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BRAIN Publication-derived

Meningioma Brain Tumouroids (2021)

Full SOP with Academic or a plan

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

Authors: Henry Siu Cheung Chan, Ho Keung Ng, Aden Ka-Yin Chan, Sau Ha Cheng, Chit Chow, Nathalie Wong, George Kwok Chu Wong Duration: 14 days 10 phases Cell source: 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

Phase 1 · Tissue Collection and Cell Isolation Day 0
  1. a Obtain informed consent and collect meningioma tissue
  2. b Transport tissue in HBSS medium
  3. c Mechanical dissociation of tumor tissue
Phase 2 · Enzymatic Digestion (Method 1: Collagenase/Dispase) Day 0
  1. a Prepare digestion buffer
  2. b Enzymatic digestion with collagenase/dispase
  3. c Centrifuge and resuspend cells
Phase 3 · Enzymatic Digestion (Method 2: Trypsin) Day 0
  1. a Enzymatic digestion with trypsin
  2. b Discard supernatant and resuspend
Phase 4 · Cell Preparation and Straining Day 0
  1. a Filter cells through 70 μm cell strainer
  2. b Centrifuge filtrate and prepare organoid culture medium
Phase 5 · Organoid Embedding and Initial Culture Day 0-1
  1. a Embed cells in Matrigel
  2. b Incubate Matrigel droplets for polymerization
  3. c Add culture medium to organoid droplets
Phase 6 · Live/Dead Cell Viability Assay Day 1-3
  1. a Prepare LIVE/DEAD staining solution
  2. b Harvest organoid from Matrigel
  3. c Wash organoid with PBS
  4. d Incubate organoid with LIVE/DEAD staining solution
  5. e Wash stained organoid
  6. f Observe and analyze under microscope
Phase 7 · Organoid Culture and Morphological Assessment Day 1-14
  1. a Observe organoid morphology at Day 1
  2. b Observe organoid morphology at Day 7
  3. c Observe organoid morphology at Day 14
  4. d Replace or refresh culture medium every 2-3 days
Phase 8 · Organoid Fixation and Paraffin Processing Day 14
  1. a Harvest organoid at Day 14
  2. b Transfer organoid to microcentrifuge tube
  3. c Fix organoid in paraformaldehyde
  4. d Prepare molten histogel mold
  5. e Transfer fixed organoid to histogel mold
  6. f Place in embedding cassette for FFPE processing
  7. g Embed in paraffin block
Phase 9 · Hematoxylin and Eosin (H&E) Staining After paraffin embedding
  1. a Section paraffin block and mount slides
  2. b Deparaffinize slides
  3. c Rehydrate slides with ethanol
  4. d Stain with hematoxylin
  5. e Stain with eosin
  6. f Dehydrate and mount slides
  7. g Observe H&E-stained sections
Phase 10 · Immunohistochemistry Staining (EMA and Ki67) After paraffin embedding
  1. a Deparaffinize and rehydrate slides
  2. b Perform heat-induced antigen retrieval
  3. c Block non-specific antibody binding
  4. d Incubate with primary antibody
  5. e Wash slides with PBS
  6. f Apply secondary antibody and detection reagent
  7. g Counter-stain with hematoxylin
  8. h Dehydrate and mount slides
  9. i Observe immunostained sections

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Attribution

This SOP was written by Organthis based on the published method in Chan et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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