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BRAIN Publication-derived

Patient-derived glioblastoma organoids as real-time avatars for assessing responses to clinical CAR-T cell therapy

Source Logun et al., 2025 · University of Pennsylvania Perelman School of Medicine · 10.1016/j.stem.2024.11.010

👤 Meghan Logun, Xin Wang, Yusha Sun, Stephen J. Bagley, Guo-li Ming, Hongjun Song, Donald M. O'Rourke ⏱ 35 days 📋 9 phases 🧫 Patient-derived GBM tumor tissue (recurrent glioblastoma)

Abstract

This protocol describes the rapid generation of patient-derived glioblastoma organoids (GBOs) from fresh surgical tissue and their use in real-time co-culture with autologous CAR-T cells (EGFR-IL13Ra2) to assess CAR-T cell bioactivity in a phase 1 clinical trial. GBOs are cultured in parallel with patient treatment to correlate ex vivo tumor cytolysis and cytokine release with clinical CAR-T cell engraftment and patient outcomes.

Cell source
Patient-derived GBM tumor tissue (recurrent glioblastoma)
Application
Disease modeling and CAR-T cell bioactivity assessment

Protocol overview

35 steps across 9 phases

Patient-Derived GBO Generation Days -35 to -17 (pre-treatment)
  1. 1 Tissue collection and transport
  2. 2 Tissue dissection and preparation
  3. 3 Culture initiation in GBO medium
  4. 4 GBO maturation and expansion
Immunohistochemical Validation of CAR Target Antigens Day 21 (concurrent with GBO generation)
  1. 1 Tissue sectioning and slide preparation
  2. 2 Antigen retrieval and blocking
  3. 3 Primary antibody incubation
  4. 4 Secondary antibody incubation and mounting
  5. 5 Confocal imaging and analysis
CAR-T Cell Product Collection and Characterization Day 0 (day of clinical CAR-T infusion)
  1. 1 Collect patient CAR-T cell product
  2. 2 Flow cytometric analysis of CAR expression
  3. 3 Cell viability and count determination
GBO and CAR-T Cell Co-Culture and Cytolysis Assay Days 0–6 (concurrent with clinical treatment)
  1. 1 Prepare CytoView impedance plates
  2. 2 Plate GBOs and monitor baseline adherence
  3. 3 Calculate and add CAR-T cells at defined E:T ratio
  4. 4 Real-time impedance monitoring for 6 days
  5. 5 Calculate percent cytolysis
Immunofluorescence Analysis of GBO Cytolysis and T Cell Activation Day 6 (end of co-culture)
  1. 1 Fix GBOs and prepare tissue sections
  2. 2 Immunostaining for cleaved-caspase 3 and CD3
  3. 3 Confocal imaging and quantification of cCas3
T Cell Activation Marker Analysis (Ki-67, Granzyme B, CD69) Day 6 (end of co-culture)
  1. 1 Immunostaining for Ki-67 and CD3
  2. 2 Immunostaining for Granzyme B and CD69
  3. 3 Confocal imaging and quantification
Target Antigen Reduction Analysis Day 6 (end of co-culture)
  1. 1 Immunofluorescence staining for EGFR and IL13Ra2
  2. 2 Confocal imaging and quantification of antigen levels
  3. 3 Flow cytometric analysis of antigen expression
Cytokine Quantification in GBO Co-Culture Media Days 1–6 (daily collection during co-culture)
  1. 1 Daily media collection and refresh
  2. 2 ELISA for interferon-gamma (IFN-γ)
  3. 3 ELISA for interleukin-2 (IL-2)
  4. 4 ELISA for tumor necrosis factor-alpha (TNF-α)
  5. 5 Data processing and temporal analysis
Statistical Analysis and Correlation with Patient Data Post-assay (data analysis)
  1. 1 Compare GBO cytolysis to patient CSF CAR-T engraftment
  2. 2 Statistical comparison of treated vs. untreated GBOs
  3. 3 Multiple comparison correction for cytokine analysis
  4. 4 Summarize clinical correlations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Logun et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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