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BRAIN Publication-derived

Glioblastoma and Cerebral Organoids: Development and Analysis of an In Vitro Model for Glioblastoma Migration (GLICO Coculture)

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

👤 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 ⏱ 90 days 📋 13 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fedorova et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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