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Generation of spatially patterned human neural tube-like structures using microfluidic gradient devices

Nature Protocols (2025)Cite this article

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Abstract

The functional complexity and anatomical organization of the nervous system are established during regional patterning of its embryonic precursor—the neural tube. Human pluripotent stem (hPS) cell-based models have emerged as valuable complements to animal models for studying neural development. Here we present the design and implementation of a microfluidic gradient device for modeling human neural tube formation and regional patterning with hPS cells. The microfluidic device enables the formation of tubular or spherical colonies of hPS cells at prescribed locations within microfluidic channels, allowing the cell colonies to form lumenal structures while being exposed to well-controlled chemical gradients for rostral–caudal and/or dorsal–ventral patterning, resulting in the formation of a microfluidic neural tube-like structure (μNTLS) or a forebrain-like structure (μFBLS). The μNTLS recapitulates important hallmarks of early human neural development, including well-defined lumenal morphologies, spatially organized regional marker expression, emergence of secondary signaling centers and the development of neural crest cells. The dorsal–ventral patterned μFBLS further recapitulates spatially segregated dorsal and ventral regions, as well as the layered segregation of early neurons from neural progenitors, mimicking human forebrain pallium and subpallium development. Both the μNTLS and μFBLS are compatible with long-term culture, live imaging, immunofluorescence staining and single-cell sequencing, serving as robust systems for studying human neurodevelopment and disease. This protocol can be implemented by a researcher with polydimethylsiloxane soft lithography and cell culture experience and takes ~8–41 d to complete, depending on the types of neural structure to model and their developmental stages, with an option for prolonged culture to promote neuronal maturation.

Key points

  • This protocol describes the fabrication and implementation of a microfluidic gradient device-based culture system to generate spatially patterned, human neural tube- and forebrain-like structures. This device enables the controllable formations of orthogonal chemical gradients, which are imposed on the human pluripotent stem cell colonies.

  • This allows the recapitulation of several key aspects of neural patterning in both brain and spinal cord along both rostral–caudal and dorsal–ventral axes in three dimensions.

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Fig. 1: Design of microfluidic devices and stamps.
Fig. 2: Generation of micropatterned hPS cell colonies in the microfluidic devices.
Fig. 3: Common issues encountered during implementation of the protocol.
Fig. 4: Generation of an R–C patterned microfluidic μNTLS.
Fig. 5: Generation of an R–C and D–V patterned microfluidic μNTLS.
Fig. 6: Generation of a D–V patterned microfluidic μFBLS.

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Data availability

All data supporting the development of this protocol are included in the figures or in the supporting primary research article18.

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Acknowledgements

This work is supported by the Michigan–Cambridge Collaboration Initiative (J.F.), the University of Michigan Mcubed Fund (J.F.), the 21st Century Jobs Trust Fund received through the Michigan Strategic Fund from the State of Michigan (grant no. CASE-315037; J.F.), a University of Michigan Mid-career Biosciences Faculty Achievement Recognition Award (J.F.), the National Science Foundation of the United States (I-Corps grant no. 2112458, CBET grant no. 1901718 and EFMA grant no. 2422149 to J.F.), the NIH of the United States (grant nos. R21 NS113518, R21 NS127983, R01 GM143297 and R01 NS129850 to J.F.) and the startup funding from Cincinnati Children’s Research Foundation (X.X.).

Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA

    Xufeng Xue, Shiyu Sun, Jeyoon Bok & Jianping Fu

  2. Division of Developmental Biology, Center for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Xufeng Xue & Omar M. Rahman

  3. Greenhills School, Ann Arbor, MI, USA

    Aoife Tang

  4. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA

    Jianping Fu

  5. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Jianping Fu

Authors
  1. Xufeng Xue
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  2. Omar M. Rahman
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Contributions

X.X. and J.F. conceived and initiated the projects; X.X. designed, performed and quantified the experiments. O.M.R., S.S., J.B. and A.T. assisted with the experiments. O.M.R. and J.B. independently repeated the experiments. A.T. contributed to the literature search. J.F. supervised the study. All authors edited and approved the manuscript.

Corresponding authors

Correspondence to Xufeng Xue or Jianping Fu.

Ethics declarations

Competing interests

The University of Michigan, Ann Arbor, has filed a patent application describing microfluidic devices and methods for the development of neural tube-like tissues and neural spheroids (PCT/US2021/058090), with J.F. and X.X. as co-inventors. The other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Giorgia Quadrato, Guohao Dai, Peter Serles and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference

Xue, X. et al. Nature 628, 391–399 (2024): https://doi.org/10.1038/s41586-024-07204-7

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Data 1

An AutoCAD file for the photomask used for microfabrication of the microfluidic device to generate R–C patterned μNTLS. The transparent areas in the AutoCAD file will be etched away during the DRIE process.

Supplementary Data 2

An AutoCAD file for the photomask used for microfabrication of the microfluidic device to generate R–C and D–V patterned μNTLS and D–V patterned μFBLS. The transparent areas in the AutoCAD file will be etched away during the DRIE process.

Supplementary Data 3

An AutoCAD file for the photomask used for microfabrication of the stamp to generate R–C patterned μNTLS. The transparent areas in the AutoCAD file will be etched away during the DRIE process.

Supplementary Data 4

An AutoCAD file for the photomask used for microfabrication of the stamp to generate R–C and D–V patterned μNTLS. The transparent areas in the AutoCAD file will be etched away during the DRIE process.

Supplementary Data 5

An AutoCAD file for the photomask used for microfabrication of the stamp to generate D–V patterned μFBLS. The transparent areas in the AutoCAD file will be etched away during the DRIE process.

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Xue, X., Rahman, O.M., Sun, S. et al. Generation of spatially patterned human neural tube-like structures using microfluidic gradient devices. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01266-1

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