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BRAIN Publication-derived

Congenital Cytomegalovirus Infection Cortical Organoids

Full SOP with Academic or a plan

Source Sison et al., 2019 · Medical College of Wisconsin · 10.1128/jvi.00954-19

Authors: Samantha L. Sison, Benjamin S. O'Brien, Amanda J. Johnson, Emily R. Seminary, Scott S. Terhune, Allison D. Ebert Duration: 60 days 8 phases Cell source: Human iPSC

Abstract

This protocol investigates HCMV infection of human iPSC-derived neural progenitor cells and cortical organoids to model congenital CMV-induced microcephaly. The protocol measures calcium signaling dysfunction in infected neurons and astrocytes using live-cell imaging and assesses organoid structure and neural differentiation markers under viral infection with and without antiviral treatment.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

22 steps across 8 phases

Phase 1 · Viral Stock Preparation and Cell Culture Setup Day -30 to Day 0
  1. a Prepare HCMV TB40/E-eGFP Viral Stock
  2. b Culture MRC-5 Fibroblasts
  3. c Generate iPSC-Derived Neural Progenitor Cells (NPCs)
Phase 2 · NPC and Fibroblast Infection with HCMV Day 1-28
  1. a Infect NPCs with HCMV
  2. b Infect MRC-5 Fibroblasts with HCMV
Phase 3 · Live-Cell Calcium Imaging Day 2-14 (relative to infection)
  1. a Prepare Cells for Calcium Imaging
  2. b Baseline Calcium Measurement
  3. c ATP Stimulation for Purinergic Response
  4. d KCl Stimulation for Voltage-Gated Calcium Channel Response
  5. e Data Analysis and Quantification
Phase 4 · Thapsigargin Treatment (ER Calcium Depletion Control) Day 3 (fibroblasts) or Day 14 (NPCs)
  1. a Treat Cells with Thapsigargin
Phase 5 · Cortical Organoid Generation Day 0-60
  1. a Embryoid Body (EB) Formation
  2. b Neural Induction (Days 6-10)
  3. c Organoid Maturation in Matrigel (Days 11-14)
  4. d Organoid Maturation with Vitamin A (Days 15-60)
Phase 6 · Cortical Organoid Infection Day 30 or Day 60
  1. a Organoid Infection with HCMV
  2. b Monitor Viral Infection Progress
Phase 7 · Organoid Dissociation and Neural Cell Calcium Imaging Day 14 (postinfection)
  1. a Dissociate Organoids
  2. b Perform Calcium Imaging on Organoid-Derived Cells
Phase 8 · RNA and Immunofluorescence Analysis of Organoids Day 14 (postinfection)
  1. a RNA Isolation and RT-PCR
  2. b Organoid Fixation and Cryosectioning
  3. c Immunofluorescence Staining

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Attribution

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

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