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BRAIN Publication-derived

Meningioma Cerebral Organoids (2020)

Full SOP with Academic or a plan

Source Magill et al., 2020 · University of California San Francisco · 10.1038/s41467-020-18582-7

Authors: Stephen T. Magill, Harish N. Vasudevan, Kyounghee Seo, Javier E. Villanueva-Meyer, Abrar Choudhury, S. John Liu, Melike Pekmezci, Sarah Findakly, Stephanie Hilz, Sydney Lastella, Benjamin Demaree, Steve E. Braunstein, Nancy Ann Oberheim Bush, Manish K. Aghi, Philip V. Theodosopoulos, Penny K. Sneed, Adam R. Abate, Mitchel S. Berger, Michael W. McDermott, Daniel A. Lim, Erik M. Ullian, Joseph F. Costello, David R. Raleigh Duration: 360 days 4 phases Cell source: Human iPSC-derived astrocytes; Patient-derived meningioma cells

Abstract

This protocol describes the development and culture of human cerebral organoids containing predifferentiated astrocytes cocultured with patient-derived meningioma cell lines to model meningioma formation in a physiologic microenvironment. The system enables investigation of meningioma cell interactions with astrocytes and identification of gene expression programs driving meningioma proliferation and invasion.

Cell source
Human iPSC-derived astrocytes; Patient-derived meningioma cells
Application
Disease modeling; Mechanistic interrogation of meningioma tumorigenesis

Protocol overview

22 steps across 4 phases

Phase 1 · Primary Meningioma Cell Line Derivation Days 0-7
  1. a Tissue preparation and cell line establishment
  2. b Cell culture expansion to confluence
  3. c DNA methylation analysis and CNV verification
  4. d Fluorescent protein transduction
Phase 2 · Human Cerebral Organoid Generation and Astrocyte Differentiation Days 0-360
  1. a Neuroepithelial aggregate formation
  2. b Rosette formation and expansion
  3. c Astroglial differentiation induction
  4. d Green fluorescent protein transduction
  5. e Organoid formation from dissociated astrocytes
  6. f Organoid transfer to spheroid microplate
Phase 3 · Meningioma Cell and Cerebral Organoid Coculture Days 0-14
  1. a Meningioma cell preparation and organoid seeding
  2. b Live imaging of meningioma-organoid interactions
  3. c Coculture maintenance and medium changes
  4. d Terminal imaging and quantification at day 14
  5. e Sample collection for single-cell RNA sequencing
Phase 4 · CRISPR Interference and Pharmacologic Inhibition Studies Days 0-10
  1. a Generation of dCas9-KRAB meningioma cells
  2. b sgRNA cloning and lentiviral production
  3. c sgRNA transduction and selection
  4. d Gene repression verification by qRT-PCR
  5. e 2D proliferation assay with CRISPR or pharmacologic inhibition
  6. f Ki-67 immunofluorescence and proliferation quantification
  7. g 3D coculture proliferation inhibition assay

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Attribution

This SOP was written by Organthis based on the published method in Magill et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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