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Fibrous polyisocyanide hydrogels for 3D cell culture applications

Nature Protocols volume 20, pages 3339–3360 (2025)Cite this article

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Abstract

Three-dimensional (3D) cell culture models based on hydrogels are rapidly evolving into a prominent tool for tissue engineering, mechanobiology, disease modeling and drug screening. While a vast variety of synthetic gels have emerged in recent years, they fail to penetrate the market substantially for two major reasons: they poorly mimic the extracellular matrix or they are difficult to use in gel formation and cell extraction. Mimicking the complexity of nature is challenging: the extracellular matrix plays a crucial role in cell development and function, which goes well beyond simple mechanical support. Recently, we introduced polyisocyanide (PIC) hydrogels for 3D cell culture applications. The fibrous architecture and associated (non)linear mechanical behavior closely mimic the physical properties of biogels such as collagen and fibrin. As fully synthetic materials, PIC gels benefit from high tailorability and reproducibility. Moreover, the thermoresponsive properties of PIC gels make them easy to handle in the lab; the gels form instantly at 37 °C and cells are easily extracted after cooling to 5 °C. The potential of PIC gels has been demonstrated in a quickly expanding library of papers discussing different cell lines, primary cells and organoids, as well as in vivo experiments. This manuscript provides protocols on how to handle PIC gels in the chemistry and cell biology laboratories. Material preparation requires 72 h. Cell encapsulation takes 1 h and the time for downstream analysis depends on the (commercial) methods used. The protocols described are suitable for researchers with expertise in cell culture and molecular biology.

Key points

  • Protocol describing the use of polyisocyanide (PIC) as a model matrix for 3D cell culture. PIC hydrogels closely mimic the physical properties of biogels such as collagen and fibrin and, as fully synthetic materials, benefit from a high reproducibility.

  • The linear and nonlinear mechanics of PIC hydrogel can be controlled by tuning the polymer length or concentration, and the polymer can be biofunctionalized through azide-based click chemistry.

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Fig. 1: Overview of PIC hydrogel features.
Fig. 2: PIC design parameters.
Fig. 3: Expected physical properties of the PIC gel.
Fig. 4: Representative cell culture results using PIC.
Fig. 5: Representative results of organoid culture using PIC.

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Data availability

The authors declare that the data in this protocol are available through the supporting papers17,18,19,23,27,34,43,47,65. Reasonable requests for materials can be addressed to P.H.J.K. at Radboud University.

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Acknowledgements

We sincerely mourn the recent loss of R.J.M. Nolte and express our heartfelt gratitude for his instrumental contributions to the field of PIC polymers. We thank A. E. Rowan and all others who contributed to the development of PIC hydrogels, and W. Bonger for repeating the rheology experiments. In addition, the authors gratefully acknowledge the funding support from the National Key Research and Development Program of China (2023YFF1205500, H.Y.), the National Natural Science Foundation of China (32471368, H.Y; 32201097, K.L.), the Excellent Young Scientist Fund of the Natural Science Foundation of Hebei Province (B2022202027, H.Y.), the Shenzhen International Science and Technology Cooperation Project (grant no. GJHZ20240218112602004, K.L.), the Mainland–Hong Kong Joint Funding Scheme (grant no. 2023YFE0210500, K.L.), the Fonds Wetenschappelijk Onderzoek (12A2423N, H.Y.) and the funding from Dutch Organization for knowledge and innovation in health, healthcare and well-being (ZonMw, 01142052310003, TOP grant 91218030, P.H.J.K.), the Dutch Research Council (NWO, Demonstrator 19358, Take Off I 20941, P.H.J.K.). Figures were created with support of BioRender.

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Author notes

  1. These authors contributed equally: Hongbo Yuan, Kaizheng Liu.

Authors and Affiliations

  1. Innovation and Research Institute of Hebei University of Technology in Shijiazhuang, Shijiazhuang, China

    Hongbo Yuan & Yuying Bao

  2. Molecular Imaging and Photonics, Chemistry Department, KU Leuven, Leuven, Belgium

    Hongbo Yuan & Susana Rocha

  3. Research Center for Human Tissue and Organ Degeneration, Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Kaizheng Liu

  4. Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands

    Melissa J. J. van Velthoven, Jyoti Kumari & Paul H. J. Kouwer

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  1. Hongbo Yuan
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  2. Kaizheng Liu
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  3. Melissa J. J. van Velthoven
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  7. Paul H. J. Kouwer
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Contributions

All authors contributed to developing and writing the protocols in this manuscript. H.Y. and K.L. initiated the project, and drafted and edited the manuscript. P.H.J.K. supervised the project and edited the manuscript. H.Y. and K.L. contributed equally to this manuscript.

Corresponding authors

Correspondence to Hongbo Yuan, Kaizheng Liu or Paul H. J. Kouwer.

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Competing interests

H.Y., K.L. and P.H.J.K. have commercialized PIC polymers, which are available at SBMatrices B.V. (worldwide, under the commercial name Fybrix,) and at GTI Shenzhen Ltd. (in China). The other authors declare no competing interests.

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Key references

Kouwer, P. H. J. et al. Nature 493, 651–655 (2013): https://doi.org/10.1038/nature11839

Yuan, H. et al. Proc. Natl Acad. Sci. USA 120, e2216934120 (2023): https://doi.org/10.1073/pnas.2216934120

Van Velthoven, M. J. J. et al. Adv. Healthc. Mater. 12, 2301109 (2023): https://doi.org/10.1002/adhm.202301109

Zhang, Y. et al. Adv. Sci. 7, 2001797 (2020): https://doi.org/10.1002/advs.202001797

Liu, K. et al. Biomacromolecules 20, 826–834 (2019): https://doi.org/10.1021/acs.biomac.8b01445

Supplementary information

Supplementary Video 1

Overview of PIC preparation and cell encapsulation.

Supplementary Video 2

Overview of the procedure of fluorescence labeling PIC, matrix and cell encapsulation.

Supplementary Video 3

Overview of the cell retrieval procedure.

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Yuan, H., Liu, K., van Velthoven, M.J.J. et al. Fibrous polyisocyanide hydrogels for 3D cell culture applications. Nat Protoc 20, 3339–3360 (2025). https://doi.org/10.1038/s41596-025-01159-3

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