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BRAIN Publication-derived

Methadone Treatment and Transcriptional Analysis in Human Cortical Organoids

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

👤 Ila Dwivedi, Andrew B. Caldwell, Dan Zhou, Wei Wu, Shankar Subramaniam, Gabriel G. Haddad ⏱ 120 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dwivedi et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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