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BRAIN Publication-derived

Brain Ischaemia Cortical Organoids

Full SOP with Academic or a plan

Source De Paola et al., 2023 · Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy · 10.1177/0271678x231152023

Authors: Massimiliano De Paola, Francesca Pischiutta, Davide Comolli, Alessandro Mariani, Joe Kelk, Ilaria Lisi, Milica Cerovic, Stefano Fumagalli, Gianluigi Forloni, Elisa R Zanier Duration: 72 days 11 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of three-dimensional human cortical organoids (hCO) from induced pluripotent stem cells to model brain ischemic injury through oxygen-glucose deprivation (OGD). The self-assembling organoids recapitulate neuronal-glial interactions and display measurable neurotoxicity proportional to OGD duration, enabling the assessment of neuroprotective strategies and biomarker release relevant to ischemic stroke.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

47 steps across 11 phases

Phase 1 · iPSC Culture and Maintenance Ongoing until neural induction
  1. a Culture episomal iPSC in feeder-free conditions
Phase 2 · Neural Progenitor Cell (NPC) Generation and Expansion Days 0-15 (up to passage 15)
  1. a Induce neural differentiation via SMAD inhibition
  2. b Dissociate and plate primitive NPC at day 7
  3. c Expand NPC in feeder-free conditions
Phase 3 · Human Cortical Organoid (hCO) Generation Days 0-60 (minimum 60 days culture before injury)
  1. a Prepare NPC single-cell suspension
  2. b Seed NPC in ultra-low attachment plates with gyratory shaking
  3. c Change medium to hCO complete medium
  4. d Maintain organoids under continuous gyratory shaking and perform half-medium changes
  5. e Culture organoids to maturity
Phase 4 · Ischemic Injury Induction via Oxygen-Glucose Deprivation (OGD) Day 60 (at maturity)
  1. a Prepare hypoxic conditions and deoxygenated medium
  2. b Expose organoids to OGD for 2 or 8 hours
  3. c Return organoids to normoxic conditions and restore normal medium
  4. d Maintain control organoids under normoxic conditions
Phase 5 · Cell Death Assessment – Lactate Dehydrogenase (LDH) Assay Before OGD, immediately after, and 24 and 48 hours post-OGD (timepoints: Tpre, T0, T24, T48)
  1. a Collect conditioned media at defined timepoints
  2. b Measure LDH activity with bioluminescent assay
  3. c Normalize LDH levels and compare between groups
Phase 6 · Cell Death Visualization – Propidium Iodide (PI) Incorporation 48 hours post-OGD (T48) or beyond
  1. a Incubate organoids with propidium iodide
  2. b Acquire serial focal plane images
  3. c Analyze PI distribution with concentric region segmentation
Phase 7 · Biomarker Measurement – Neurofilament Light Chain (NfL) and GFAP in Culture Media 48 hours post-OGD (T48)
  1. a Collect conditioned media at T48
  2. b Dilute samples in diluent buffer
  3. c Measure NfL with simoa NfL advantage assay
  4. d Measure GFAP with simoa GFAP discovery assay
  5. e Perform quality control by duplicate analysis
Phase 8 · Immunohistochemistry – Whole-Mount Staining for GFAP and MAP-2 72 hours post-OGD (T72)
  1. a Fix organoids with formaldehyde
  2. b Dehydrate organoids with methanol series
  3. c Permeabilize organoids
  4. d Incubate with primary antibodies
  5. e Incubate with secondary antibodies
  6. f Stain nuclei with HOECHST 33258
  7. g Dehydrate organoids for clearing
  8. h Clear tissue with Visikol HISTO-M
Phase 9 · Confocal Microscopy and Quantitative Image Analysis Following whole-mount immunostaining
  1. a Acquire overview images on confocal microscope
  2. b Acquire high-resolution fields for GFAP and MAP-2 quantification
  3. c Quantify GFAP and MAP-2 positive voxels
  4. d Analyze MAP-2 network complexity
Phase 10 · Gene Expression Analysis – Real-Time PCR 3 days post-OGD (T72) for injury samples; 14 and 60 DIV for maturation characterization
  1. a Collect and freeze samples
  2. b Extract total RNA
  3. c Treat with DNase and reverse-transcribe
  4. d Perform real-time reverse transcription PCR
  5. e Analyze and normalize gene expression
Phase 11 · Electrophysiological Characterization (Optional) 60 DIV organoids, at room temperature during recording
  1. a Transfer organoid to recording chamber
  2. b Prepare extracellular and intracellular solutions
  3. c Visualize cells and form whole-cell configuration
  4. d Record evoked action potentials in current-clamp mode
  5. e Record whole-cell currents in voltage-clamp mode
  6. f Analyze recordings with Clampfit software

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Attribution

This SOP was written by Organthis based on the published method in De Paola et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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