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BRAIN Publication-derived

Establishment and characterization of meningioma patient-derived organoid

Source Chan et al., 2021 · Division of Neurosurgery, Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong · 10.1016/j.jocn.2021.10.035

👤 Henry Siu Cheung Chan, Ho Keung Ng, Aden Ka-Yin Chan, Sau Ha Cheng, Chit Chow, Nathalie Wong, George Kwok Chu Wong ⏱ 14 days 📋 10 phases 🧫 Patient-Derived iPSC (Meningioma)

Abstract

This protocol describes the isolation, culture, and characterization of patient-derived meningioma cells in a three-dimensional organoid system. Meningioma tissues are mechanically and enzymatically dissociated, embedded in Matrigel, and cultured to establish 3D organoids that better preserve tumor architecture and cell-cell interactions compared to 2D culture, enabling disease modeling and therapeutic testing.

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

Protocol overview

46 steps across 10 phases

Tissue Collection and Cell Isolation Day 0
  1. 1 Obtain informed consent and collect meningioma tissue
  2. 2 Transport tissue in HBSS medium
  3. 3 Mechanical dissociation of tumor tissue
Enzymatic Digestion (Method 1: Collagenase/Dispase) Day 0
  1. 1 Prepare digestion buffer
  2. 2 Enzymatic digestion with collagenase/dispase
  3. 3 Centrifuge and resuspend cells
Enzymatic Digestion (Method 2: Trypsin) Day 0
  1. 1 Enzymatic digestion with trypsin
  2. 2 Discard supernatant and resuspend
Cell Preparation and Straining Day 0
  1. 1 Filter cells through 70 μm cell strainer
  2. 2 Centrifuge filtrate and prepare organoid culture medium
Organoid Embedding and Initial Culture Day 0–1
  1. 1 Embed cells in Matrigel
  2. 2 Incubate Matrigel droplets for polymerization
  3. 3 Add culture medium to organoid droplets
Live/Dead Cell Viability Assay Day 1–3
  1. 1 Prepare LIVE/DEAD staining solution
  2. 2 Harvest organoid from Matrigel
  3. 3 Wash organoid with PBS
  4. 4 Incubate organoid with LIVE/DEAD staining solution
  5. 5 Wash stained organoid
  6. 6 Observe and analyze under microscope
Organoid Culture and Morphological Assessment Day 1–14
  1. 1 Observe organoid morphology at Day 1
  2. 2 Observe organoid morphology at Day 7
  3. 3 Observe organoid morphology at Day 14
  4. 4 Replace or refresh culture medium every 2–3 days
Organoid Fixation and Paraffin Processing Day 14
  1. 1 Harvest organoid at Day 14
  2. 2 Transfer organoid to microcentrifuge tube
  3. 3 Fix organoid in paraformaldehyde
  4. 4 Prepare molten histogel mold
  5. 5 Transfer fixed organoid to histogel mold
  6. 6 Place in embedding cassette for FFPE processing
  7. 7 Embed in paraffin block
Hematoxylin and Eosin (H&E) Staining After paraffin embedding
  1. 1 Section paraffin block and mount slides
  2. 2 Deparaffinize slides
  3. 3 Rehydrate slides with ethanol
  4. 4 Stain with hematoxylin
  5. 5 Stain with eosin
  6. 6 Dehydrate and mount slides
  7. 7 Observe H&E-stained sections
Immunohistochemistry Staining (EMA and Ki67) After paraffin embedding
  1. 1 Deparaffinize and rehydrate slides
  2. 2 Perform heat-induced antigen retrieval
  3. 3 Block non-specific antibody binding
  4. 4 Incubate with primary antibody
  5. 5 Wash slides with PBS
  6. 6 Apply secondary antibody and detection reagent
  7. 7 Counter-stain with hematoxylin
  8. 8 Dehydrate and mount slides
  9. 9 Observe immunostained sections

Full SOP

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Attribution

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

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