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INTESTINAL Publication-derived

Organoid Culture from Metastatic Colorectal Cancer Biopsies with Genetic Validation

Source Weeber et al., 2015 · The Netherlands Cancer Institute · 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 ⏱ 150 days 📋 6 phases 🧫 Patient-Derived Colorectal Cancer (Metastatic)

Abstract

This protocol describes the derivation and culture of 3D organoids from metastatic colorectal cancer biopsies with a reported success rate of 71%. The method includes tissue embedding, selective culture medium optimization, and validates that organoid cultures preserve the mutational and copy number landscape of the original tumor, making them suitable for personalized cancer screening and treatment selection.

Cell source
Patient-Derived Colorectal Cancer (Metastatic)
Application
Disease modeling

Protocol overview

20 steps across 6 phases

Biopsy Collection and Patient Enrollment Baseline
  1. 1 Patient Consent and IRB Approval
  2. 2 Tumor Biopsy Collection
  3. 3 Blood Draw for Germline Control
Organoid Culture Establishment Days 1–10
  1. 1 Tissue Dissection and Matrigel Embedding
  2. 2 Overlay with Growth Medium
  3. 3 Initial Culture Period
Organoid Maintenance and Passaging Days 7–60+
  1. 1 Medium Changes
  2. 2 Organoid Passaging — Mechanical and Enzymatic Disruption
  3. 3 Continue Culture to 2–3 Months
DNA Isolation and Sample Preparation Day 60–90
  1. 1 DNA Isolation from Organoids
  2. 2 DNA Isolation from Tumor Biopsy and Blood
  3. 3 Quantify and Assess DNA Quality
Library Preparation and Sequencing Day 90–120
  1. 1 Generate Barcoded Fragment Libraries
  2. 2 Genomic Enrichment Using SureSelect
  3. 3 Sequence Using SOLiD 5500xl System
  4. 4 Mapping, Variant Calling, and Annotation
DNA Copy Number Analysis Day 120–150
  1. 1 Generate Copy Number Profiles from BAM Files
  2. 2 Normalize and Analyze Read Counts
  3. 3 Circular Binary Segmentation (CBS) and CGHcall Analysis
  4. 4 Calculate Correlation and Overlap 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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