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BRAIN Publication-derived

Detailed Cerebral Organoid Fusion Method

Source <UNKNOWN> · 10.1038/nmeth.4304

👤 <UNKNOWN> ⏱ 80 days 📋 5 phases 🧫 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

Embryoid Body (EB) Formation and Early Culture Days 0–3
  1. 1 Prepare single-cell suspension from hPSCs
  2. 2 Count cell density and assess viability
  3. 3 Seed EBs in low-attachment plates
  4. 4 First media exchange
Neural Induction and Brain Regional Patterning Days 5–11
  1. 5 Initiate neural induction and small-molecule patterning
  2. 6 Continue small-molecule patterning
Embedding EBs in Matrigel and Organoid Fusion Day 12
  1. 7 Thaw matrigel on ice
  2. 8 Transfer EBs to differentiation medium
  3. 9 Prepare parafilm wells
  4. 10 Transfer one dorsal and one ventral EB to each parafilm well
  5. 11 Remove excess media
  6. 12 Embed EBs in matrigel droplets
  7. 13 Position EBs in matrigel droplet
  8. 14 Solidify matrigel by incubation
  9. 15 Remove embedded EBs from parafilm and add differentiation medium
  10. 16 Transfer to culture incubator
Differentiation and Maturation of Cerebral Organoid Fusions Days 16–40+
  1. 17 First differentiation medium exchange
  2. 18 Transfer to orbital shaker
  3. 19 Continue differentiation media exchanges
  4. 20 Prepare matrigel-supplemented media for maturation phase
  5. 21 Continue maturation culture with matrigel-supplemented media
Creating Organoid Fusion Slice Cultures Variable (typically starting at day 30–80)
  1. 1 Prepare low-melt agarose solution
  2. 2 Aliquot agarose into microcentrifuge tubes
  3. 3 Transfer organoid fusion to tissue embedding mold
  4. 4 Remove excess media
  5. 5 Embed organoid fusion in agarose
  6. 6 Add additional agarose
  7. 7 Solidify agarose by chilling
  8. 8 Attach agarose block to vibratome
  9. 9 Fill vibratome chamber with ice-cold PBS
  10. 10 Section organoid fusion tissue with vibratome
  11. 11 Transfer tissue sections to differentiation medium
  12. 12 Select slices containing both tissue components
  13. 13 Plate slice cultures on organotypic cell culture inserts
  14. 14 First media exchange for slice cultures
  15. 15 Continue maintenance of slice cultures

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in the published method. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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