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BRAIN Publication-derived

Opioid Use Disorder Cortical Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Dwivedi et al., 2023 · University of California, San Diego · 10.1038/s41398-023-02397-3

Authors: Ila Dwivedi, Andrew B. Caldwell, Dan Zhou, Wei Wu, Shankar Subramaniam, Gabriel G. Haddad Duration: 120 days 7 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived cortical organoids (hCOs) and their chronic treatment with methadone (1 μM) for 50 days to model the effects of prenatal methadone exposure on early cortical development. Bulk mRNA-sequencing reveals that methadone alters transcriptional programs associated with synapse formation, extracellular matrix remodeling, and ciliary function, providing insights into the molecular mechanisms underlying neurocognitive deficits linked to maternal opioid addiction treatment.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

23 steps across 7 phases

Phase 1 · iPSC Culture and Organoid Generation Days -7 to 8
  1. a iPSC Culture
  2. b Cortical Organoid Generation
Phase 2 · Methadone Treatment Preparation Day 8
  1. a Methadone Stock Solution Preparation
  2. b Vehicle Control Preparation
Phase 3 · Neural Proliferation I and Methadone Treatment Initiation Days 9-15
  1. a Neural Proliferation Medium with Methadone
Phase 4 · Extended Neural Proliferation and Maturation with Methadone Days 16-60
  1. a Neural Proliferation II Medium (Days 16-22)
  2. b Neural Maturation Medium (Days 28-60)
Phase 5 · Cortical Organoid Collection and RNA Isolation Day 60
  1. a Organoid Harvest and Pellet Formation
  2. b PBS Wash and Second Centrifugation
  3. c Snap Freezing
  4. d Storage
  5. e RNA Extraction
Phase 6 · RNA Quality Assessment and Library Preparation Post-extraction
  1. a RNA Integrity Assessment
  2. b PolyA+ Library Preparation
  3. c Multiplexing and Sequencing
Phase 7 · Protein Validation by Western Blot (Optional, for TSP1 quantification) Days 1-120 (parallel to RNA-seq organoid generation)
  1. a Organoid Generation for Western Blot
  2. b Organoid Collection at 4 Months
  3. c Protein Extraction and Quantification
  4. d SDS-PAGE and Transfer
  5. e Blocking and Antibody Incubation
  6. f Secondary Antibody Incubation and Detection
  7. g Band Intensity Analysis
  8. h Statistical Analysis

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Attribution

This SOP was written by Organthis based on the published method in Dwivedi et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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