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BRAIN Publication-derived

Traumatic Brain Injury Organoids

Full SOP with Academic or a plan

Source Lai et al., 2024 · Keck School of Medicine, University of Southern California · 10.1016/j.stem.2024.03.004

Authors: Jesse D. Lai, Joshua E. Berlind, Gabriella Fricklas, Cecilia Lie, Jean-Paul Urenda, Kelsey Lam, Naomi Sta Maria, Russell Jacobs, Violeta Yu, Zhen Zhao, Justin K. Ichida Duration: 62 days 9 phases Cell source: Human iPSC, Patient-derived iPSC (C9ORF72 ALS/FTD)

Abstract

This protocol describes the generation of human iPSC-derived cortical organoids and their mechanical injury via high-intensity focused ultrasound (HIFU) to model traumatic brain injury (TBI) pathophysiology. The model recapitulates key TBI hallmarks including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. Through genome-wide CRISPRi screening, KCNJ2 inhibition is identified as protective against injury-induced neuronal death in both healthy and C9ORF72 ALS/FTD organoids.

Cell source
Human iPSC, Patient-derived iPSC (C9ORF72 ALS/FTD)
Application
Disease modeling, traumatic brain injury and ALS pathology in organoids

Protocol overview

37 steps across 9 phases

Phase 1 · iPSC maintenance and cortical organoid generation Day 0-45
  1. a Prepare iPSC spheroids
  2. b Neural induction (Days 1-5)
  3. c Neural expansion phase (Days 6-25)
  4. d Transition to bioreactor and maturation (Days 20-43)
  5. e Prepare organoids for injury experiments
Phase 2 · Lentiviral transduction for cell tracking Day 57-62 (5 days prior to injury)
  1. a Prepare lentiviral vector (SYN1::EGFP)
  2. b Infect organoids with SYN1::EGFP lentivirus
  3. c Verify GFP expression and prepare for immobilization
Phase 3 · Organoid immobilization and mechanical injury via HIFU Day 62 (day 0 of injury protocol)
  1. a Immobilize organoid in Matrigel
  2. b Calibrate HIFU apparatus
  3. c Deliver mechanical injury via HIFU at 0.6 MPa
  4. d Transfer injured organoid to culture dishes
Phase 4 · Live imaging of neuronal survival Day 0-7 post-injury (7 days post-injury; dpi)
  1. a Longitudinal imaging of GFP+ neurons
  2. b Track single-neuron survival from day 0 to day 7
  3. c End-point fixation at day 7 post-injury
Phase 5 · Immunofluorescence analysis of TDP-43 and neuronal markers Day 7 post-injury (tissue processing and staining)
  1. a Tissue rehydration and permeabilization
  2. b Primary antibody staining for TDP-43 and neuronal markers
  3. c Secondary antibody labeling and DAPI counterstaining
  4. d Confocal imaging of TDP-43 localization
Phase 6 · Western blot analysis of neuronal injury markers Day 7 post-injury (protein preparation and blotting)
  1. a Organoid lysis and protein extraction
  2. b Protein quantification
  3. c SDS-PAGE gel preparation and protein separation
  4. d Protein transfer and membrane blocking
  5. e Primary and secondary antibody incubation
  6. f Membrane imaging and quantification
Phase 7 · Tau phosphorylation biomarker analysis (supernatant) Day 0-7 post-injury (sample collection and ELISA)
  1. a Collect organoid supernatant
  2. b Measure phospho-tau (Thr231) and total tau by ELISA
  3. c Statistical analysis of tau biomarkers
Phase 8 · Optional: Chemical inhibition of KCNJ2 (ML133 treatment) 2 h prior to injury through day 7 post-injury
  1. a Prepare ML133 solution
  2. b Pre-treat organoids with ML133
  3. c Perform HIFU injury (as in Phase 3)
  4. d Analyze neuroprotective effect of ML133
Phase 9 · Optional: CRISPRi-based genetic screening for mechanosensitive genes NGN2-spheroid generation through day 7 post-injury
  1. a Generate NGN2-inducible spheroids
  2. b Transduce NGN2-spheroids with CRISPRi library
  3. c Divide spheroids into HIFU-injured and sham control groups
  4. d Culture injured and sham spheroids for 7 days; harvest for sequencing
  5. e Analyze sgRNA enrichment and identify protective hits

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Attribution

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

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