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BRAIN Publication-derived

Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids

Source Azzarelli et al., 2021 · Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge · 10.1242/bio.056416

👤 Roberta Azzarelli, Michela Ori, Anna Philpott, Benjamin D. Simons ⏱ 42 days 📋 5 phases 🧫 Human iPSC (BobC, FSPS-13B, IMR-90) and Patient-Derived GSCs (GCGR-E27, GCGR-E35)

Abstract

This protocol describes a method for generating three-dimensional glioblastoma models by co-culturing patient-derived glioblastoma stem cells (GSCs) with human iPSC-derived cerebral organoids. The method combines organoid generation, GSC fluorescent labeling via lentiviral infection, and optimized co-culture conditions to study GSC integration, differentiation, and heterogeneity in a complex microenvironment.

Cell source
Human iPSC (BobC, FSPS-13B, IMR-90) and Patient-Derived GSCs (GCGR-E27, GCGR-E35)
Application
Disease modeling

Protocol overview

18 steps across 5 phases

hiPSC culture and maintenance Ongoing (every 5-7 days)
  1. 1 Prepare wells with growth substrate
  2. 2 Culture hiPSCs in growth medium
  3. 3 Dissociate and passage hiPSCs
Generation of cerebral organoids (Days 0-10) Days 0-10
  1. 1 Prepare and seed hiPSCs for neural induction
  2. 2 Feed organoids on Day 2 and Day 4
  3. 3 Transfer aggregates and begin neural induction (Day 5)
  4. 4 Embed organoids in Matrigel (Day 7)
  5. 5 Change medium to maturation medium (Day 10)
Organoid maturation and expansion (Days 10-42) Days 10-42
  1. 1 Remove Matrigel from organoids (Day 14)
  2. 2 Culture organoids on orbital shaker
  3. 3 Add Matrigel to medium after Day 30 (optional enhancement)
GSC expansion and lentiviral labeling (Days 1-3 pre-co-culture) Preparation phase (typically 2-3 weeks before co-culture)
  1. 1 Culture patient-derived GSCs in growth medium
  2. 2 Produce lentiviral particles encoding H2B-GFP
  3. 3 Infect GSCs with H2B-GFP lentivirus
GSC-organoid co-culture (Days 0-7) Days 0-7 post-co-culture initiation
  1. 1 Prepare 42-day-old organoids and GSCs
  2. 2 Perform co-culture initiation (Day 0)
  3. 3 Transfer co-culture to standard culture dishes
  4. 4 Maintain glioblastoma-organoid co-culture (GOC) for 7 days

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Azzarelli et al., 2021. 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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