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REPRODUCTIVE Publication-derived

Derivation and Long-Term Expansion of Ovarian Cancer Organoids

Source Kopper et al., 2019 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and UMC Utrecht · 10.1038/s41591-019-0422-6

👤 Oded Kopper, Chris J. de Witte, Kadi Lõhmussaar, Jose Espejo Valle-Inclan, Hans Clevers ⏱ 14 days 📋 4 phases 🧫 Patient-Derived Tumor (Ovarian Cancer)

Abstract

This protocol enables efficient derivation and long-term in vitro expansion of ovarian cancer (OC) organoids from primary tumors and metastatic lesions. OC organoids recapitulate histological and genomic features of the original tumor, maintain heterogeneity, and can be used for drug-sensitivity assays and genetic manipulation, supporting both research and personalized medicine approaches.

Cell source
Patient-Derived Tumor (Ovarian Cancer)
Application
Disease modeling and drug screening

Protocol overview

24 steps across 4 phases

Tissue Collection and Processing Day 0-1
  1. 1 Obtain and prepare OC tissue
  2. 2 Mince and wash tissue
  3. 3 Perform red blood cell lysis if needed
  4. 4 Enzymatic digestion of tissue
  5. 5 Shear and filter digested tissue
  6. 6 Final washing and concentration
  7. 7 Process ascites/pleural effusion samples (if applicable)
Organoid Culture Initialization Day 1-5
  1. 1 Prepare BME-cell suspension
  2. 2 Solidify BME drops
  3. 3 Add organoid culture medium
  4. 4 Optional HGF supplementation
  5. 5 Monitor early organoid formation
Medium Maintenance and Passaging Day 5 onwards (long-term culture)
  1. 1 Change medium every 3–4 days
  2. 2 Monitor organoid growth and determine passaging interval
  3. 3 Mechanical dissociation for passaging
  4. 4 Enzymatic dissociation for dense organoids (if needed)
  5. 5 Resuspend in BME and replate
  6. 6 Alternative: Transfer to repellent plates for floating organoids
  7. 7 Cryopreservation of organoid lines
Quality Control and Characterization Days 7-14 and periodically thereafter
  1. 1 Histological assessment (H&E staining)
  2. 2 Immunohistochemical validation
  3. 3 Metaphase spread analysis for ploidy assessment
  4. 4 Ki67 proliferation assessment
  5. 5 Scanning electron microscopy (SEM) for morphological validation

Full SOP

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Attribution

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

This wording is awaiting legal review.

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