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INTESTINAL Publication-derived

Prospective Derivation of a Living Organoid Biobank of CRC

Source van de Wetering et al. · Hubrecht Institute and University Medical Centre Utrecht, Utrecht, Netherlands · 10.1016/j.cell.2015.03.053

👤 van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, van Houdt W, van Gorp J, Taylor-Weiner A, Kester L, McLaren-Douglas A, Blokker J, Jaksani S, Penber S, Stange DE, Vries RG, de Jong H, van de Ven M, Nuber G, Odom DT, Garnett MJ, Clevers H ⏱ 30 days 📋 5 phases 🧫 Patient-Derived (CRC Surgical Resection and Matched Normal)

Abstract

Established the first large-scale living organoid biobank from colorectal cancer patients with matched tumor and normal tissue pairs. Comprehensive genomic characterization and high-throughput drug screening demonstrated that organoids faithfully recapitulate tumor heterogeneity and can predict drug sensitivity.

Cell source
Patient-Derived (CRC Surgical Resection and Matched Normal)
Application
Cancer Drug Screening

Protocol overview

24 steps across 5 phases

Tissue Isolation and Crypt Extraction Day 0
  1. 1 Normal Tissue Preparation
  2. 2 Chelation Solution Washing
  3. 3 EDTA Treatment and Crypt Isolation
  4. 4 Crypt Collection and Washing
  5. 5 Tumor Tissue Dissociation
  6. 6 Tumor Cell Pellet Preparation
Organoid Establishment in Basement Membrane Extract Day 0–1
  1. 7 Basement Membrane Extract Preparation and Organoid Plating
  2. 8 Addition of Culture Medium for Normal Organoids
  3. 9 Addition of Culture Medium for Tumor Organoids
Organoid Maintenance and Expansion Day 1–30+
  1. 10 Routine Medium Refreshment
  2. 11 Organoid Passaging – Mechanical Disruption
  3. 12 Trypsin Dissociation
  4. 13 Enzyme Inactivation and Centrifugation
  5. 14 Re-plating in Fresh BME
  6. 15 Addition of Medium with Y27632
  7. 16 Cryopreservation of Master Cell Bank
High-Throughput Drug Sensitivity Screening Day 1–8 of screen
  1. 17 Organoid Preparation for Drug Screen
  2. 18 Drug Compound Dispensing
  3. 19 Drug Incubation
  4. 20 Cell Viability Measurement
  5. 21 Dose-Response Curve Fitting and IC50 Determination
Genomic and Transcriptomic Characterization Throughout study
  1. 22 Genomic DNA Extraction
  2. 23 Whole-Exome Sequencing Library Preparation
  3. 24 RNA Extraction and Microarray Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in van de Wetering et al.. 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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