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BRAIN Publication-derived

Brain 2D Cell Cultures for Drug Screening

Full SOP with Academic or a plan

Source Xu et al., 2016 · National Center for Advancing Translational Sciences, National Institutes of Health; Johns Hopkins University School of Medicine; Florida State University · 10.1038/nm.4184

Authors: Miao Xu, Emily M. Lee, Zhexing Wen, Yichen Cheng, Wei-Kai Huang, Xuyu Qian, Julia TCW, Jennifer Kouznetsova, Sarah C. Ogden, Christy Hammack, Fadi Jacob, Ha Nam Nguyen, Misha Itkin, Catherine Hanna, Paul Shinn, Chase Allen, Samuel G. Michael, Anton Simeonov, Wenwei Huang, Kimberly M. Christian, Alison Goate, Kristen J. Brennand, Ruili Huang, Menghang Xia, Guo-li Ming, Wei Zheng, Hongjun Song, Hengli Tang Duration: 24 days 7 phases Cell source: Human iPSC-derived forebrain-specific neural progenitor cells (hNPCs) and astrocytes

Abstract

This protocol describes a high-throughput drug repurposing screen of ~6,000 compounds to identify inhibitors of Zika virus infection and caspase-3-mediated cell death in human neural cells. The screen identified Emricasan as a pan-caspase inhibitor providing neuroprotection, ten structurally distinct CDK inhibitors showing antiviral activity, and Niclosamide (an FDA-approved anthelmintic) as an inhibitor of ZIKV replication. Combination treatments of neuroprotective and antiviral compounds enhanced protection of human neural progenitors and astrocytes from ZIKV-induced cell death.

Cell source
Human iPSC-derived forebrain-specific neural progenitor cells (hNPCs) and astrocytes
Application
Disease modeling and drug screening for Zika virus infection and neuroprotection

Protocol overview

38 steps across 7 phases

Phase 1 · Cell culture and differentiation 0-24
  1. a Prepare human iPSC colonies
  2. b Detach iPSC colonies and form embryoid bodies (EBs)
  3. c Transition to neural induction
  4. d Maintain rosettes and form neurospheres
  5. e Generate monolayer hNPCs
  6. f Differentiate hNPCs to astrocytes
Phase 2 · Brain organoid generation 0-23
  1. a Initiate organoid formation
  2. b Transition to induction medium
  3. c Embed organoids in Matrigel
  4. d Dissociate organoids from Matrigel and transfer to bioreactor
  5. e Prepare organoids for viral infection
  6. f Test compound toxicity on organoids
Phase 3 · ZIKV preparation and cell infection Variable
  1. a Obtain and amplify ZIKV stocks
  2. b Determine viral titer
  3. c Prepare cells for infection
  4. d Add compound and virus to cells
Phase 4 · High-throughput compound screening Variable
  1. a Prepare compound libraries
  2. b Seed cells for primary screening
  3. c Transfer test compounds
  4. d Incubate compounds with cells
  5. e Caspase-3/7 activity assay
  6. f ATP content cell viability assay
  7. g Normalize and analyze screening data
Phase 5 · Hit confirmation and counter-screening Variable
  1. a Select primary hits
  2. b Confirm caspase-3 activity
  3. c Confirm cell viability protection
  4. d Assess compound cytotoxicity
Phase 6 · Secondary assays and mechanism of action studies Variable
  1. a Western blot analysis of ZIKV protein
  2. b Quantify intracellular ZIKV RNA by qRT-PCR
  3. c Measure infectious ZIKV production by FFU assay
  4. d Perform time-of-addition experiments
  5. e NS1 ELISA for secreted protein quantification
Phase 7 · Immunocytochemistry and immunofluorescence Variable
  1. a Fix cells
  2. b Permeabilize and block
  3. c Apply primary antibodies
  4. d Wash and apply secondary antibodies
  5. e Mount and image
  6. f EdU proliferation assay

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Attribution

This SOP was written by Organthis based on the published method in Xu et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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