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BRAIN Publication-derived

Recurrent Glioblastoma Brain Organoids

Full SOP with Academic or a plan

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

Authors: Meghan Logun, Xin Wang, Yusha Sun, Stephen J. Bagley, Guo-li Ming, Hongjun Song, Donald M. O'Rourke Duration: 35 days 9 phases Cell source: 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

Phase 1 · Patient-Derived GBO Generation Days -35 to -17 (pre-treatment)
  1. a Tissue collection and transport
  2. b Tissue dissection and preparation
  3. c Culture initiation in GBO medium
  4. d GBO maturation and expansion
Phase 2 · Immunohistochemical Validation of CAR Target Antigens Day 21 (concurrent with GBO generation)
  1. a Tissue sectioning and slide preparation
  2. b Antigen retrieval and blocking
  3. c Primary antibody incubation
  4. d Secondary antibody incubation and mounting
  5. e Confocal imaging and analysis
Phase 3 · CAR-T Cell Product Collection and Characterization Day 0 (day of clinical CAR-T infusion)
  1. a Collect patient CAR-T cell product
  2. b Flow cytometric analysis of CAR expression
  3. c Cell viability and count determination
Phase 4 · GBO and CAR-T Cell Co-Culture and Cytolysis Assay Days 0-6 (concurrent with clinical treatment)
  1. a Prepare CytoView impedance plates
  2. b Plate GBOs and monitor baseline adherence
  3. c Calculate and add CAR-T cells at defined E:T ratio
  4. d Real-time impedance monitoring for 6 days
  5. e Calculate percent cytolysis
Phase 5 · Immunofluorescence Analysis of GBO Cytolysis and T Cell Activation Day 6 (end of co-culture)
  1. a Fix GBOs and prepare tissue sections
  2. b Immunostaining for cleaved-caspase 3 and CD3
  3. c Confocal imaging and quantification of cCas3
Phase 6 · T Cell Activation Marker Analysis (Ki-67, Granzyme B, CD69) Day 6 (end of co-culture)
  1. a Immunostaining for Ki-67 and CD3
  2. b Immunostaining for Granzyme B and CD69
  3. c Confocal imaging and quantification
Phase 7 · Target Antigen Reduction Analysis Day 6 (end of co-culture)
  1. a Immunofluorescence staining for EGFR and IL13Ra2
  2. b Confocal imaging and quantification of antigen levels
  3. c Flow cytometric analysis of antigen expression
Phase 8 · Cytokine Quantification in GBO Co-Culture Media Days 1-6 (daily collection during co-culture)
  1. a Daily media collection and refresh
  2. b ELISA for interferon-gamma (IFN-γ)
  3. c ELISA for interleukin-2 (IL-2)
  4. d ELISA for tumor necrosis factor-alpha (TNF-α)
  5. e Data processing and temporal analysis
Phase 9 · Statistical Analysis and Correlation with Patient Data Post-assay (data analysis)
  1. a Compare GBO cytolysis to patient CSF CAR-T engraftment
  2. b Statistical comparison of treated vs. untreated GBOs
  3. c Multiple comparison correction for cytokine analysis
  4. d Summarize clinical correlations

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Attribution

This SOP was written by Organthis based on the published method in Logun et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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