Abstract
Organoids are in vitro miniaturized cellular models of organs that offer opportunities for studying organ development, disease mechanisms and drug screening. Understanding the complex processes governing organoid development and function requires methods suitable for the continuous, long-term monitoring of cell activities (for example, electrophysiological and mechanical activity) at single-cell resolution throughout the entire three-dimensional (3D) structure. Cyborg organoid technology addresses this need by seamlessly integrating stretchable mesh nanoelectronics with tissue-like properties, such as tissue-level flexibility, subcellular feature size and mesh-like networks, into 3D organoids through a 2D-to-3D tissue reconfiguration process during organogenesis. This approach enables longitudinal, tissue-wide, single-cell functional mapping, thereby overcoming the limitations of existing techniques including recording duration, spatial coverage, and the ability to maintain stable contact with the tissue during organoid development. This protocol describes the fabrication and characterization of stretchable mesh nanoelectronics, their electrical performance, their integration with organoids and the acquisition of long-term functional organoid activity requiring multimodal data analysis techniques. Cyborg organoid technology represents a transformative tool for investigating organoid development and function, with potential for improving in vitro disease models, drug screening and personalized medicine. The procedure is suitable for users within a multidisciplinary team with expertise in stem cell biology, tissue engineering, nanoelectronics fabrication, electrophysiology and data science.
Key points
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The procedure includes the fabrication of stretchable mesh nanoelectronics, the nanoelectronics’ integration within developing organoids and the long-term electrical measurements of organoid function.
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Alternatives include imaging-based techniques, intracellular recordings and extracellular recordings using multielectrode arrays. Cyborg organoids provide stable 3D bioelectronics interfaces and enable the continuous monitoring of electrophysiological activity during development.
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Data availability
The authors declare that the main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1021/acs.nanolett.9b02512, https://doi.org/10.1002/adma.202106829, https://doi.org/10.1126/sciadv.ade8513, and https://doi.org/10.1016/j.cell.2023.03.023, https://doi.org/10.1101/2024.03.18.585551).
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Acknowledgements
We acknowledge the valuable discussions with A.ML., X.Z. and H.S. J.Liu acknowledges the support of NIH/NIMH 1RF1MH123948; NIH/NIDDK 1DP1DK130673; NSF/ECCS-2038603; NIH/NLM 5R01LM014465.
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Z.L., W.W., R.L., Q.L. and J.Liu. developed the protocol and drafted the manuscript with input from C.H. and J.Lee. All authors read, edited and approved the final manuscript.
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J. Liu is a co-founder of Axoft, Inc.
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Nature Protocols thanks Gi Doo Cha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
Li, Q. et al. Nano Lett. 19, 5781–5789 (2019): https://doi.org/10.1021/acs.nanolett.9b02512
Lin, Z. et al. Sci. Adv. 9, eade8513 (2023): https://doi.org/10.1126/sciadv.ade8513
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Le Floch, P. et al. Adv. Mater. 34, e2106829 (2022): https://doi.org/10.1002/adma.202106829
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Supplementary Information
Supplementary Figures 1-2.
Supplementary Data 1
Photomask of mesh nanoelectronics.
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Lin, Z., Wang, W., Liu, R. et al. Cyborg organoids integrated with stretchable nanoelectronics can be functionally mapped during development. Nat Protoc 20, 2528–2559 (2025). https://doi.org/10.1038/s41596-025-01147-7
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DOI: https://doi.org/10.1038/s41596-025-01147-7
