EDTA-mediated dissociation
  4. d Stop dissociation with serum and settle tissue
  5. e Mechanical dissociation of tissue pieces
  6. f Filter and collect dissociated fragments
  7. g Centrifuge and wash fragments
  8. h Resuspend fragments in Matrigel
Phase 2 · Mouse organoid culture initiation and expansion Day 0-10 (Passage 0)
  1. a Plate organoid fragments in Matrigel drops
  2. b Prepare and add culture medium
  3. c Culture organoids in incubator
  4. d Refresh culture medium every 2 days
  5. e Monitor organoid development
  6. f Passage organoids (7-10 days)
  7. g Enzymatically dissociate organoids
  8. h Centrifuge and wash cells
  9. i Disperse cells and resuspend in Matrigel
  10. j Final centrifugation and Matrigel embedding
  11. k Add ROCK inhibitor and culture medium
  12. l Continue culture and monitor passage number
Phase 3 · Human endometrium tissue dissociation and preparation Day 0
  1. a Obtain and prepare human endometrial biopsy
  2. b Mince human endometrial tissue
  3. c Enzymatic dissociation with collagenase IV
  4. d Mechanical dissociation during enzymatic incubation
  5. e Centrifuge and wash dissociated tissue
  6. f Secondary enzymatic dissociation with TrypLE
  7. g Centrifuge and prepare cell pellet
  8. h Resuspend cells in Matrigel
Phase 4 · Human organoid culture initiation and expansion Day 0-14 (Passage 0)
  1. a Plate human cells in Matrigel drops
  2. b Prepare human organoid culture medium
  3. c Add culture medium to wells
  4. d Incubate organoids
  5. e Refresh medium every 2 days
  6. f Monitor organoid morphology
  7. g Passage organoids (14 days)
  8. h Enzymatically dissociate organoids with TrypLE
  9. i Centrifuge and wash cells
  10. j Disperse cells and resuspend in Matrigel
  11. k Embed cells in Matrigel and plate
  12. l Add culture medium and continue culture
  13. m Repeat passaging and long-term culture
Phase 5 · Hormone treatment of mouse organoids Varies (2-4 days total per experiment)
  1. a Prepare estrogen (E₂) treatment
  2. b Treat organoids with E₂ for 2 days
  3. c Switch to progesterone (Prog) treatment
  4. d Harvest treated organoids for analysis
Phase 6 · Hormone treatment of human organoids to mimic menstrual cycle 18-25 days total
  1. a Prepare and apply estrogen (E₂) treatment
  2. b Add progesterone while reducing E₂ concentration
  3. c Withdraw hormones to mimic menstrual phase
  4. d Harvest organoids for analysis

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Attribution

This SOP was written by Organthis based on the published method in Boretto et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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