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INTESTINAL Publication-derived

T Cell Co-Culture for Adoptive Immunotherapy

Source Dijkstra et al. · Netherlands Cancer Institute (NKI), Amsterdam, Netherlands · 10.1016/j.cell.2018.07.009

👤 Dijkstra KK, Cattaneo CM, Weeber F, Chalabi M, van de Haar J, Fanchi LF, Slagter M, van der Velden DL, Kaing S, Kelderman S, van Rooij N, van Leerdam ME, Depla A, Smit EF, Haanen JBAG, Schumacher TN, Voest EE ⏱ 28 days 📋 8 phases 🧫 Primary (Human CRC Tumor Organoids + Autologous Peripheral Blood T Cells)

Abstract

Developed a tumor organoid-autologous T cell co-culture platform that expands tumor-reactive T cells capable of specific tumor organoid killing. This enables personalized immunotherapy development including neoantigen-reactive T cell identification without prior knowledge of HLA restriction.

Cell source
Primary (Human CRC Tumor Organoids + Autologous Peripheral Blood T Cells)
Application
Adoptive Cell Therapy

Protocol overview

26 steps across 8 phases

Organoid Preparation and Characterization Days 1–14
  1. 1 Tissue Collection and Processing
  2. 2 Organoid Embedding in Geltrex
  3. 3 Organoid Culture and Passaging
  4. 4 MHC Class I and PD-L1 Expression Assessment
PBMC Isolation and Pre-culture Days 1–2
  1. 1 PBMC Isolation via Density Gradient Separation
  2. 2 PBMC Thawing and Benzonase Treatment
Tumor Organoid–PBMC Co-culture (Induction Phase) Days 1–14
  1. 1 Plate Preparation with Anti-CD28 Coating
  2. 2 Organoid Pre-stimulation with IFNγ
  3. 3 Organoid Dissociation and Co-culture Initiation
  4. 4 Co-culture Maintenance and Weekly Re-stimulation
T Cell Reactivity Assessment Day 14
  1. 1 T Cell Restimulation for Flow Cytometry
  2. 2 Flow Cytometry Staining for IFNγ and CD107a
  3. 3 CD137 Expression Assessment (Alternative)
T Cell Expansion (Optional) Days 14–28
  1. 1 CD137-Based Enrichment of Tumor-Reactive T Cells (Optional)
  2. 2 Rapid Expansion Protocol
Tumor Organoid Killing Assay Day 21
  1. 1 Plate Preparation and Organoid Pre-stimulation
  2. 2 Organoid Isolation and Cell Quantification
  3. 3 T Cell–Organoid Co-culture for Killing Assay
  4. 4 Live Organoid Quantification via Flow Cytometry
  5. 5 Live Imaging of T Cell-Mediated Killing (Optional)
Specificity Controls: Normal Organoid and Tumor Digest Testing Day 10–14
  1. 1 Establishment of Normal Colon/Lung Organoids
  2. 2 T Cell Restimulation with Control Organoids
  3. 3 Testing Against Tumor Digest (If Available)
Geltrex-Reactivity Control (CD4+ T Cell Validation) Day 10–14
  1. 1 Isolation of Organoids from Geltrex
  2. 2 Monocyte-Derived Dendritic Cell (moDC) Preparation
  3. 3 moDC Loading with Geltrex or Organoid Cells

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dijkstra 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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