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INTESTINAL Publication-derived

Establishment and Genetic Characterization of Patient-Derived Tumor Organoids from Colorectal Cancer Metastases

Source Weeber et al., 2015 · The Netherlands Cancer Institute, Amsterdam · 10.1073/pnas.1516689112

👤 Fleur Weeber, Marc van de Wetering, Marlous Hoogstraat, Krijn K. Dijkstra, Oscar Krijgsman, Thomas Kuilman, Christa G. M. Gadellaa-van Hooijdonk, Daphne L. van der Velden, Daniel S. Peeper, Edwin P. J. G. Cuppen, Robert G. Vries, Hans Clevers, Emile E. Voest ⏱ 90 days 📋 5 phases 🧫 Patient-Derived Tumor Biopsy (Metastatic Colorectal Cancer)

Abstract

This protocol describes the establishment of 3D organoid cultures from fresh tumor biopsies of patients with metastatic colorectal cancer, with subsequent genetic characterization by SOLiD sequencing and copy number analysis. The method achieves a 71% culture success rate and preserves the mutational landscape and copy number profiles of the original metastatic tumor.

Cell source
Patient-Derived Tumor Biopsy (Metastatic Colorectal Cancer)
Application
Disease modeling and personalized drug screening

Protocol overview

22 steps across 5 phases

Biopsy Collection and Specimen Preparation Day 0
  1. 1 Obtain patient consent and clinical approval
  2. 2 Collect tumor biopsies
  3. 3 Collect blood for control DNA
Organoid Culture Establishment Day 0 to Day 70–90
  1. 1 Dissect and embed tumor biopsy
  2. 2 Prepare culture medium
  3. 3 Overlay Matrigel with growth medium
  4. 4 Maintain organoid cultures with regular medium changes
  5. 5 Passage organoids mechanically and enzymatically
  6. 6 Continue culture expansion
DNA Isolation Day 60–90 (after 2–3 months of culture)
  1. 1 Harvest organoid cultures for DNA extraction
  2. 2 Isolate DNA from organoid cultures
  3. 3 Isolate DNA from matched tumor biopsy
  4. 4 Isolate DNA from blood control
  5. 5 Quantify and assess DNA quality
DNA Sequencing Day 90+
  1. 1 Prepare barcoded fragment libraries
  2. 2 Perform targeted genomic enrichment
  3. 3 Sequence samples on 5500xl SOLiD system
  4. 4 Perform mapping, variant calling, and annotation
Copy Number Analysis Day 90+
  1. 1 Generate copy number profiles from sequencing data
  2. 2 Normalize copy number data
  3. 3 Perform circular binary segmentation (CBS) and copy number calling
  4. 4 Calculate correlation metrics

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Weeber et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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