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ESOPHAGEAL Publication-derived

Oesophageal Organoids for Drug Screening

Full SOP with Academic or a plan

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

Authors: 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 Duration: 7 days 8 phases Cell source: 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

Phase 1 · Tissue sample preparation and enzymatic digestion Day 0-1
  1. a Tissue collection and wash
  2. b Tissue mincing
  3. c Collagenase digestion
  4. d Filtration to remove undigested fragments
  5. e Cell pellet collection and washing
Phase 2 · Organoid seeding and initial culture establishment Day 1-3
  1. a Preparation of BME-2 culture matrix
  2. b Plating cells in BME-2 droplets
  3. c BME-2 polymerization
  4. d Initial culture incubation
Phase 3 · Complete culture medium composition and maintenance Day 1 onwards
  1. a Complete culture medium preparation
  2. b Medium refreshment schedule
Phase 4 · Organoid passaging and long-term culture Day 7 onwards (as needed)
  1. a BME-2 disassociation
  2. b Organoid collection and enzyme treatment
  3. c Vigorous manual shake and centrifugation
  4. d Cell pellet resuspension and re-plating
Phase 5 · Organoid characterization – Histology and immunohistochemistry As scheduled for analysis
  1. a Organoid fixation and embedding
  2. b Paraffin sectioning
  3. c Hematoxylin and Eosin (H&E) staining
  4. d Immunohistochemistry (IHC) staining
Phase 6 · Organoid characterization – Immunofluorescence As scheduled for analysis
  1. a Organoid fixation for immunofluorescence
  2. b Permeabilization
  3. c Quenching and primary antibody incubation
  4. d Secondary antibody and marker staining
  5. e Confocal imaging
Phase 7 · Chromosome analysis – Spectral karyotyping (SKY) As scheduled for analysis
  1. a Colcemid treatment to arrest metaphase cells
  2. b Cell harvest and dissociation
  3. c Hypotonic treatment
  4. d Fixation and storage
  5. e M-FISH probe preparation and hybridization
  6. f Imaging and karyotyping
Phase 8 · Drug sensitivity testing Day of screening (6-day assay)
  1. a Organoid preparation and plating
  2. b Drug compound dispensing
  3. c Drug incubation
  4. d Cell viability assessment
  5. e Luminescence measurement and data analysis

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Attribution

This SOP was written by Organthis based on the published method in Li et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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