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BRAIN Publication-derived

Methamphetamine Exposure Cerebral Organoids

Full SOP with Academic or a plan

Source Dang et al., 2021 · University of California San Diego · 10.1038/s41380-020-0676-x

Authors: Jason Dang, Shashi Kant Tiwari, Tariq M. Rana Duration: 11 days 8 phases Cell source: Human ESC (H9)

Abstract

This protocol describes the generation of self-organizing human cerebral organoids from embryonic stem cells and their characterization using single-cell RNA sequencing to study methamphetamine-induced neuroinflammation and glial cell diversity. Organoids treated with METH display marked transcriptional changes in astrocytes and neural progenitor cells, including upregulation of neuroinflammatory, immune response, and apoptosis-related genes.

Cell source
Human ESC (H9)
Application
Disease modeling (prenatal methamphetamine exposure, neuroinflammation)

Protocol overview

42 steps across 8 phases

Phase 1 · H9 hESC Culture and Embryoid Body Formation Days 0-6
  1. a Prepare H9 hESCs from feeder layer
  2. b Form embryoid bodies using hanging drop method
  3. c Transfer embryoid bodies to petri dishes
  4. d Transition to neural induction media
Phase 2 · Cerebral Organoid Differentiation in Matrigel and Bioreactor Culture Days 11-METH treatment (variable)
  1. a Embed organoids in Matrigel droplets
  2. b Transfer organoids to stir flask bioreactors
  3. c Perform regular media changes during organoid maturation
Phase 3 · METH Treatment of Cerebral Organoids Day treatment varies (10-month-old organoids treated for 1 week)
  1. a Prepare METH treatment solution
  2. b Administer METH to cerebral organoids
  3. c Collect supernatants for ELISA analysis
Phase 4 · Organoid Dissociation and Single-Cell RNA Sequencing (scRNA-seq) End of treatment period
  1. a Dissociate organoids into single-cell suspension
  2. b Filter and assess cell viability
  3. c Load cells into 10x Chromium microfluidic system
  4. d Perform reverse transcription in GEMs
  5. e Recover and amplify cDNA
  6. f Construct sequencing libraries
  7. g Analyze library quality and quantify
  8. h Sequence libraries on HiSeq4000
Phase 5 · Immunohistochemistry and Immunofluorescence Analysis End of treatment period or in parallel
  1. a Prepare organoids for immunostaining
  2. b Prepare organoid tissue sections
  3. c Block non-specific antibody binding
  4. d Incubate with primary antibodies
  5. e Wash away primary antibody
  6. f Incubate with secondary antibodies
  7. g Final washing and mounting
Phase 6 · BrdU Incorporation and Proliferation Analysis During neural stem cell culture
  1. a Pulse cells with BrdU
  2. b Denature DNA for BrdU detection
  3. c Block and incubate with anti-BrdU antibody
  4. d Detect BrdU with fluorescent secondary antibody
Phase 7 · Protein Analysis by Immunoblotting End of treatment period
  1. a Prepare organoid and astrocyte lysates
  2. b Quantify total protein
  3. c Separate proteins by SDS-PAGE
  4. d Block and incubate with primary antibodies
  5. e Wash and incubate with secondary antibody
  6. f Detect immunoreactive proteins
  7. g Analyze and quantify protein bands
Phase 8 · ELISA for IL-6 Detection During and after METH treatment
  1. a Coat ELISA plate with capture antibody
  2. b Block and prepare wells
  3. c Add standards and samples
  4. d Wash and add detection antibody
  5. e Add Avidin-HRP and substrate
  6. f Read and analyze ELISA results

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Attribution

This SOP was written by Organthis based on the published method in Dang et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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