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BRAIN Publication-derived

3D Spheroid Culture of SHH Medulloblastoma for Growth, Drug Response, and Migration Studies

Source Roper et al., 2021 · Children's Brain Tumour Research Centre, Biodiscovery Institute, University of Nottingham · 10.1038/s41598-021-83809-6

👤 Sophie J. Roper, Franziska Linke, Paul J. Scotting, Beth Coyle ⏱ 21 days 📋 7 phases 🧫 Human Medulloblastoma Cell Lines (SHH subgroup: DAOY, ONS76, UW228-3, HD-MB03, D458)

Abstract

This protocol establishes a robust, reproducible 3D spheroid culture method for SHH medulloblastoma using ultra-low attachment (ULA) round-bottom 96-well plates. SHH medulloblastoma cell lines form tight, compact spheroids with optimal diameter of 250–350 µm by day 4 when cultured in neurosphere medium, developing pathophysiological oxygen gradients. These 3D spheroids recapitulate tumor biology, exhibit increased drug resistance compared to 2D monolayers, and can be used to model metastatic dissemination across hyaluronan hydrogels, enabling improved pre-clinical drug screening and mechanistic studies of tumor migration.

Cell source
Human Medulloblastoma Cell Lines (SHH subgroup: DAOY, ONS76, UW228-3, HD-MB03, D458)
Application
Disease modeling and drug screening

Protocol overview

51 steps across 7 phases

Cell Preparation and Spheroid Generation Day 0–4
  1. 1 Harvest and Dissociate Cells
  2. 2 Seed Cells into Ultra-Low Attachment (ULA) Plates
  3. 3 Prepare Outer Wells with HBSS
  4. 4 Culture Plates in Incubator
  5. 5 Perform Medium Replenishment
Spheroid Characterization (Morphology and Growth Monitoring) Day 4–21
  1. 1 Image Spheroids on Day 4
  2. 2 Calculate Coefficient of Variation (CV)
  3. 3 Monitor Growth Over 3 Weeks
  4. 4 Assess Spheroid Morphology
Immunohistochemical Characterization of Spheroid Physiology Day 7, 14, 21
  1. 1 Harvest Spheroids
  2. 2 Resuspend Spheroids in HistoGel
  3. 3 Transfer to Cryomold and Cool
  4. 4 Tissue Processing: Fixation and Washing
  5. 5 Tissue Processing: Dehydration
  6. 6 Tissue Processing: Paraffin Infiltration and Embedding
  7. 7 Section Paraffin Blocks
  8. 8 Immunohistochemical Staining for Ki67 (Proliferation Marker)
  9. 9 Immunohistochemical Staining for CA9 (Hypoxia Marker)
  10. 10 Counterstaining and Mounting
  11. 11 Image and Analyze Stained Sections
Drug Response Studies (2D vs. 3D Comparison) Day 4–7
  1. 1 Prepare 2D Monolayer Cells
  2. 2 Prepare 3D Spheroids in Black-Walled ULA Plates
  3. 3 Prepare Drug Stock Solutions
  4. 4 Treat 2D Monolayers with Drug
  5. 5 Treat 3D Spheroids with Drug
  6. 6 Assess 2D Drug Response by CellTiter-Glo 3D Viability Assay (Endpoint)
  7. 7 Assess 3D Drug Response by CellTiter-Glo 3D Viability Assay (Endpoint)
  8. 8 Calculate Drug Response and Generate Dose–Response Curves
  9. 9 Optional: Assess 3D Drug Response by Spheroid Volume Measurement
Dual-Inhibitor Drug Combination Studies Day 4–7
  1. 1 Prepare Drug Combination Stocks
  2. 2 Treat 3D Spheroids with Single Agents and Drug Combinations
  3. 3 Measure Spheroid Volume After 72 h Treatment
  4. 4 Perform Continued Monitoring of Drug Combination Effects
  5. 5 Analyze Statistical Differences
3D Spheroid Migration Assay on Hyaluronan Hydrogels Day 0–7
  1. 1 Prepare Hyaluronan (HA) Hydrogel Solution
  2. 2 Optional: Supplement HA Hydrogel with ECM Components
  3. 3 Coat 96-Well Flat-Bottom Plates with HA Hydrogel
  4. 4 Incubate Hydrogel-Coated Plates
  5. 5 Transfer Day 4 Spheroids to Hydrogel-Coated Plates
  6. 6 Allow Spheroids to Settle on Matrix
  7. 7 Image Spheroids at Multiple Time Points
  8. 8 Quantify Spheroid Outgrowth
  9. 9 Analyze Statistical Differences in Migration Between Matrices
RNA Sequencing and Gene Expression Analysis Day 4–7
  1. 1 Harvest Spheroids for RNA Extraction
  2. 2 Extract RNA
  3. 3 Assess RNA Quality and Quantity
  4. 4 Submit RNA Samples for Next-Generation Sequencing (NGS)
  5. 5 Perform Differential Gene Expression Analysis
  6. 6 Perform Gene Ontology (GO) and Pathway Enrichment Analysis
  7. 7 Identify Fibronectin-Associated Genes
  8. 8 Correlate Gene Expression with Patient Outcomes

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Roper et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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