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BRAIN Publication-derived

Tauopathy Brain Assembloids

Full SOP with Academic or a plan

Source Rickner et al., 2022 · Boston University · 10.1038/s41467-022-34005-1

Authors: Hannah Drew Rickner, Lulu Jiang, Rui Hong, Nicholas K. O'Neill, Chromewell A. Mojica, Benjamin J. Snyder, Lushuang Zhang, Dipan Shaw, Maria Medalla, Benjamin Wolozin, Christine S. Cheng Duration: 21 days 5 phases Cell source: Human iPSC

Abstract

The AstTau system combines iPSC-derived neurons and astrocytes in a 3D spheroidal culture to rapidly model tauopathy pathology induced by seeding with human tau oligomers. Over 21 days of culture, the system recapitulates key pathological features of human tau-mediated neurodegeneration including tau misfolding, phosphorylation, aggregation, neuronal loss, and reactive astrogliosis, enabling high-throughput mechanistic studies and drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

25 steps across 5 phases

Phase 1 · Oligomeric Tau Extraction and Preparation Preparation before cell culture
  1. a Collect brain tissue from PS19 P301S transgenic mice
  2. b Homogenize tissue in extraction buffer
  3. c First centrifugation step
  4. d Second centrifugation step (S1p isolation)
  5. e Resuspend and aliquot S1p fraction
  6. f Quantify total tau concentration by immunoblot
  7. g Normalize and prepare oTau working stock
Phase 2 · Neuronal and Astrocytic Differentiation (2D culture) Days 0-21 (2D phase)
  1. a Maintain and passage neural progenitor cells (NPCs)
  2. b Differentiate NPCs into neuronal cells (hiNCs) via NGN2 overexpression
  3. c Differentiate NPCs into astrocytic cells (hiACs) via small-molecule differentiation
  4. d Mature astrocyte precursor cells
  5. e Treat hiNCs with oligomeric tau (oTau) before 3D assembly
Phase 3 · 3D Asteroid Assembly and Culture (DIV3D) Days 0-21 (3D phase)
  1. a Prepare single-cell suspensions of hiNCs and hiACs
  2. b Prepare asteroid culture media and coat AggreWell plates
  3. c Seed cells into AggreWell microwells
  4. d Incubate asteroids in AggreWell plate
  5. e Transfer asteroids to ultra-low-attachment plates
  6. f Maintain asteroids in suspension culture
Phase 4 · Optional Drug Treatment (PU-H71 or Control) Days 19-21 DIV3D
  1. a Prepare PU-H71 treatment stock
  2. b Apply PU-H71 treatment to asteroids
Phase 5 · Sample Collection and Fixation At 7, 14, and 21 DIV3D
  1. a Collect asteroids for fixation (immunofluorescence)
  2. b Wash fixed asteroids
  3. c Collect asteroids for protein lysates (immunoblotting)
  4. d Collect conditioned media (LDH assay)
  5. e Collect asteroids for single-cell RNA-seq

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Attribution

This SOP was written by Organthis based on the published method in Rickner et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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