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BRAIN Publication-derived

Brain Organoids for Disease Modelling (2024)

Full SOP with Academic or a plan

Source Hendriks et al., 2024 · Princess Máxima Center for Pediatric Oncology; Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) · 10.1016/j.cell.2023.12.012

Authors: Delilah Hendriks, Anna Pagliaro, Francesco Andreatta, Ziliang Ma, Joey van Giessen, Simone Massalini, Carmen López-Iglesias, Gijs J.F. van Son, Jeff DeMartino, J. Mirjam A. Damen, Iris Zoutendijk, Nadzeya Staliarova, Annelien L. Bredenoord, Frank C.P. Holstege, Peter J. Peters, Thanasis Margaritis, Susana Chuva de Sousa Lopes, Wei Wu, Hans Clevers, Benedetta Artegiani Duration: 30 days 7 phases Cell source: Human fetal brain tissue (gestational weeks 12-15)

Abstract

This protocol describes the establishment and culture of human fetal brain organoids (FeBOs) derived from small tissue fragments (1-2 mm diameter) of healthy human fetal central nervous system tissue. FeBOs self-organize in vitro into long-term expanding 3D structures that recapitulate cellular heterogeneity, regional identity, and tissue-like extracellular matrix niche properties of developing human brain, enabling studies of neural development, regional specification, and disease mechanisms.

Cell source
Human fetal brain tissue (gestational weeks 12-15)
Application
Developmental study; Disease modeling; Cancer modeling

Protocol overview

22 steps across 7 phases

Phase 1 · Tissue Isolation and Fragment Preparation Day 0
  1. a Obtain human fetal CNS tissue
  2. b Dissect and cut tissue into small fragments
  3. c Verify cellular composition
Phase 2 · FeBO Establishment and Initial Culture Days 0-8
  1. a Plate tissue fragments in expansion medium
  2. b Place cultures on orbital shaker
  3. c Monitor organoid formation
Phase 3 · FeBO Expansion and Passaging Days 20-30 (initial passage); every 2 weeks thereafter
  1. a Allow initial FeBO growth
  2. b Monitor organoid size
  3. c Passage organoids by mechanical cutting
  4. d Plate cut organoid pieces
  5. e Repeat passaging every 2 weeks
Phase 4 · Optional: FeBO Maturation 10 days maturation pulse
  1. a Switch to maturation medium
  2. b Culture in maturation medium
Phase 5 · Optional: Morphogen Treatment 7 days of exposure
  1. a Prepare morphogen treatment conditions
  2. b Expose organoids to BMP4 + CHIR-99021
Phase 6 · Optional: ECM Perturbation Studies Experimental duration varies
  1. a Perform ECM perturbation experiments
  2. b Analyze growth and organization changes
Phase 7 · Optional: CRISPR-Based Genetic Engineering for Disease Modeling Variable; engineering to clonal line establishment typically 3 months
  1. a Prepare organoids for electroporation
  2. b Perform whole-organoid electroporation
  3. c Culture engineered organoids and monitor clonal expansion
  4. d Establish isogenic clonal organoid lines
  5. e Perform mutation-drug sensitivity assays

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Attribution

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

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