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


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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Figure 1: Building a SpinΩ bioreactor.
Figure 2: Schematic diagram of brain region–specific organoid protocols.
Figure 3: Progression and quality control of forebrain organoid development.
Figure 4: Immunofluorescence characterization of brain region–specific organoids.


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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




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.

Corresponding author

Correspondence to Guo-li Ming.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Purchased bioreactor parts. (XLSX 10 kb)

Supplementary Data 1

Bioreactor CAD zip file. (ZIP 179 kb)

Supplementary Data 2

SpinΩ 3D PDF. (ZIP 451 kb)

Supplementary Data 3

SpinΩ 3D PDF, exploded view. (ZIP 451 kb)

SpinΩ assembly. (MOV 18655 kb)

Embedding Matrigel. (MOV 27987 kb)

Breaking Matrigel. (MOV 19796 kb)

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Qian, X., Jacob, F., Song, M. et al. Generation of human brain region–specific organoids using a miniaturized spinning bioreactor. Nat Protoc 13, 565–580 (2018).

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