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BRAIN Publication-derived

Cerebral Organoids for Disease Modelling with Vasculature (2024)

Full SOP with Academic or a plan

Source Lau et al., 2024 · University of Toronto, Department of Molecular Genetics · 10.1098/rsob.180216

Authors: Tsz Yin Charmaine Lau, Jeff Wrana, Jason Moffat, Cindi Peterson Duration: 12 days 10 phases Cell source: Human hESC (H9)

Abstract

This protocol develops vascularized cerebral organoids (vCOs) from human embryonic stem cells for modeling glioblastoma multiforme (GBM) and minimal residual disease. The vCOs incorporate endothelial cells, fibroblasts, and pericytes within a fibrin-collagen hydrogel to provide stromal components and vasculature lacking in conventional organoids. Patient-derived GBM cell lines are co-cultured with vCOs to study tumor-vascular interactions and treatment response in a human-specific 3D microenvironment.

Cell source
Human hESC (H9)
Application
Disease modeling - Glioblastoma Multiforme and minimal residual disease

Protocol overview

36 steps across 10 phases

Phase 1 · hESC Culture and MEF Conditioned Media Production Ongoing maintenance
  1. a Maintain H9 hESC culture
  2. b Prepare irradiated MEF plates for conditioned media production
  3. c Condition media on MEFs
Phase 2 · Embryoid Body (EB) Formation (Day 0-2) D0-D2
  1. a Dissociate and singularize hESCs
  2. b Neutralize TrypLE and collect cells
  3. c Count cells and plate in v-bottom 96-well plate
  4. d Feed and select EBs (D2)
Phase 3 · Neural Induction (Day 4-7) D4-D7
  1. a Transfer EBs and initiate neural induction
  2. b Feed neural induction cultures
Phase 4 · Neuroepithelial Expansion in Matrigel (Day 8-11) D8-D11
  1. a Embed EBs in Growth Factor-Reduced Matrigel
  2. b Feed matrigel-embedded organoids
Phase 5 · Cerebral Organoid Maturation in Spinning Culture (Day 12 onwards) D12 to endpoint (typically 1-3 months for characterization)
  1. a Extract organoids from matrigel and transfer to spinning culture
  2. b Maintain organoids in spinning culture
Phase 6 · Vascularized Cerebral Organoid (vCO) Generation (Day 1-12 post-vascularization) vCO generation and culture (1-1.5-month-old COs used)
  1. a Prepare fibrin hydrogel components
  2. b Prepare and count vascular cell types
  3. c Prepare organoids and assemble vCO
  4. d Allow hydrogel polymerization
  5. e Feed vascularized organoids
Phase 7 · GBM Cell Culture and Maintenance Ongoing maintenance
  1. a Prepare culture plates with basement membrane coating
  2. b Culture three GBM lines derived from patient material: J8-P, J5-P, J3-R.
  3. c Passage GBM cells
Phase 8 · Vascular Cell Culture (BMVE, NHLF, HBVP) Ongoing maintenance for vCO generation
  1. a Culture brain microvascular endothelial cells (BMVE)
  2. b Culture normal human lung fibroblasts (NHLF)
  3. c Culture human brain vascular pericytes (HBVP)
Phase 9 · Hematopoietic Progenitor Cell (HPC) Generation D-1 to D12
  1. a Prepare H9 hESCs at varying densities for HPC differentiation
  2. b Follow STEMdiff Hematopoietic Kit protocol
  3. c Isolate CD43+ HPCs via FACS on D12
  4. d Cryopreserve sorted HPCs
Phase 10 · Immunohistochemistry and Sample Processing Upon endpoint or fixation
  1. a Fix samples in paraformaldehyde
  2. b Cryoprotect organoids and prepare for cryosectioning
  3. c Cryosection organoids
  4. d Rehydrate slides and prepare for staining
  5. e Block and permeabilize samples
  6. f Incubate with primary antibodies
  7. g Incubate with secondary antibodies
  8. h Final washes and mounting

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Attribution

This SOP was written by Organthis based on the published method in Lau et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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