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BRAIN Publication-derived

Buprenorphine and methadone differentially alter early brain development in human cortical organoids

Source Yao et al., 2023 · University of California, San Diego · 10.1016/j.neuropharm.2023.109683

👤 Hang Yao, Daisy Hu, Juan Wang, Wei Wu, Helen H. Zhao, Lu Wang, Joe Gleeson, Gabriel G. Haddad ⏱ 24 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and treatment of human cortical organoids (hCOs) from induced pluripotent stem cells to model the differential neurodevelopmental effects of buprenorphine and methadone. The organoids are used to assess opioid-induced changes in organoid growth and neuronal activity via multi-electrode array recordings, providing a human-relevant model to compare the pharmacological impacts of medication-assisted treatment drugs on early fetal brain development.

Cell source
Human iPSC
Application
Disease modeling; Drug screening

Protocol overview

39 steps across 8 phases

iPSC Culture and Embryoid Body Formation Days 0–1
  1. 1 Daily iPSC feeding
  2. 2 iPSC dissociation
  3. 3 Cell centrifugation and resuspension
  4. 4 Embryoid body formation in suspension
Neural Differentiation and Media Transitions Days 2–21
  1. 5 Media1 application (Days 3–10)
  2. 6 Media2 with FGF2 application (Week 2)
  3. 7 Media2 with FGF2 and EGF application (Week 3)
  4. 8 Media3 application (Week 4)
  5. 9 Long-term maintenance in Media2
Drug Treatment and Organoid Growth Monitoring Days 1–24
  1. 10 Addition of methadone or buprenorphine
  2. 11 Daily organoid imaging
  3. 12 Organoid diameter measurement
Pre-treatment Studies (nBNI and U69593 antagonist/agonist experiments) Day 0–16
  1. 13 nBNI pre-treatment (1 hour)
  2. 14 Combined nBNI + MTD treatment (16 days)
  3. 15 nBNI single-treatment control (16 days)
  4. 16 U69593 κ-agonist treatment (16 days)
  5. 17 BUP pre-treatment and combined BUP + MTD treatment (16 days)
Immunofluorescence Staining (10 weeks post-seeding) Week 10
  1. 18 Organoid plating on coated dishes
  2. 19 Paraformaldehyde fixation
  3. 20 Permeabilization
  4. 21 Blocking and antibody incubation
  5. 22 Secondary antibody incubation
  6. 23 DAPI counterstaining
  7. 24 Coverslip application and microscopy
Multi-Electrode Array (MEA) Recordings (12-week-old organoids) Week 12 to week 14
  1. 25 MEA plate seeding
  2. 26 Pre-recording stabilization (1–2 weeks)
  3. 27 Baseline recording (10 minutes)
  4. 28 Drug treatment initiation (after baseline recording)
  5. 29 Post-treatment recording (10 minutes)
  6. 30 Pharmacological probe recordings (during week 2)
  7. 31 Repeated recordings every 3 days (2-week period)
  8. 32 Spike detection and active electrode classification
Receptor Antagonist and Agonist Functional Studies Day 7 of MEA recordings
  1. 33 nBNI κ-antagonist application (chronic)
  2. 34 Picrotoxin (PTX) GABAA antagonist application
  3. 35 NBQX AMPA antagonist application
  4. 36 APV NMDA antagonist application
Data Analysis and Statistical Evaluation Post-recording
  1. 37 Firing rate (FR) normalization
  2. 38 Statistical testing (one-way or two-way ANOVA)
  3. 39 Summary visualization and reporting

Full SOP

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Attribution

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

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