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BRAIN Publication-derived

Glioblastoma Brain Organoids (2021)

Full SOP with Academic or a plan

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

Authors: Roberta Azzarelli, Michela Ori, Anna Philpott, Benjamin D. Simons Duration: 42 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Azzarelli et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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