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Generation of human brain region–specific organoids using a miniaturized spinning bioreactor

Nature Protocols volume 13, pages 565580 (2018) | Download Citation


Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14–84 d to complete (depending on the type of brain region–specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

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We thank K.M. Christian for feedback on the manuscript, members of Ming and Song laboratories for discussions, and L. Liu, Y. Cai and D.G. Johnson for technical assistance. The research was supported by grants from the National Institutes of Health (R37NS047344, U19MH106434, P01NS097206 and R01AG057497 to H.S.; R21NS095348, R01MH105128, R35NS097370 and U19AI131130 to G.M.), the Simons Foundation (to H.S. and G.M.) and The Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to G.M.).

Author information

Author notes

    • Xuyu Qian
    •  & Fadi Jacob

    These authors contributed equally to this work.


  1. Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Xuyu Qian
    • , Fadi Jacob
    • , Hongjun Song
    •  & Guo-li Ming
  2. Biomedical Engineering Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Xuyu Qian
  3. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Fadi Jacob
  4. Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

    • Mingxi Max Song
  5. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Ha Nam Nguyen
  6. The Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Hongjun Song
    •  & Guo-li Ming


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X.Q., F.J., M.M.S., H.N.N., H.S. and G.M. designed, built and tested the systems and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Guo-li Ming.

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Purchased bioreactor parts.

Zip files

  1. 1.

    Supplementary Data 1

    Bioreactor CAD zip file.

  2. 2.

    Supplementary Data 2

    SpinΩ 3D PDF.

  3. 3.

    Supplementary Data 3

    SpinΩ 3D PDF, exploded view.


  1. 1.

    SpinΩ assembly.

  2. 2.

    Embedding Matrigel.

  3. 3.

    Breaking Matrigel.

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