Skip to content
← Back to browse
BRAIN Publication-derived

A 3D human brain–like tissue model of herpes-induced Alzheimer's disease

Source Cairns et al., 2020 · Department of Biomedical Engineering, Tufts University · 10.1126/sciadv.aay8828

👤 Dana M. Cairns, Nicolas Rouleau, Rachael N. Parker, Katherine G. Walsh, Lee Gehrke, David L. Kaplan ⏱ 35 days 📋 7 phases 🧫 Human fibroblast-derived induced neural stem cells (hiNSCs)

Abstract

This protocol describes a 3D bioengineered human brain tissue model using hiNSCs to study herpes simplex virus type 1 (HSV-1)-induced Alzheimer's disease pathology. The model recapitulates key AD hallmarks including amyloid-beta plaque-like formations, neurofibrillary tangles, reactive gliosis, neuroinflammation, and reduced neural network functionality without exogenous AD mediators.

Cell source
Human fibroblast-derived induced neural stem cells (hiNSCs)
Application
Disease modeling

Protocol overview

62 steps across 7 phases

hiNSC Generation and Propagation Days 0–30+
  1. 1 Plate human foreskin fibroblasts
  2. 2 Culture fibroblasts in fibroblast media
  3. 3 Transduce fibroblasts with lentiviral vector
  4. 4 Switch to hiNSC media
  5. 5 Trypsinize and plate on feeder layers
  6. 6 Feed hiNSC colonies on feeder layers
  7. 7 Pick and expand individual colonies
  8. 8 Enzymatically passage and freeze stocks
HSV-1 Virus Production and Titration Days 1–3
  1. 1 Prepare HSV-1 McIntyre strain
  2. 2 Infect Vero monolayers with HSV-1
  3. 3 Harvest virus after 48 hours
  4. 4 Centrifuge to remove cell debris
  5. 5 Measure virus titer by plaque assay
hiNSC Differentiation and 2D HSV-1 Infection Days 1–7
  1. 1 Trypsinize hiNSC colonies from feeder layers
  2. 2 Filter cell suspension through cell strainer
  3. 3 Culture dissociated hiNSCs on gelatin-coated plates
  4. 4 Prepare HSV-1 inoculum at defined MOI
  5. 5 Infect hiNSCs with HSV-1
  6. 6 Prepare valacyclovir HCl treatment
  7. 7 Add VCV at Day 0 or Day 1
  8. 8 Culture infected hiNSCs for endpoint analysis
3D Cortical Brain Tissue Model Construction Days 1–30
  1. 1 Prepare silk protein sponges
  2. 2 Punch silk sponges into donut-shaped scaffolds
  3. 3 Autoclave scaffolds
  4. 4 Coat scaffolds with laminin
  5. 5 Seed hiNSCs into silk scaffolds
  6. 6 Allow cell adhesion overnight
  7. 7 Prepare type I rat tail collagen gel
  8. 8 Infuse scaffolds with collagen gel
  9. 9 Culture 3D constructs in neurobasal media
  10. 10 Maintain 3D culture for 4 weeks prior to infection
3D HSV-1 Infection and Antiviral Treatment Days 28–35
  1. 1 Prepare HSV-1 inoculum for 3D constructs
  2. 2 Infect mature 3D constructs with HSV-1
  3. 3 Culture infected 3D constructs for 1 week
  4. 4 Optional: Add valacyclovir HCl to 3D constructs
Molecular and Cellular Analyses Endpoint (Day 3–7 for 2D; Day 7 for 3D)
  1. 1 Fix samples in paraformaldehyde
  2. 2 Prepare blocking buffer for immunostaining
  3. 3 Block samples with buffer
  4. 4 Incubate with primary antibodies
  5. 5 Wash samples with PBS
  6. 6 Incubate with secondary antibodies
  7. 7 Wash and counterstain with DAPI
  8. 8 Mount samples for microscopy
  9. 9 Image samples
  10. 10 Perform Thioflavin T staining (optional)
  11. 11 Harvest RNA for qPCR
  12. 12 Synthesize complementary DNA
  13. 13 Perform quantitative reverse transcription PCR
  14. 14 Perform Alzheimer's disease gene array (optional)
  15. 15 Harvest conditioned media for ELISA
  16. 16 Measure amyloid-beta isoforms by ELISA
Scanning Electron Microscopy and Electrophysiology Endpoint (Day 7 for 3D constructs)
  1. 1 Prepare 3D constructs for SEM
  2. 2 Gold-sputter-coat samples
  3. 3 Image with scanning electron microscope
  4. 4 Prepare samples for local field potential recording
  5. 5 Warm samples to physiological temperature
  6. 6 Secure samples to prevent movement artifacts
  7. 7 Fabricate recording electrodes
  8. 8 Measure electrode impedance
  9. 9 Position electrode within construct
  10. 10 Record local field potentials
  11. 11 Analyze electrophysiological data

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Cairns et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence