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BRAIN Publication-derived

Intravital Multimodal Imaging of Human Cortical Organoids for Chronic Stroke Treatment in Mice (MIPOT)

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

👤 Jinghui Wang, Guanda Qiao, Honglin Tan, Mengyang Jacky Li, Kexin Wang, Jiadi Xu, Miroslaw Janowski, Tian-Ming Fu, Piotr Walczak, Yajie Liang ⏱ 56 days 📋 11 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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