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Manufacturing synthetic viscoelastic antigen-presenting cells for immunotherapy

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Abstract

This protocol details the preparation and functionalization of viscoelastic synthetic antigen-presenting cells (APCs) for T cell activation, designed to enhance immunotherapeutic efficacy. Using a high-throughput microfluidic system and post-processing, we create cell-sized sodium alginate microbeads with tunable stiffness, viscoelasticity and surface chemistry, enabling them to better mimic the physical and activation properties of natural APCs. The protocol includes fabrication of synthetic cells with defined sizes, crosslinking strategies to achieve desirable mechanical properties, surface functionalization via click chemistry for attaching activation molecules, and characterization methods for mechanical and biochemical properties. Compared with traditional matrices or rigid microbeads, this approach allows precise control over the mechanical and biochemical features of synthetic APCs, ensuring optimal T cell activation. The resulting synthetic cells support robust T cell activation and expansion, enhance the CD8/CD4 T cell ratio, promote T memory stem cell (TMSC) formation and improve chimeric antigen receptor transduction efficiency, leading to superior tumor-killing efficacy in vitro and in vivo. Additionally, these synthetic cells can be efficiently removed from T cells after activation using simple centrifugation or calcium chelation, preserving the activated T cells. The complete protocol, including fabrication, functionalization and quality assessment, requires ~1 week to complete. Users should have experience in microfluidics, biomaterial handling, bioconjugation techniques and basic cell culture. This platform can be adapted for broader applications in immune cell engineering.

Key points

  • Protocol describing the fabrication of synthetic viscoelastic antigen-presenting cells and their application in T cell engineering, including crosslinking strategies to achieve desirable mechanical properties, surface functionalization via click chemistry for attaching activation molecules, and characterization methods for mechanical and biochemical properties.

  • These synthetic cells support robust T cell expansion, which contributes to longer in vivo persistence, stronger antitumor immune responses and improved tumor control than those expanded with conventional methods.

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Fig. 1: Workflow of CAR T cell generation and the role of synthetic viscoelastic APCs (synthetic cells) in enhancing cancer immunotherapy.
Fig. 2: Workflow for the preparation and application of synthetic cells in T cell activation and functional characterization.
Fig. 3: The engineering design of the microfluidic chip used in this protocol and mechanical characterization of synthetic cells.
Fig. 4: Cross-disciplinary workflow for the development, manufacturing and application of synthetic cells.
Fig. 5: Comparison of CAR-T cell expansion and characterization using synthetic cells versus Dynabeads.
Fig. 6: CAR-T cell expansion and tumor-killing efficacy using synthetic cells, elastic beads and Dynabeads.

Data availability

The genomics (scRNA-seq) data were obtained from the public repository Gene Expression Omnibus (GEO) database (accession code GSE242531; related to Fig. 5d–f). Additional information and materials will be made available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We extend our gratitude to the UCLA Division of Laboratory Animal Medicine (DLAM) for assistance with animal studies, the UCLA BSCRC Flow Cytometry Core Facility for cell sorting, and the UCLA TCGB for scRNA-seq services. The UCLA CFAR Virology Core is acknowledged for providing human PBMCs, and the Advanced Light Microscopy/Spectroscopy Laboratory along with the Leica Microsystems Center at the California NanoSystems Institute (CNSI) for support with imaging. Additionally, we thank the NIH Tetramer Facility for providing tetramers utilized in this research. L.Y. is a member of UCLA Parker Institute for Cancer Immunotherapy (PICI). Y.-R.L. is supported by a UCLA Chancellor’s Award for Postdoctoral Research and a UCLA Goodman–Luskin Microbiome Center Collaborative Research Fellowship.

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Author notes

  1. These authors contributed equally: Zeyang Liu, Yan-Ruide Li, Youcheng Yang.

Authors and Affiliations

  1. Department of Bioengineering, University of California Los Angeles, Los Angeles, CA, USA

    Zeyang Liu, Yan-Ruide Li, Youcheng Yang, Enbo Zhu, Haochen Nan, Yue Yan, Bo Zhang, Guorui Chen, Nicolas Pedroncelli, Zibai Lyu, Jason Lin, Jennifer Soto, Lili Yang & Song Li

  2. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Yan-Ruide Li, Zibai Lyu & Lili Yang

  3. Eli and Edythe Broad Centre of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang & Song Li

  4. Jonsson Comprehensive Cancer Centre, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang & Song Li

  5. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

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Z. Liu, Y.-R.L. and Y. Yang developed and optimized the protocol. Z. Liu, Y.-R.L., Y. Yang, E.Z., H.N., Y. Yan, B.Z., G.C., N.P. and Z.L. performed the protocol validation and optimization experiments. Z. Liu, Y.-R.L., Y. Yang and E.Z. analyzed and compiled the data. Z. Liu, Y.-R.L., Y. Yang, E.Z. and J.L. documented and prepared the protocol steps and procedures. L.Y. and S.L. supervised the protocol development and provided critical insights throughout the study. Z. Liu, Y.-R.L., Y. Yang, L.Y. and S.L. wrote and revised the manuscript, incorporating feedback from all authors.

Corresponding authors

Correspondence to Lili Yang or Song Li.

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Competing interests

Z. Liu, Y.-R.L, L.Y. and S.L. filed a patent application (PCT/US24/22516) on synthetic cell as inventors. The other authors declare no competing interests.

Peer review

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Nature Protocols thanks Xiao Huang, Paolo Provenzano, Qinghe Zeng, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference

Liu, Z. et al. Nat. Biomed. Eng. 8, 1615–1633 (2024): https://doi.org/10.1038/s41551-024-01272-w

Supplementary information

Source data

Source data Figs. 5 and 6

Statistical source data.

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Liu, Z., Li, YR., Yang, Y. et al. Manufacturing synthetic viscoelastic antigen-presenting cells for immunotherapy. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01265-2

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