Bioink design for organ-scale projection-based 3D bioprinting

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Bioink design for organ-scale projection-based 3D bioprinting

Abstract

Projection-based three-dimensional bioprinting offers an approach for manufacturing biomimetic tissues with complex spatial structures and bioactivity, presenting potential for creating implantable organs or organoids to test drug response. Nevertheless, the extended printing times required for organ-scale manufacturing represents a challenge. Here we provide step-by-step instructions to manufacture organ-scale structures using bioinks while preserving high bioactivity. This approach incorporates Ficoll 400 to mitigate the heterogeneity of bioink with respect to refractive index and density, while 4-(2-aminoethyl)benzenesulfonyl fluoride and oil-sealing ensure the stability of the bioink components, thereby allowing extended printing times. This procedure also enables high-cell-viability printing via the calibration of the pH value of the bioink. This Protocol is appropriate for users with basic laboratory skills and fundamental knowledge in biotechnology to manufacture organ-scale structures for utilization in a wide variety of experimental designs. The approach is generalizable, as demonstrated by the successful printing of corpora cavernosa structures with a cell density of 10 million per milliliter, measuring 10 mm × 10 mm × 10 mm. After 7 d of culture, the cell viability was measured at 82.5%, highlighting the potential applicability in tissue engineering. All bioink preparation and printing steps are expected to take 5 h, while the development of printed structures requires 7 d of continuous culture.

Key points

  • We provide a versatile process for the fabrication of organ-scale structures with complex spatial architectures and high bioactivity using bioinks. We use Ficoll 400 to reduce bioink heterogeneity, while 4-(2-aminoethyl)benzenesulfonyl fluoride and oil-sealing ensure the components’ stability, enabling extended printing time.

  • This Protocol permits organ-scale printing without the need to modify existing 3D bioprinting equipment. It makes this Protocol an economical, stable and easily scalable 3D bioprinting solution.

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Data availability

All remaining data generated or analyzed during this protocol are included in this published article and its supplementary files.

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Acknowledgements

This work was sponsored by the National Natural Science Foundation of China (grant nos. 52235007, T2121004 and 52325504) and Key R&D Program of Zhejiang (grant no. 2024SSYS0027).

Author information

Authors and Affiliations

  1. State Key Laboratory of Fluid Power and Mechatronic Systems and Liangzhu Laboratory, School of Mechanical Engineering, Zhejiang University, Hangzhou, China

    Tianhong Qiao  (乔天鸿), Chaofan He  (何超凡), Guofeng Liu  (刘国峰), Yuan Sun  (孙元) & Yong He  (贺永)

  2. The Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou, China

    Tianhong Qiao  (乔天鸿), Chaofan He  (何超凡), Guofeng Liu  (刘国峰), Yuan Sun  (孙元) & Yong He  (贺永)

  3. Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, College of Mechanical Engineering, Zhejiang University, Hangzhou, China

    Tianhong Qiao  (乔天鸿), Chaofan He  (何超凡), Guofeng Liu  (刘国峰), Yuan Sun  (孙元) & Yong He  (贺永)

  4. Department of General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China

    Pengcheng Xia  (夏鹏程)

  5. The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China

    Miao Sun  (孙苗) & Mengfei Yu  (俞梦飞)

  6. Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, China

    Miao Sun  (孙苗) & Mengfei Yu  (俞梦飞)

  7. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

    Yi Wang  (王毅) & Yiyu Cheng  (程翼宇)

Authors

  1. Tianhong Qiao  (乔天鸿)
  2. Chaofan He  (何超凡)
  3. Pengcheng Xia  (夏鹏程)
  4. Guofeng Liu  (刘国峰)
  5. Yuan Sun  (孙元)
  6. Miao Sun  (孙苗)
  7. Yi Wang  (王毅)
  8. Yiyu Cheng  (程翼宇)
  9. Mengfei Yu  (俞梦飞)
  10. Yong He  (贺永)

Contributions

These authors contributed equally: T.Q. and C.H. T.Q., C.H., M.Y. and Y.H. designed the experiments. T.Q., C.H., P.X., G.L., Y.S., M.S. and Y.H. contributed to the experimental design. T.Q., P.X. and C.H. performed the experiments and analyzed the data. T.Q., C.H., M.Y., Y.C. and Y.H. wrote and reviewed the manuscript.

Corresponding authors

Correspondence to Yiyu Cheng  (程翼宇), Mengfei Yu  (俞梦飞) or Yong He  (贺永).

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Competing interests

The authors declare no competing interests.

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Nature Protocols thanks Hyungseok Lee, Yeong-Jin Choi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

He, C. F. et al. Research 8, 0613 (2025): https://doi.org/10.34133/research.0613

He, C. F. et al. Adv. Funct. Mater. 33, 2301209 (2023): https://doi.org/10.1002/adfm.202301209

Yu, K. et al. Bioact. Mater. 11, 254–267 (2022): https://doi.org/10.1016/j.bioactmat.2021.09.021

Sun, Y. et al. Biofabrication 13, 035032 (2021): https://doi.org/10.1088/1758-5090/aba413

Extended data

Extended Data Fig. 1 The adaptability of bioink formulations.

a) Photographs of organ-scale 3D structures printed using SilMA (DS = 30%) as a substitute for GelMA. b) Photographs of organ-scale 3D structures printed using hydrogel precursor solutions within the acceptable DS range (4% GelMA + 5% PEGDA, with GelMA having a DS of 40%; the compressive modulus of bioink is 18.00 kPa). c) Photographs of organ-scale 3D structures printed using a higher concentration (7.5%) of hydrogel precursor solution. d) Photographs of organ-scale 3D structures printed using a higher (10%) concentration of hydrogel precursor solution. e) Compressive properties of bioinks with different concentrations. f) Compressive moduli of bioinks with different concentrations. Data are displayed as mean ± SD, n = 3.

Extended Data Fig. 3 Cell viability among the layers of the printed structures.

a) Micrographs showing live (green)/dead (red) staining of cells encapsulated in the printed rectangular structures. b) Photographs of the printed rectangular structures. c) Results of the live/dead assays showing cell viability among the layers of the printed rectangular structures. d) Results of the live/dead assays showing cell viability of the printed rectangular structures. Data are displayed as mean ± SD, n = 4.

Extended Data Fig. 4

Immunofluorescence images showing staining for DAPI (blue), α-SMA (green), Phalloidin (red) of the cells encapsulated in the printed structures after 3 days of culture.

Extended Data Fig. 5

High-magnification Immunofluorescence images showing staining of the cells after 7 days of culture.

Extended Data Fig. 6

Immunofluorescence images showing HUVEC (red), USMC (green), and RS1 (blue) within the printed structures after 0, 2, 4, 6 days of culture.

Extended Data Fig. 8

Low-magnification Immunofluorescence images showing staining of the cells after 7 days of culture.

Extended Data Fig. 9 Scale expansion of organ-scale bioprinting.

a) Photographs of printed structures (14mm×14mm×14mm) after culturing 7 days. b) Immunofluorescence images showing staining for DAPI (blue), α-SMA (green), Phalloidin (red) of the cells encapsulated in the printed structures (14mm×14mm×14mm) after 7 days of culture. c) Photographs of printed structures (18mm × 18mm × 18mm) after culturing 7 days.

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Qiao, T., He, C., Xia, P. et al. Bioink design for organ-scale projection-based 3D bioprinting. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01221-0

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