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ENDOMETRIUM Publication-derived

Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening

Source Boretto et al., 2019 · KU Leuven, Laboratory of Tissue Plasticity in Health and Disease · 10.1038/s41556-019-0360-z

👤 Matteo Boretto, Nina Maenhoudt, Xinlong Luo, Aurélie Hennes, Bram Boeckx, Bich Bui, Ruben Heremans, Lisa Perneel, Hiroto Kobayashi, Indra Van Zundert, Hilde Brems, Benoit Cox, Marc Ferrante, Hiroshi Uji-i, Kian Peng Koh, Thomas D'Hooghe, Arne Vanhie, Ignace Vergote, Christel Meuleman, Carla Tomassetti, Diether Lambrechts, Joris Vriens, Dirk Timmerman, Hugo Vankelecom ⏱ 30 days 📋 17 phases 🧫 Patient-Derived Endometrial Tissue (Healthy and Endometrial Diseases: Endometriosis, Hyperplasia, Endometrial Cancer)

Abstract

This protocol describes the derivation and long-term expansion of three-dimensional organoids from patient endometrial biopsies spanning healthy and diseased endometrium including endometriosis, hyperplasia, and endometrial cancer. The organoids maintain genomic and transcriptomic stability, reproduce disease-associated phenotypes both in vitro and in vivo, and are suitable for drug screening applications.

Cell source
Patient-Derived Endometrial Tissue (Healthy and Endometrial Diseases: Endometriosis, Hyperplasia, Endometrial Cancer)
Application
Disease modeling and drug screening

Protocol overview

70 steps across 17 phases

Tissue Preparation and Dissociation Day 0–1
  1. 1 Tissue Mincing and Rinsing
  2. 2 Enzymatic Dissociation with Collagenase IV
  3. 3 TrypLE Treatment
  4. 4 Digestion Stop and Centrifugation
Organoid Seeding and Initial Culture Day 0–7 (organoids form within 7–14 days)
  1. 5 Matrigel Droplet Preparation
  2. 6 Standard Organoid Medium (SOM) Addition
  3. 7 Initial Culture and Organoid Monitoring
Organoid Expansion and Passaging Day 14 onwards (every 10–20 days per passage)
  1. 8 Organoid Recovery from Matrigel
  2. 9 TrypLE Dissociation
  3. 10 Replating in Matrigel
  4. 11 Cryopreservation for Biobanking
Clonogenic Capacity Assessment (Optional) Day 0–30
  1. 12 Single-Cell Dissociation and Filtration
  2. 13 Low-Density Single-Cell Seeding
  3. 14 Organoid Formation and Counting
Medium Optimization Studies (For EC-O) 7–20 days per condition
  1. 15 Preparation of Modified Media
  2. 16 Culture of Dissociated Organoid Cells in Modified Media
  3. 17 Organoid Quantification
WNT Pathway Interrogation (Optional) 7–10 days
  1. 18 Culture in WNT-Modified Media
  2. 19 Organoid Counting and RNA Extraction
In Vivo Transplantation Day 0 (transplant); assess after 1–2 months
  1. 20 Organoid Preparation for Transplantation
  2. 21 Subrenal Transplantation (For Initial Feasibility)
  3. 22 Intraperitoneal Injection (For Endometriosis Models)
  4. 23 Uterine Orthotopic Transplantation (For Endometrial Cancer Models)
  5. 24 Graft Assessment and Tissue Collection
Immunohistochemical and Histological Characterization Concurrent with organoid culture and post-transplantation
  1. 25 Organoid Fixation and Embedding
  2. 26 Haematoxylin and Eosin (H&E) Staining
  3. 27 Immunohistochemical (IHC) Staining
  4. 28 Periodic Acid–Schiff (PAS) Staining
  5. 29 Immunofluorescence (IF) Staining
Transmission Electron Microscopy (TEM) Processing time: 3–5 days
  1. 30 Organoid Preparation for TEM
  2. 31 Heavy Metal Staining
  3. 32 Dehydration and Resin Embedding
  4. 33 Ultramicrotomy and TEM Imaging
Two-Photon Excitation Microscopy for 3D Imaging Concurrent with organoid culture
  1. 34 Organoid Fixation and Labeling
  2. 35 Two-Photon Confocal Microscopy
Genomic and Transcriptomic Characterization (aCGH, WES, RNA-seq) 10–30 days (processing time varies)
  1. 36 DNA Extraction
  2. 37 Array Comparative Genomic Hybridization (aCGH)
  3. 38 Whole-Exome Sequencing (WES) Library Preparation
  4. 39 Whole-Genome Library Preparation for Shallow Sequencing
  5. 40 Sequencing on HiSeq4000
  6. 41 WES Data Analysis and Variant Calling
  7. 42 Shallow-Seq and Copy Number Analysis
  8. 43 TP53 Sequencing Analysis
  9. 44 Microsatellite Instability (MSI) Assessment
RNA Sequencing and Gene Expression Analysis 10–20 days (processing time)
  1. 45 RNA Extraction
  2. 46 RNA Amplification
  3. 47 RNA-Seq Library Preparation
  4. 48 NextSeq500 Sequencing
  5. 49 RNA-Seq Read Alignment and Quantification
  6. 50 Differential Gene Expression Analysis
  7. 51 Gene Ontology and Pathway Analysis
  8. 52 SNP Variant Calling from RNA-seq
Reverse Transcription–Quantitative PCR (RT-qPCR) 1 day (per experiment)
  1. 53 RNA Extraction and Reverse Transcription
  2. 54 qPCR Setup
  3. 55 qPCR Data Analysis
Drug Screening and Chemosensitivity Testing 10–20 days (per drug panel)
  1. 56 Organoid Dissociation and Seeding
  2. 57 Drug Preparation and Addition
  3. 58 Cell Viability Assessment (XTT Assay)
  4. 59 Dose–Response Curve Generation and IC50 Calculation
Ion Channel Functionality Analysis 1–3 days (per experiment)
  1. 60 Organoid Dissociation and Cell Seeding
  2. 61 Calcium Indicator Loading
  3. 62 Ion Channel Agonist Stimulation
  4. 63 Calcium Imaging and Data Analysis
  5. 64 Ion Channel Blocker Cocktail Treatment (Optional)
Targeted Sequencing for Mismatch Repair Genes (Lynch Syndrome) 1–2 days
  1. 65 PCR Amplification of Target Regions
  2. 66 Mutation Confirmation
Quality Control and Statistical Analysis Throughout protocol execution
  1. 67 Genomic Stability Assessment
  2. 68 Transcriptomic Stability Verification
  3. 69 Statistical Analysis
  4. 70 Reproducibility and Replication

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Boretto et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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