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BRAIN Publication-derived

Cerebral Malaria Brain Organoids

Full SOP with Academic or a plan

Source Harbuzariu et al., 2019 · Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology · 10.1038/s41598-019-55631-8

Authors: Adriana Harbuzariu, Sidney Pitts, Juan Carlos Cespedes, Keri Oxendine Harp, Annette Nti, Andrew P. Shaw, Mingli Liu, Jonathan K. Stiles Duration: 40 days 10 phases Cell source: Human iPSC (CD34+ umbilical cord blood-derived)

Abstract

This protocol describes the generation and characterization of human brain cortical organoids derived from CD34+ umbilical cord blood-derived iPSCs to model heme-mediated brain injury associated with cerebral malaria. The organoids are exposed to heme to recapitulate pathogenic mechanisms and tested for neuroprotective effects of neuregulin-1 (NRG-1) treatment.

Cell source
Human iPSC (CD34+ umbilical cord blood-derived)
Application
Disease modeling

Protocol overview

41 steps across 10 phases

Phase 1 · iPSC Characterization and Culture Passage 3-4
  1. a iPSC Morphology Assessment
  2. b Flow Cytometry Assessment of Pluripotency Markers
  3. c iPSC Culture Maintenance
Phase 2 · Embryoid Body Formation and Neural Induction Days 0-10
  1. a Embryoid Body (EB) Initiation
  2. b EB Maturation
  3. c Neural Induction
Phase 3 · Neuroepithelial Expansion in Matrigel Days 10-14
  1. a Matrigel Embedding
  2. b Transfer to Culture Plates
  3. c Formation of Ventricular-like Cavities
Phase 4 · Organoid Maturation Days 14-40+
  1. a Matrix Removal and Transfer
  2. b Orbital Shaker Culture
  3. c Medium Changes
  4. d Developmental Assessment
Phase 5 · Heme and NRG-1 Treatment of Organoids Days 20-40 (treatment window)
  1. a Heme Treatment Preparation
  2. b Organoid Heme Exposure
  3. c NRG-1 Co-treatment
  4. d Control Treatments
Phase 6 · Apoptosis and Cell Viability Assessment in Organoids After heme and NRG-1 treatment (hours 26-28)
  1. a Real-Time Glo Annexin V Apoptosis/Necrosis Assay Setup
  2. b Apoptosis Detection (Chemiluminescence Readout)
  3. c Necrosis Detection (Fluorescence Readout)
  4. d Fluorescence Microscopy of Necrotic Cells
Phase 7 · Histological Analysis and Immunohistochemistry After treatment (day 20 or 40)
  1. a Organoid Fixation
  2. b Tissue Sectioning and Preparation
  3. c Hematoxylin and Eosin (H&E) Staining
  4. d Epitope Unmasking
  5. e Blocking and Primary Antibody Incubation
  6. f Secondary Antibody and Fluorescence Staining
  7. g Mounting and Confocal Imaging
  8. h Quantification of Marker Expression
Phase 8 · iPSC Viability and Apoptosis Studies Single timepoint experiments
  1. a CCK-8 Cell Viability Assay
  2. b Optical Density Measurement
  3. c Annexin V Apoptosis/Necrosis Assay
  4. d Flow Cytometry Data Analysis
Phase 9 · Pluripotency Marker Analysis Single timepoint experiments
  1. a Heme-Induced Differentiation Assay Setup
  2. b Flow Cytometry Assessment of Pluripotency Markers
  3. c Flow Cytometry Data Acquisition and Analysis
Phase 10 · ERBB4 and NRG-1 Expression Analysis in iPSCs Single timepoint experiments
  1. a ERBB4 Flow Cytometry Assessment
  2. b Western Blot Sample Preparation
  3. c SDS-PAGE Separation
  4. d Antibody Incubation
  5. e Chemiluminescent Detection and Quantification

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Attribution

This SOP was written by Organthis based on the published method in Harbuzariu et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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