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BRAIN Publication-derived

Chronic Stroke Cortical Organoids

Full SOP with Academic or a plan

Source Wang et al., 2025 · University of Maryland School of Medicine · 10.1101/2025.07.05.663297

Authors: Jinghui Wang, Guanda Qiao, Honglin Tan, Mengyang Jacky Li, Kexin Wang, Jiadi Xu, Miroslaw Janowski, Tian-Ming Fu, Piotr Walczak, Yajie Liang Duration: 56 days 11 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of human induced pluripotent stem cell (hiPSC)-derived cortical organoids (COs) and their transplantation into chronic stroke lesions in mice, combined with a multimodal imaging platform (MIPOT) integrating MRI, bioluminescence imaging, light microscopy, and two-photon fluorescence microscopy to track graft viability, location, and functional integration in vivo over time.

Cell source
Human iPSC
Application
Disease modeling; stroke repair and organoid engraftment tracking

Protocol overview

59 steps across 11 phases

Phase 1 · hiPSC Culture and Maintenance Days 1-14 (ongoing)
  1. a Culture hiPSCs on vitronectin-coated plates
  2. b Passage hiPSCs every 4-6 days
Phase 2 · Lentiviral Transduction and Selection of hiPSCs Days 1-14 (post-transduction)
  1. a Transduce hiPSCs with lentiviral reporter vector
  2. b Replace medium and incubate cells
  3. c Perform single-cell cloning by limiting dilution
  4. d Select and expand fluorescent-positive clones
Phase 3 · Generation of Human Cortical Organoids (Days 0-56) Days 1-56
  1. a Form organoid spheroids (Days 0-5)
  2. b Induce neuroectodermal fate (Days 1-5)
  3. c Promote neural progenitor expansion (Days 6-24)
  4. d Induce cortical specification and maturation (Days 25-56)
Phase 4 · Lentiviral Transduction of Cortical Organoids (Days 40-56) Days 40-56
  1. a Prepare organoids for transduction
  2. b Incubate organoids with lentiviral vectors
  3. c Transfer organoids and overnight incubation
  4. d Wash and transfer to 24-well plates
  5. e Monitor transgene expression
  6. f Select organoids for transplantation
Phase 5 · Photothrombotic Stroke Induction (Day 0, relative to stroke) Day 0 (stroke induction)
  1. a Anesthetize mouse
  2. b Prepare surgical field
  3. c Inject photosensitive dye
  4. d Position fiber optic cable and target area
  5. e Illuminate target area with laser
  6. f Post-operative care
Phase 6 · Infarct Maturation and Preparation for Transplantation (Days 1-10 post-stroke) Days 1-10 post-stroke
  1. a Allow infarct to mature
Phase 7 · Organoid Transplantation (Day 10 post-stroke) Day 10 post-stroke
  1. a Anesthetize mouse and perform craniotomy
  2. b Aspirate necrotic tissue and create cavity
  3. c Prepare organoid for transplantation
  4. d Load organoid piece into glass pipette
  5. e Calibrate pipette depth and define z = 0
  6. f Inject organoid at three discrete depths
  7. g Retract pipette slowly
  8. h Maintain hemostasis and irrigation
  9. i Establish cranial window
  10. j Seal surgical site and close scalp
  11. k Provide post-operative analgesia and recovery
  12. l Initiate immunosuppression
Phase 8 · In Vivo MRI Monitoring (Days –3, 1, 14 post-transplantation) Days –3 to 14 post-transplantation
  1. a Acquire T2-weighted MRI images
  2. b Perform diffusion tensor imaging (DTI) at day 14
  3. c Analyze MRI images
Phase 9 · Bioluminescence Imaging (BLI) Monitoring (Days 1-14 post-transplantation) Days 1-14 post-transplantation
  1. a Anesthetize mouse and prepare for BLI
  2. b Inject D-luciferin substrate
  3. c Acquire bioluminescent images
  4. d Quantify photon flux
  5. e Perform serial BLI at multiple timepoints
Phase 10 · Two-Photon Fluorescence Microscopy (TPFM) Imaging (Day 14 post-transplantation) Day 14 post-transplantation
  1. a Prepare mouse for intravital TPFM imaging
  2. b Set up two-photon microscope and optimize imaging parameters
  3. c Acquire three-dimensional z-stack images
  4. d Monitor imaging session duration
  5. e Post-imaging recovery
  6. f Process and analyze two-photon imaging data
Phase 11 · Tissue Collection and Immunohistochemical Analysis (Days 14, 28, 56 post-transplantation) Days 14-56 post-transplantation (terminal timepoints)
  1. a Deeply anesthetize and perfuse mouse
  2. b Post-fix and cryoprotect brain tissue
  3. c Prepare organoids for immunostaining
  4. d Embed tissue in OCT compound and cryosection
  5. e Block and prepare for immunostaining
  6. f Incubate with primary antibodies
  7. g Incubate with secondary antibodies
  8. h Counterstain nuclei and mount
  9. i Image immunostained sections
  10. j Analyze immunostaining data

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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