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BRAIN Publication-derived

Brain Organoids for Infection Studies (2019)

Full SOP with Academic or a plan

Source Xu et al., 2019 · State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences · 10.1038/s41422-019-0152-9

Authors: Yan-Peng Xu, Yang Qiu, Boya Zhang, Guilai Chen, Qi Chen, Miao Wang, Fan Mo, Jiuyue Xu, Jin Wu, Rong-Rong Zhang, Meng-Li Cheng, Na-Na Zhang, Bao Lyu, Wen-Liang Zhu, Meng-Hua Wu, Qing Ye, Da Zhang, Jiang-Hong Man, Xiao-Feng Li, Jie Cui, Zhiheng Xu, Baoyang Hu, Xi Zhou, Cheng-Feng Qin Duration: 72 days 13 phases Cell source: Human ESC (H9), Patient-Derived hNPC (line 15167)

Abstract

This protocol describes the differentiation of human ESCs into neural progenitor cells (hNPCs) and the generation of brain organoids to model Zika virus (ZIKV) infection and study RNAi-mediated antiviral immunity. The protocol includes methods for ZIKV infection, small RNA sequencing, RNAi pathway manipulation, and therapeutic intervention with enoxacin to prevent ZIKV-induced microcephalic phenotypes in brain organoids.

Cell source
Human ESC (H9), Patient-Derived hNPC (line 15167)
Application
Disease modeling - Zika virus infection and microcephaly phenotype

Protocol overview

57 steps across 13 phases

Phase 1 · H9 ESC Maintenance and Neural Differentiation via Embryoid Bodies Days 0-15
  1. a H9 ESC Maintenance on Matrigel
  2. b Formation of Embryoid Bodies (EBs)
  3. c EB Attachment and Neural Rosette Formation
  4. d Rosette Isolation and Sphere Formation
  5. e hNPC Generation from Neuroepithelial Spheres
  6. f hNPC Plating and Culture
Phase 2 · Neuronal Differentiation from hNPCs Days 25-35
  1. a Neural Progenitor Dissociation
  2. b Neuronal Culture Substrate Preparation and Plating
  3. c Neuronal Maturation
Phase 3 · hNPC Line 15167 Culture Ongoing
  1. a hNPC Line 15167 Maintenance
Phase 4 · Brain Organoid Generation from H9 ESCs Days 0-38
  1. a H9 ESC Preparation for Organoid Formation
  2. b Formation of Pre-organoids
  3. c Pre-organoid Culture (Days 1-7)
  4. d Transition to Neural Medium (Day 8)
  5. e Early Organoid Differentiation (Days 8-22, D20 checkpoint)
  6. f Later Organoid Maturation (Day 23 onwards)
Phase 5 · Zika Virus Infection of hNPCs Single time-point (typically day 0 of infection)
  1. a Viral Stock Preparation
  2. b hNPC Infection Setup
  3. c Infection Medium Replacement
  4. d Infection Monitoring
Phase 6 · RNAi Pathway Manipulation and Analysis Variable (typically 48-72 hours post-transfection/transduction)
  1. a Dicer/AGO2 Knockdown via siRNA
  2. b CRISPR/Cas9-Mediated Dicer KO Generation
  3. c Nodamura Virus B2 Protein Expression
  4. d Synthetic vsiRNA Transfection
Phase 7 · Small RNA Sequencing and Northern Blot Analysis Days 1-3 (post-infection or post-manipulation)
  1. a Total RNA Extraction
  2. b Deep Sequencing Library Preparation
  3. c High-Throughput Sequencing
  4. d Bioinformatic Analysis of Sequencing Data
  5. e Northern Blot Assay for vsRNA Detection
Phase 8 · Enoxacin Treatment and Antiviral Efficacy Assessment Variable (typically 24-72 hours)
  1. a Enoxacin Stock Preparation
  2. b Cytotoxicity Assessment (CC50 Determination)
  3. c Enoxacin Pre-treatment
  4. d ZIKV Infection Under Enoxacin Treatment
  5. e Viral RNA and Protein Quantification
  6. f IC50 Determination
Phase 9 · Brain Organoid Infection and Enoxacin Treatment D20 or D38 organoids, 48-72 hours post-infection
  1. a D20 Brain Organoid Pre-treatment with Enoxacin
  2. b D20 Brain Organoid ZIKV Infection
  3. c D20 Organoid Morphological and Viability Assessment
  4. d D38 Brain Organoid Treatment and Infection
  5. e D38 Organoid Assessment
Phase 10 · Immunofluorescence Assay and Microscopy Post-infection or post-treatment (timing varies)
  1. a Cell Fixation
  2. b Organoid Cryoprotection and Embedding
  3. c Cryosection Rehydration and Fixation
  4. d Blocking
  5. e Primary Antibody Incubation
  6. f Secondary Antibody Incubation
  7. g Nuclear Counterstaining
  8. h Mounting and Microscopy
Phase 11 · EdU Incorporation Assay for Cell Proliferation During infection/treatment period
  1. a EdU Pulse and Detection
Phase 12 · Viral Titration by Plaque Assay Post-infection harvest
  1. a BHK-21 Cell Preparation
  2. b Viral Inoculation and Adsorption
  3. c Overlay Medium Application
  4. d Plaque Staining and Counting
Phase 13 · ZIKV NS1 ELISA and Innate Immune Gene Expression Post-infection harvest (typically 48 hpi)
  1. a NS1 ELISA Sample Preparation
  2. b NS1 ELISA Assay
  3. c Innate Immune Gene Expression Analysis
  4. d Cytokine Multiplex Analysis

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Attribution

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

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