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.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Wen, Z., Christian, K.M., Song, H. & Ming, G.L. Modeling psychiatric disorders with patient-derived iPSCs. Curr. Opin. Neurobiol. 36, 118–127 (2016).
Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).
Kadoshima, T. et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc. Natl. Acad. Sci. USA 110, 20284–20289 (2013).
Pasca, A.M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).
Jo, J. et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 19, 248–257 (2016).
Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016).
Nguyen, H.N.N., Song, H. & Ming, G. Engineering human pluripotent stem cell-derived 3D brain tissue for drug discovery. J. Transl. Neurosci. 1, 38–48 (2016).
Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).
Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell 18, 25–38 (2016).
Danjo, T. et al. Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals. J. Neurosci. 31, 1919–1933 (2011).
Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep. 10, 537–550 (2015).
Sakaguchi, H. et al. Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue. Nat. Commun. 6, 8896 (2015).
Sasai, Y. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).
Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).
Blackshaw, S. et al. Molecular pathways controlling development of thalamus and hypothalamus: from neural specification to circuit formation. J. Neurosci. 30, 14925–14930 (2010).
Byerly, M.S. & Blackshaw, S. Vertebrate retina and hypothalamus development. Wiley Interdiscip. Rev. Syst. Biol. Med. 1, 380–389 (2009).
Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).
Wen, Z. et al. Synaptic dysregulation in a human iPS cell model of mental disorders. Nature 515, 414–418 (2014).
Yoon, K.J. et al. Modeling a genetic risk for schizophrenia in iPSCs and mice reveals neural stem cell deficits associated with adherens junctions and polarity. Cell Stem Cell 15, 79–91 (2014).
Chiang, C.H. et al. Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Mol. Psychiatry 16, 358–360 (2011).
Yoon, K.J. et al. Zika-virus-encoded NS2A disrupts mammalian cortical neurogenesis by degrading adherens junction proteins. Cell Stem Cell 21, 349–358.e6 (2017).
Xu, M. et al. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat. Med. 22, 1101–1107 (2016).
Yoon, K.J. et al. Temporal control of mammalian cortical neurogenesis by m6A methylation. Cell 171, 877–889.e17 (2017).
Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).
Bagley, J.A., Reumann, D., Bian, S., Levi-Strauss, J. & Knoblich, J.A. Fused cerebral organoids model interactions between brain regions. Nat. Methods 14, 743–751 (2017).
Xiang, Y. et al. Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration. Cell Stem Cell 21, 383–398.e7 (2017).
Lui, J.H., Hansen, D.V. & Kriegstein, A.R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).
Qian, X., Nguyen, H.N., Jacob, F., Song, H. & Ming, G.L. Using brain organoids to understand Zika virus-induced microcephaly. Development 144, 952–957 (2017).
Ming, G.L., Tang, H. & Song, H. Advances in Zika virus research: stem cell models, challenges, and opportunities. Cell Stem Cell 19, 690–702 (2016).
Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015).
Chen, Y.W. et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat. Cell Biol. 19, 542–549 (2017).
Juopperi, T.A. et al. Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington′s disease patient cells. Mol. Brain 5, 17 (2012).
Lancaster, M.A. et al. Guided self-organization and cortical plate formation in human brain organoids. Nat. Biotechnol. 35, 659–666 (2017).
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.).
The authors declare no competing financial interests.
Purchased bioreactor parts. (XLSX 10 kb)
Bioreactor CAD zip file. (ZIP 179 kb)
SpinΩ 3D PDF. (ZIP 451 kb)
SpinΩ 3D PDF, exploded view. (ZIP 451 kb)
About this article
Cite this article
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). https://doi.org/10.1038/nprot.2017.152