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BRAIN Publication-derived

Traumatic Brain Injury Organoids with Electrical Pacing

Full SOP with Academic or a plan

Source Hu et al., 2024 · Academy of Medical Engineering and Translational Medicine, Tianjin University · 10.1038/s41467-024-53858-2

Authors: Nan Hu, Jian-Xin Shi, Chong Chen, Hai-Huan Xu, Zhe-Han Chang, Peng-Fei Hu, Di Guo, Xiao-Wang Zhang, Wen-Wei Shao, Xiu Fan, Jia-Chen Zuo, Dong Ming, Xiao-Hong Li 9 phases Cell source: Human ESC (H9-hESC)

Abstract

This protocol describes the generation of three-dimensional human brain organoids from H9 embryonic stem cells, their transplantation into injured mouse primary sensory cortex, and implantation of flexible microelectrodes to create organoid-brain-computer interfaces (OBCIs). The procedure enables long-term electrical stimulation and recording to promote organoid maturation, integration with host brain tissue, and functional neural circuit restoration after traumatic brain injury.

Cell source
Human ESC (H9-hESC)
Application
Disease modeling and neural repair via organoid-based brain-computer interfaces

Protocol overview

47 steps across 9 phases

Phase 1 · H9-hESC maintenance and expansion Ongoing maintenance
  1. a Culture H9-hESC in mTeSR plus medium on Matrigel-coated plates
  2. b Passage cells using EDTA dissociation
Phase 2 · Brain organoid generation in vitro (Days 0-60+) D0 to D60+
  1. a Dissociate hESCs and initiate neural induction
  2. b Prepare neural induction medium (NIM)
  3. c Change half the medium every other day (D2-D10)
  4. d Transfer EBs to 6-well plates at D10 in differentiation medium (DIM)
  5. e Culture organoids in mature medium from D20 onwards
  6. f Monitor organoid development to D90-D120 for transplantation readiness
Phase 3 · Organoid-electrode complex in vitro (D90-D150) D90 to D150
  1. a Immobilize organoid on 3D-printed scaffold
  2. b Insert dual-shank flexible electrodes into organoid
  3. c Record spontaneous neural activity from D90 to D120
  4. d Test electrical stimulation parameters (frequency and amplitude)
  5. e Validate stimulation effects on organoid differentiation and maturation
Phase 4 · Brain injury and organoid transplantation (Day 0 dpt) Day 0 post-transplantation (0 dpt)
  1. a Anesthetize NOD/SCID mice and perform stereotactic surgery
  2. b Create brain injury cavity in primary sensory cortex (S1)
  3. c Cut organoid into 1 mm diameter pieces
  4. d Transplant organoid piece into cavity
  5. e Close cranial window and skin incision
  6. f Post-operative recovery and monitoring
Phase 5 · Electrode implantation and early-stage stimulation (25-35 dpt) 25-35 days post-transplantation
  1. a Perform secondary craniotomy and identify graft location
  2. b Implant dual-shank flexible electrode into organoid and host brain
  3. c Secure reference and ground electrodes
  4. d Allow tissue to settle and stabilize (1 hour minimum)
  5. e Record baseline neural activity and verify signal quality
  6. f Initiate early-stage electrical stimulation (10 days duration)
  7. g Record electrophysiological changes during early-stage stimulation
Phase 6 · Late-stage stimulation (60 dpt onwards) 60 days post-transplantation to 180 dpt
  1. a Determine optimal late-stage stimulation frequency
  2. b Initiate late-stage electrical stimulation (10 days duration)
  3. c Record long-term electrophysiological activity (60-180 dpt)
  4. d Maintain animal health and monitor for complications
Phase 7 · Behavioral and sensory testing (60-180 dpt) 60-180 days post-transplantation
  1. a von Frey mechanical sensory test
  2. b Hot-plate test for thermal nociception
  3. c Novel object recognition test
  4. d General locomotor assessment
Phase 8 · Tissue harvest and immunohistochemistry (60, 120, 180 dpt) At 60, 120, 180 days post-transplantation
  1. a Perfuse and fix tissue
  2. b Prepare cryosections
  3. c Permeabilize and block sections
  4. d Incubate with primary antibodies
  5. e Wash and incubate with secondary antibodies
  6. f Counter-stain with DAPI and mount
  7. g Confocal microscopy and image analysis
Phase 9 · Electrophysiological data analysis Offline analysis at any time point
  1. a Pre-process raw neural recordings
  2. b Detect and analyze action potentials (spikes)
  3. c Analyze local field potentials (LFP)
  4. d Compute cross-frequency phase-amplitude coupling (PAC)
  5. e Calculate correlation and connectivity
  6. f Statistical analysis and visualization

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Attribution

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

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