Skip to content
← Back to browse
ESOPHAGEAL Publication-derived

Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics

Source Li et al., 2018 · MRC Cancer Unit, University of Cambridge · 10.1038/s41467-018-05190-9

👤 Xiaodun Li, Hayley E. Francies, Maria Secrier, Juliane Perner, Ahmad Miremadi, Núria Galeano-Dalmau, William J. Barendt, Laura Letchford, Genevieve M. Leyden, Emma K. Goffin, Andrew Barthorpe, Howard Lightfoot, Elisabeth Chen, James Gilbert, Ayesha Noorani, Ginny Devonshire, Lawrence Bower, Amber Grantham, Shona MacRae, Nicola Grehan, David C. Wedge, Rebecca C. Fitzgerald, Mathew J. Garnett ⏱ 7 days 📋 8 phases 🧫 Patient-Derived Cancer Tissue (Esophageal Adenocarcinoma)

Abstract

This protocol describes the derivation and characterization of three-dimensional esophageal adenocarcinoma (EAC) organoid cultures from primary patient tissue. The organoids recapitulate the morphological, genomic, and transcriptomic landscape of the primary tumor including clonal heterogeneity and mutational signatures, enabling studies of clonal evolution and drug sensitivity testing for precision medicine strategies.

Cell source
Patient-Derived Cancer Tissue (Esophageal Adenocarcinoma)
Application
Disease modeling and precision therapeutics

Protocol overview

35 steps across 8 phases

Tissue sample preparation and enzymatic digestion Day 0-1
  1. 1 Tissue collection and wash
  2. 2 Tissue mincing
  3. 3 Collagenase digestion
  4. 4 Filtration to remove undigested fragments
  5. 5 Cell pellet collection and washing
Organoid seeding and initial culture establishment Day 1-3
  1. 1 Preparation of BME-2 culture matrix
  2. 2 Plating cells in BME-2 droplets
  3. 3 BME-2 polymerization
  4. 4 Initial culture incubation
Complete culture medium composition and maintenance Day 1 onwards
  1. 1 Complete culture medium preparation
  2. 2 Medium refreshment schedule
Organoid passaging and long-term culture Day 7 onwards (as needed)
  1. 1 BME-2 disassociation
  2. 2 Organoid collection and enzyme treatment
  3. 3 Vigorous manual shake and centrifugation
  4. 4 Cell pellet resuspension and re-plating
Organoid characterization – Histology and immunohistochemistry As scheduled for analysis
  1. 1 Organoid fixation and embedding
  2. 2 Paraffin sectioning
  3. 3 Hematoxylin and Eosin (H&E) staining
  4. 4 Immunohistochemistry (IHC) staining
Organoid characterization – Immunofluorescence As scheduled for analysis
  1. 1 Organoid fixation for immunofluorescence
  2. 2 Permeabilization
  3. 3 Quenching and primary antibody incubation
  4. 4 Secondary antibody and marker staining
  5. 5 Confocal imaging
Chromosome analysis – Spectral karyotyping (SKY) As scheduled for analysis
  1. 1 Colcemid treatment to arrest metaphase cells
  2. 2 Cell harvest and dissociation
  3. 3 Hypotonic treatment
  4. 4 Fixation and storage
  5. 5 M-FISH probe preparation and hybridization
  6. 6 Imaging and karyotyping
Drug sensitivity testing Day of screening (6-day assay)
  1. 1 Organoid preparation and plating
  2. 2 Drug compound dispensing
  3. 3 Drug incubation
  4. 4 Cell viability assessment
  5. 5 Luminescence measurement and data analysis

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Li et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence