Skip to content
Back to browse
BRAIN Publication-derived

Glioblastoma Cerebral Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Fedorova et al., 2023 · Masaryk University, Faculty of Medicine, Department of Histology and Embryology · 10.1002/1878-0261.13389

Authors: Veronika Fedorova, Veronika Pospisilova, Tereza Vanova, Katerina Amruz Cerna, Pavel Abaffy, Jiri Sedmik, Jan Raska, Simona Vochyanova, Zuzana Matusova, Jana Houserova, Lukas Valihrach, Zdenek Hodny, Dasa Bohaciakova Duration: 90 days 13 phases Cell source: Human iPSC

Abstract

This protocol establishes a glioblastoma-cerebral organoid (GLICO) coculture model using 55-day-old human iPSC-derived cerebral organoids and U87 glioblastoma cells. The protocol encompasses iPSC maintenance, cerebral organoid differentiation, glioblastoma cell preparation, coculture under various conditions (inclined plane with or without extracellular matrix proteins), visualization and quantification of cell migration via tissue clearing and confocal microscopy, and molecular analysis of cocultured glioblastoma cells.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

49 steps across 13 phases

Phase 1 · iPSC Culture and Maintenance Ongoing (routine passage every 3-5 days)
  1. a iPSC screening and expansion
Phase 2 · Cerebral Organoid Differentiation (Days 1-55) Day 1 to Day 55
  1. a Embryoid body formation
  2. b Medium change at Day 2
  3. c Neuroinduction
  4. d Geltrex embedding and initial culture
  5. e Medium change to vitamin A-containing CODM
  6. f Regular medium changes (Days 20-55)
Phase 3 · Preparation of Thick Organoid Sections (Day 55) Day 55
  1. a Agarose embedding and vibratome sectioning
  2. b Agarose removal and section storage
Phase 4 · Glioblastoma Cell Line Preparation and Labeling Preparation phase (variable duration depending on transfection efficiency)
  1. a U87 cell line culture and mycoplasma screening
  2. b Fluorescent protein transfection
  3. c FACS sorting of positive cells
Phase 5 · U87 Spheroid Formation (24 hours prior to GLICO setup) Day −1 (24 hours before coculture)
  1. a U87 cell detachment and counting
  2. b Spheroid seeding in nonadherent plates
  3. c Centrifugation to facilitate spheroid formation
  4. d Spheroid maturation (overnight)
Phase 6 · GLICO Coculture Setup: Inclined Plane Method (Without ECM) Day 0 to Day 90+ (or analysis timepoint)
  1. a Preparation of nonadherent culture plates and medium
  2. b Spheroid and organoid positioning
  3. c Inclined plane positioning (48 hours)
  4. d Attachment verification and repositioning if needed
  5. e Transfer to standard culture conditions
  6. f Transfer to orbital shaker (Day 4 onwards)
Phase 7 · GLICO Coculture Setup: With ECM Proteins (Matrigel or Geltrex) Day 0 to Day 30-60 (depending on ECM type)
  1. a ECM protein preparation and droplet setup
  2. b ECM polymerization
  3. c Transfer to nonadherent culture dishes
  4. d Culture on orbital shaker with regular medium changes
Phase 8 · Fixation and Preparation for Analysis At analysis timepoint (Day 30, 60, or 90 depending on experimental design)
  1. a Harvesting and fixation of GLICO
  2. b PBS washing and storage
Phase 9 · Tissue Clearing and Whole-Mount Staining (CUBIC Protocol) Variable (7-10 days for complete protocol)
  1. a CUBIC1 incubation
  2. b Washing and nuclear staining with Hoechst
  3. c CUBIC2 incubation for final clearing
  4. d Embedding in mounting solution
Phase 10 · Confocal Microscopy and Image Acquisition Day of analysis
  1. a Histological section imaging (thin sections)
  2. b Cleared whole-mount tissue imaging
Phase 11 · Image Analysis and Cell Migration Quantification Post-acquisition (variable duration depending on dataset size)
  1. a Individual glioblastoma cell detection using IMARIS
  2. b Determination of migration boundary
  3. c Quantification of migration parameters
  4. d Statistical analysis and visualization
Phase 12 · GLICO Dissociation and FACS Sorting (For Molecular Analysis) At analysis timepoint (Day 20 or 40 depending on application)
  1. a GLICO tissue preparation and enzymatic dissociation
  2. b Dilution, filtering, and cell collection
  3. c FACS sorting of GFP-positive glioblastoma cells
Phase 13 · Immunohistochemistry (IHC) on Fixed Sections Post-fixation (1-3 days depending on workflow)
  1. a Tissue embedding and sectioning
  2. b Deparaffinization and rehydration
  3. c Antigen retrieval
  4. d Permeabilization
  5. e Primary antibody incubation
  6. f Washing and secondary antibody incubation
  7. g Nuclear staining and mounting
  8. h Confocal microscopy imaging

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Fedorova et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence