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BRAIN Publication-derived

Malignant Brain Tumour Organoids

Full SOP with Academic or a plan

Source Chen et al., 2022 · China Medical University Hospital, Taichung, Taiwan · 10.1038/s41598-022-20487-y

Authors: Chun-Chung Chen, Hong-Wei Li, Yuan-Liang Wang, Chuan-Chun Lee, Yi-Chun Shen, Ching-Yun Hsieh, Hung-Lin Lin, Xian-Xiu Chen, Der-Yang Cho, Ching-Liang Hsieh, Jeng-Hung Guo, Sung-Tai Wei, John Wang, Shao-Chun Wang Duration: 28 days 6 phases Cell source: Patient-derived surgical tumor tissue (glioma, meningioma, lymphoma, and brain metastases)

Abstract

This protocol describes the generation and culture of patient-derived organoids (PDOs) from surgically resected malignant brain tumor tissues, including primary gliomas, meningiomas, lymphomas, and brain metastases. Tumor tissues are dissociated and cultured in serum-free medium on ultra-low attachment plates with orbital agitation, producing three-dimensional organoids within 2 weeks that recapitulate tumor histopathology and can be used for functional drug-response screening to guide personalized therapeutic decisions.

Cell source
Patient-derived surgical tumor tissue (glioma, meningioma, lymphoma, and brain metastases)
Application
Disease modeling and drug screening for personalized precision treatment

Protocol overview

31 steps across 6 phases

Phase 1 · Tumor tissue collection and processing Day 0
  1. a Obtain surgical tumor specimens
Phase 2 · PDO culture initiation and growth phase Days 1-14
  1. a Prepare ultra-low attachment culture plates with PDO medium
  2. b Seed tumor tissue pieces into culture plates
  3. c Set up orbital shaker in incubator
  4. d Perform first medium change
  5. e Continue medium changes every 48 hours
  6. f Monitor organoid development
Phase 3 · PDO characterization by histology and immunohistochemistry Days 14-16
  1. a Collect organoid samples for H&E staining
  2. b Embed organoids in paraffin
  3. c Cut thin tissue sections
  4. d Perform hematoxylin and eosin (H&E) staining
  5. e Perform immunohistochemistry (IHC) for GFAP
  6. f Examine and compare histopathological features
Phase 4 · PDO propagation and passaging Days 14 onwards (as needed for drug testing)
  1. a Assess organoid size and viability
  2. b Dissect organoids into smaller pieces
  3. c Transfer dissected organoid pieces to fresh medium
  4. d Continue culture maintenance
Phase 5 · BrdU incorporation assay for drug response assessment Days 21-28 (or as needed for drug testing)
  1. a Prepare organoids for drug testing
  2. b Add test drug to organoid cultures
  3. c Add BrdU labeling reagent
  4. d Centrifuge and dry microplate
  5. e Fix organoids in FixDenat solution
  6. f Incubate with anti-BrdU-POD antibody
  7. g Wash wells and add substrate
  8. h Terminate reaction and measure absorbance
  9. i Normalize BrdU data by organoid diameter
Phase 6 · Cell viability and death detection by propidium iodide and Hoechst staining Days 21-28 (as needed to confirm drug-induced cell death)
  1. a Treat organoids with drug and incubate
  2. b Wash organoids with PBS
  3. c Stain with Hoechst 33342 and propidium iodide
  4. d Image organoids by fluorescence microscopy
  5. e Quantify and analyze cell death

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Attribution

This SOP was written by Organthis based on the published method in Chen et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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