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BRAIN Publication-derived

Meningioma Brain Organoids (2025)

Full SOP with Academic or a plan

Source Zohdy et al., 2025 · Emory University, Department of Neurosurgery · 10.3390/cancers17030526

Authors: Youssef M. Zohdy, Arman Jahangiri, Fadi Jacob, Aliaksandr Aksionau, Ali M. Alawieh, Amelia Tong, Bethany Chern, Justin Maldonado, Kimberly Hoang, Edjah Nduom, Brian M. Howard, Daniel L. Barrow, Stewart G. Neill, Zhexing Wen, Gustavo Pradilla, Tomas Garzon-Muvdi Duration: 80 days 10 phases Cell source: Patient-Derived Meningioma Tissue

Abstract

This protocol describes the establishment and maintenance of patient-derived meningioma organoids (MEN-Os) from surgically resected tumor tissue. MEN-Os are cultured in a serum-free, growth-factor-free medium and preserve the three-dimensional architecture, cellular heterogeneity, tumor microenvironment, and transcriptomic signatures of the original meningiomas, providing a faithful model for studying meningioma biology and therapeutic testing.

Cell source
Patient-Derived Meningioma Tissue
Application
Disease modeling and tumor pathophysiology study

Protocol overview

51 steps across 10 phases

Phase 1 · Tissue Collection and Quality Control Day 0 (Surgery day)
  1. a Patient Sample Collection
  2. b Immediate Tissue Transport
  3. c Histopathological Quality Assessment
Phase 2 · Tissue Processing and Preparation Day 0 (following quality control)
  1. a Transfer to Culture-Grade Medium
  2. b Tissue Dissection and Mincing
  3. c Red Blood Cell Lysis
  4. d Sample Preparation for Downstream Analysis
Phase 3 · Meningioma Organoid Establishment Days 0-14
  1. a Prepare Organoid Culture Medium
  2. b Plate Tissue Pieces
  3. c Initiate Culture with ROCK Inhibitor
  4. d Medium Changes (First 48 Hours Post-Plating)
  5. e Ongoing Culture Maintenance
  6. f Sampling for Analysis
Phase 4 · Growth Analysis and Characterization Days 0, 14, 28
  1. a Brightfield Microscopy Image Capture
  2. b Size Quantification
  3. c Circularity Assessment
Phase 5 · Histological and Immunohistochemical Analysis Days 7-28 (sample-dependent)
  1. a H&E Staining of Paraffin-Embedded Tissue
  2. b Immunohistochemistry (IHC) Staining
  3. c Microscopic Analysis and Interpretation
Phase 6 · Immunofluorescence Staining (Cryosections) Days 7-28 (sample-dependent)
  1. a Cryostat Sectioning
  2. b Tissue Preparation and Blocking
  3. c Primary Antibody Incubation
  4. d Secondary Antibody and Dapi Staining
  5. e Washing, Mounting, and Sealing
  6. f Fluorescence Microscopy Imaging
Phase 7 · RNA Extraction, Purification, and Sequencing Library Preparation Days 7-28 (sample-dependent)
  1. a RNA Sample Thawing and Extraction Initiation
  2. b RNA Extraction and Purification
  3. c RNA Quality and Concentration Assessment
  4. d RNA Storage
  5. e RNA-Seq Library Preparation
  6. f Library Size and Concentration Verification
  7. g RNA-Seq on Illumina NovaSeq6000
  8. h Raw Read Quality Assessment
Phase 8 · RNA Sequencing Data Processing and Analysis Days 30-60 (post-sequencing)
  1. a Read Trimming
  2. b Read Alignment to Reference Genome
  3. c Gene Expression Quantification
  4. d Differential Gene Expression Analysis
  5. e Principal Component Analysis (PCA)
  6. f Transcriptome-Wide Correlation Analysis
  7. g Gene Set Analysis (Optional)
Phase 9 · Bulk RNA Deconvolution Analysis Days 45-75 (post-sequencing)
  1. a Single-Cell Reference Data Acquisition
  2. b Single-Cell Data Processing and Cell Type Annotation
  3. c Reference Matrix Construction
  4. d Bulk RNA Normalization
  5. e CIBERSORTx Deconvolution
  6. f Cellular Composition Comparison
Phase 10 · Transcriptomic Mutations Analysis Days 50-80 (post-sequencing)
  1. a Prepare Aligned and Sorted Reads
  2. b RNA Mutation Calling Pipeline
  3. c Mutation Variant Comparison
  4. d Mutant Transcript Fraction Analysis
  5. e Mutational Concordance Visualization

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Attribution

This SOP was written by Organthis based on the published method in Zohdy et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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