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BRAIN Publication-derived

Establishment of a patient-derived 3D in vitro meningioma model in xeno-free hydrogel for clinical applications

Source Andersen et al., 2025 · Department of Neurosurgery, Odense University Hospital, University of Southern Denmark · 10.1186/s40478-025-02008-w

👤 Mikkel Schou Andersen, Aaraby Yoheswaran Nielsen, Martin Wirenfeldt, Jeanette Krogh Petersen, Morten Winkler Møller, Christopher L. Powell, Anavaleria Castro, Grayson Herrgott, Tiit Mathiesen, Charlotte Aaberg Poulsen, Birgitte Brinkmann Olsen, Henning Bünsow Boldt, Christian Bonde Pedersen, Bo Halle, Frantz Rom Poulsen ⏱ 210 days 📋 6 phases 🧫 Patient-Derived iPSC (Meningioma)

Abstract

This protocol establishes patient-derived three-dimensional meningioma models in a xeno-free VitroGel hydrogel. The 3D models retain primary tumor morphology, immunohistochemical markers (EMA, SSTR2, PR, Ki-67), and genome-wide DNA methylation patterns, enabling personalized drug screening and treatment development for individual meningioma patients.

Cell source
Patient-Derived iPSC (Meningioma)
Application
Disease modeling

Protocol overview

25 steps across 6 phases

Patient tissue collection and primary culture establishment Day 0–21 (3 passages × 7 days per passage)
  1. 1 Meningioma tissue procurement and transport
  2. 2 Tissue washing and mechanical dissociation
  3. 3 Primary culture initiation in T75 flasks
  4. 4 Passaging to third passage
3D model establishment from fresh third-passage cells Day 22–112 (up to 3 months of culture)
  1. 1 Prepare cell suspension from third-passage cells
  2. 2 Mix cells with xeno-free hydrogel
  3. 3 Disperse hydrogel–cell mixture into 24-well plates
  4. 4 Culture 3D models for three months
3D model extraction and sample preparation for analysis Day 113–120
  1. 1 Extract 3D models from hydrogel
  2. 2 Prepare samples for histology and immunohistochemistry
  3. 3 Prepare samples for DNA extraction and methylation analysis
Histological and immunohistochemical staining Day 121–130
  1. 1 Perform H&E staining
  2. 2 Perform immunohistochemical staining for marker panels
  3. 3 Digitalize and assess stained slides
DNA methylation analysis Day 131–180
  1. 1 Extract DNA from 3D models
  2. 2 Extract DNA from primary tumor tissue (FFPE)
  3. 3 Verify DNA quality and perform bisulfite conversion
  4. 4 Restore converted DNA and prepare for amplification
  5. 5 Perform DNA amplification
  6. 6 Perform EPICv2 array and data acquisition
Computational analysis of DNA methylation data Day 181–210
  1. 1 Process raw IDAT files and assess probe quality
  2. 2 Generate preprocessed beta matrix and select variant CpG sites
  3. 3 Perform dimensionality reduction and visualization
  4. 4 Identify differentially methylated probes (DMPs)
  5. 5 Perform gene set enrichment analysis (GSEA)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Andersen et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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