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BRAIN Publication-derived

Patient-Derived Meningioma and Solitary Fibrous Tumor Organoid Culture and Characterization

Source Yamazaki et al., 2021 · Department of Neurosurgery, Nagoya University Graduate School of Medicine · 10.1093/neuonc/noab155

👤 Shintaro Yamazaki, Fumiharu Ohka, Masaki Hirano, Yukihiro Shiraki, Kazuya Motomura, Kuniaki Tanahashi, Takashi Tsujiuchi, Ayako Motomura, Kosuke Aoki, Keiko Shinjo, Yoshiteru Murofushi, Yotaro Kitano, Sachi Maeda, Akira Kato, Hiroyuki Shimizu, Junya Yamaguchi, Alimu Adilijiang, Toshihiko Wakabayashi, Ryuta Saito, Atsushi Enomoto, Yutaka Kondo, Atsushi Natsume ⏱ 14 days 📋 16 phases 🧫 Patient-Derived Tumor Tissue (Meningioma, Solitary Fibrous Tumor)

Abstract

This protocol describes the establishment and characterization of patient-derived organoid models from meningioma and solitary fibrous tumor tissues with 100% success rate. The organoids maintain histological and molecular features of parental tumors and enable functional studies to identify therapeutic targets such as FOXM1 inhibition for malignant meningiomas.

Cell source
Patient-Derived Tumor Tissue (Meningioma, Solitary Fibrous Tumor)
Application
Disease modeling and drug screening for meningioma

Protocol overview

51 steps across 16 phases

Tissue Preparation and Organoid Generation Days 0-7
  1. 1 Surgical Tissue Collection and Mincing
  2. 2 Cell Dissociation and Debris Removal
  3. 3 Matrigel Embedding and Culture Setup
  4. 4 Medium Refreshment
  5. 5 Organoid Formation Assessment
Organoid Passaging and Maintenance Days 14 and onwards
  1. 1 Organoid Passage Preparation
  2. 2 Matrigel Removal with Dispase
Cryopreservation and Recovery As needed
  1. 1 Organoid Cryopreservation
  2. 2 Organoid Thawing and Recovery
  3. 3 Post-Cryopreservation Validation
Organoid Fixation and Histological Preparation As needed for analysis
  1. 1 Matrigel Removal and Organoid Fixation
  2. 2 Organoid Embedding in Atelocollagen
Immunohistochemistry (IHC) Analysis After fixation and embedding
  1. 1 Tissue Sectioning and Deparaffinization
  2. 2 Antibody Incubation
  3. 3 Image Acquisition and Quantification
Molecular Profiling: DNA and RNA Extraction As needed
  1. 1 DNA Extraction from Organoids, Tumors, and Blood
  2. 2 RNA Extraction from Organoids and Tumors
  3. 3 Nucleic Acid Quality Assessment
Western Blot Analysis As needed
  1. 1 Organoid Cell Lysis
  2. 2 Protein Quantification
  3. 3 SDS-PAGE and Protein Transfer
  4. 4 Antibody Incubation
  5. 5 Detection and Image Analysis
Whole-Exome Sequencing (WES) and Variant Calling As needed
  1. 1 DNA Sample Preparation for WES
  2. 2 Library Preparation and Exome Capture
  3. 3 Sequencing and Data Generation
  4. 4 Variant Calling and Filtering
Targeted Resequencing and Validation As needed
  1. 1 PCR Amplicon Design and Generation
  2. 2 PCR Amplification
  3. 3 Sequencing on MiSeq Platform
Copy Number Variation (CNV) and Structural Variant Analysis As needed
  1. 1 WES Data Analysis for CNV Detection
  2. 2 Identification of Characteristic Alterations
DNA Methylation Analysis 2 weeks after organoid initiation or as needed
  1. 1 Bisulfite Conversion
  2. 2 Methylation Microarray Analysis
  3. 3 Methylation Data Analysis
RNA-Seq Library Preparation and Analysis 2 weeks after organoid initiation or as needed
  1. 1 cDNA Library Preparation
  2. 2 RNA-Seq Clustering and Comparison
  3. 3 Differential Gene Expression Analysis
FOXM1 Gene Knockdown Studies After organoid establishment (typically weeks 3–4)
  1. 1 siRNA Transfection for FOXM1 Knockdown
  2. 2 Assessment of Knockdown Effects on Organoid Proliferation
  3. 3 Ki-67 Immunostaining After Knockdown
FOXM1 Gene Overexpression Studies After organoid establishment (typically weeks 3–4)
  1. 1 FOXM1 Expression Vector Construction
  2. 2 FOXM1 Overexpression Transfection
  3. 3 Assessment of Overexpression Effects on Organoid Proliferation
  4. 4 Ki-67 Immunostaining After Overexpression
Reverse Transcription and Gene Expression Validation As needed
  1. 1 Reverse Transcription of RNA
  2. 2 Gene Expression Analysis by qRT-PCR
Drug Treatment and Radiation Therapy Studies Weeks 3–4 after organoid establishment
  1. 1 Thiostrepton Treatment Optimization
  2. 2 Radiation Therapy Treatment Optimization
  3. 3 Combination Therapy: Thiostrepton and Radiation
  4. 4 Assessment of Treatment Effects on Proliferation

Full SOP

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Attribution

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

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