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INTESTINAL Publication-derived

Intestinal Organoids for Regenerative Medicine with Immune Cells

Full SOP with Academic or a plan

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

Authors: 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 Duration: 28 days 8 phases Cell source: 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

Phase 1 · Organoid Preparation and Characterization Days 1-14
  1. a Tissue Collection and Processing
  2. b Organoid Embedding in Geltrex
  3. c Organoid Culture and Passaging
  4. d MHC Class I and PD-L1 Expression Assessment
Phase 2 · PBMC Isolation and Pre-culture Days 1-2
  1. a PBMC Isolation via Density Gradient Separation
  2. b PBMC Thawing and Benzonase Treatment
Phase 3 · Tumor Organoid–PBMC Co-culture (Induction Phase) Days 1-14
  1. a Plate Preparation with Anti-CD28 Coating
  2. b Organoid Pre-stimulation with IFNγ
  3. c Organoid Dissociation and Co-culture Initiation
  4. d Co-culture Maintenance and Weekly Re-stimulation
Phase 4 · T Cell Reactivity Assessment Day 14
  1. a T Cell Restimulation for Flow Cytometry
  2. b Flow Cytometry Staining for IFNγ and CD107a
  3. c CD137 Expression Assessment (Alternative)
Phase 5 · T Cell Expansion (Optional) Days 14-28
  1. a CD137-Based Enrichment of Tumor-Reactive T Cells (Optional)
  2. b Rapid Expansion Protocol
Phase 6 · Tumor Organoid Killing Assay Day 21
  1. a Plate Preparation and Organoid Pre-stimulation
  2. b Organoid Isolation and Cell Quantification
  3. c T Cell–Organoid Co-culture for Killing Assay
  4. d Live Organoid Quantification via Flow Cytometry
  5. e Live Imaging of T Cell-Mediated Killing (Optional)
Phase 7 · Specificity Controls: Normal Organoid and Tumor Digest Testing Day 10-14
  1. a Establishment of Normal Colon/Lung Organoids
  2. b T Cell Restimulation with Control Organoids
  3. c Testing Against Tumor Digest (If Available)
Phase 8 · Geltrex-Reactivity Control (CD4+ T Cell Validation) Day 10-14
  1. a Isolation of Organoids from Geltrex
  2. b Monocyte-Derived Dendritic Cell (moDC) Preparation
  3. c moDC Loading with Geltrex or Organoid Cells

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Attribution

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

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