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BRAIN Publication-derived

Brain Embryo Models for Developmental Studies

Full SOP with Academic or a plan

Source 10.1038/nmeth.4304

Duration: 80 days 5 phases Cell source: Human iPSC; Human ESC

Abstract

This protocol describes a method to generate three-dimensional cerebral organoid fusions by embedding independently patterned embryoid bodies in matrigel and fusing dorsal and ventral forebrain-like tissues. The approach produces reliable combinations of multiple defined brain regions within a single organoid tissue for studying brain development and regional integration.

Cell source
Human iPSC; Human ESC
Application
Developmental study

Protocol overview

36 steps across 5 phases

Phase 1 · Embryoid Body (EB) Formation and Early Culture Days 0-3
  1. a Prepare single-cell suspension from hPSCs
  2. b Count cell density and assess viability
  3. c Seed EBs in low-attachment plates
  4. d First media exchange
Phase 2 · Neural Induction and Brain Regional Patterning Days 5-11
  1. a Initiate neural induction and small-molecule patterning
  2. b Continue small-molecule patterning
Phase 3 · Embedding EBs in Matrigel and Organoid Fusion Day 12
  1. a Thaw matrigel on ice
  2. b Transfer EBs to differentiation medium
  3. c Prepare parafilm wells
  4. d Transfer one dorsal and one ventral EB to each parafilm well
  5. e Remove excess media
  6. f Embed EBs in matrigel droplets
  7. g Position EBs in matrigel droplet
  8. h Solidify matrigel by incubation
  9. i Remove embedded EBs from parafilm and add differentiation medium
  10. j Transfer to culture incubator
Phase 4 · Differentiation and Maturation of Cerebral Organoid Fusions Days 16-40+
  1. a First differentiation medium exchange
  2. b Transfer to orbital shaker
  3. c Continue differentiation media exchanges
  4. d Prepare matrigel-supplemented media for maturation phase
  5. e Continue maturation culture with matrigel-supplemented media
Phase 5 · Creating Organoid Fusion Slice Cultures Variable (typically starting at day 30-80)
  1. a Prepare low-melt agarose solution
  2. b Aliquot agarose into microcentrifuge tubes
  3. c Transfer organoid fusion to tissue embedding mold
  4. d Remove excess media
  5. e Embed organoid fusion in agarose
  6. f Add additional agarose
  7. g Solidify agarose by chilling
  8. h Attach agarose block to vibratome
  9. i Fill vibratome chamber with ice-cold PBS
  10. j Section organoid fusion tissue with vibratome
  11. k Transfer tissue sections to differentiation medium
  12. l Select slices containing both tissue components
  13. m Plate slice cultures on organotypic cell culture inserts
  14. n First media exchange for slice cultures
  15. o Continue maintenance of slice cultures

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Attribution

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

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