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BRAIN Publication-derived

Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation

Source Qian et al., 2020 · Department of Neuroscience and Mahoney Institute for Neurosciences, University of Pennsylvania, Philadelphia, PA · 10.1016/j.stem.2020.02.002

👤 Xuyu Qian, Yijing Su, Christopher D. Adam, Andre U. Deutschmann, Sarshan R. Pather, Ethan M. Goldberg, Kenong Su, Shiying Li, Lu Lu, Fadi Jacob, Phuong T.T. Nguyen, Sooyoung Huh, Ahmet Hoke, Sarah E. Swinford-Jackson, Zhexing Wen, Xiaosong Gu, R. Christopher Pierce, Hao Wu, Lisa A. Briand, H. Isaac Chen, John A. Wolf, Hongjun Song, Guo-li Ming ⏱ 150 days 📋 2 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of sliced neocortical organoids (SNOs) from human iPSCs that overcome diffusion limitations to enable sustained neurogenesis and formation of distinct upper and deep cortical layers resembling third-trimester human neocortex. SNOs maintain organized progenitor zones and support long-term culture (>150 days) with reduced interior hypoxia and cell death.

Cell source
Human iPSC
Application
Disease modeling and developmental study of human cortical layer formation

Protocol overview

15 steps across 2 phases

iPSC Maintenance and Forebrain Organoid Generation Day 0 to Day 45
  1. 1 Maintain human iPSCs in stem cell medium
  2. 2 Detach iPSC colonies for organoid formation
  3. 3 Initiate organoid formation with dual SMAD inhibition
  4. 4 Apply forebrain patterning signals
  5. 5 Embed organoids in Matrigel
  6. 6 Transfer organoids to spinning bioreactor
  7. 7 Supplement medium with ECM proteins
  8. 8 Switch to maturation medium
Sliced Neocortical Organoid (SNO) Generation and Culture Day 45 to Day 150+
  1. 1 Embed Day 45 organoids in agarose
  2. 2 Slice organoids using vibratome
  3. 3 Separate organoid slices from agarose
  4. 4 Culture slices on orbital shaker
  5. 5 Switch to maturation medium after Day 70
  6. 6 Perform repeated slicing every 4 weeks
  7. 7 Maintain culture and monitor growth

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Qian et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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