ProtocolPublished: 30 July 2025Tianhong Qiao (乔天鸿) ORCID: orcid.org/0009-0009-8578-60331,2,3,Chaofan He (何超凡) ORCID: orcid.org/0000-0003-3574-72971,2,3,Pengcheng Xia (夏鹏程)4,Guofeng Liu (刘国峰)1,2,3,Yuan Sun (孙元)1,2,3,Miao Sun (孙苗)5,6,Yi Wang (王毅)7,Yiyu Cheng (程翼宇)7,Mengfei Yu (俞梦飞)5,6 &…Yong He (贺永) ORCID: orcid.org/0000-0002-9099-08311,2,3 Nature Protocols (2025)Cite this articleSubjectsTissue cultureTissue engineeringAbstractProjection-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 pointsWe 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.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to full article PDFBuy nowPrices may be subject to local taxes which are calculated during checkoutFig. 1: Schematic diagram of organ-scale PBBP research framework.Fig. 2: Schematic diagram of the procedure for the organ-scale PBBP process.Fig. 3: Schematic diagram of DBC testing.Fig. 4: Photo-response properties of bioink.Fig. 5: Stability of bioink.Fig. 6: Oil-seal printing process reduces bioink evaporation.Fig. 7: Impact of Bioink pH on encapsulated cell viability.Fig. 8: Biocompatibility analyses of bioink.Fig. 9: 3D bioprinting of organ-scale corpora cavernosa structures.Data availabilityAll remaining data generated or analyzed during this protocol are included in this published article and its supplementary files.ReferencesZandrini, T., Florczak, S., Levato, R. & Ovsianikov, A. 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Virol. 82, 1128–1135 (2008).CAS Google Scholar Download referencesAcknowledgementsThis 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 informationAuthors and AffiliationsState Key Laboratory of Fluid Power and Mechatronic Systems and Liangzhu Laboratory, School of Mechanical Engineering, Zhejiang University, Hangzhou, ChinaTianhong Qiao (乔天鸿), Chaofan He (何超凡), Guofeng Liu (刘国峰), Yuan Sun (孙元) & Yong He (贺永)The Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou, ChinaTianhong Qiao (乔天鸿), Chaofan He (何超凡), Guofeng Liu (刘国峰), Yuan Sun (孙元) & Yong He (贺永)Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, College of Mechanical Engineering, Zhejiang University, Hangzhou, ChinaTianhong Qiao (乔天鸿), Chaofan He (何超凡), Guofeng Liu (刘国峰), Yuan Sun (孙元) & Yong He (贺永)Department of General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, ChinaPengcheng Xia (夏鹏程)The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, Zhejiang University, Hangzhou, ChinaMiao Sun (孙苗) & Mengfei Yu (俞梦飞)Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, ChinaMiao Sun (孙苗) & Mengfei Yu (俞梦飞)College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, ChinaYi Wang (王毅) & Yiyu Cheng (程翼宇)AuthorsTianhong Qiao (乔天鸿)View author publicationsSearch author on:PubMed Google ScholarChaofan He (何超凡)View author publicationsSearch author on:PubMed Google ScholarPengcheng Xia (夏鹏程)View author publicationsSearch author on:PubMed Google ScholarGuofeng Liu (刘国峰)View author publicationsSearch author on:PubMed Google ScholarYuan Sun (孙元)View author publicationsSearch author on:PubMed Google ScholarMiao Sun (孙苗)View author publicationsSearch author on:PubMed Google ScholarYi Wang (王毅)View author publicationsSearch author on:PubMed Google ScholarYiyu Cheng (程翼宇)View author publicationsSearch author on:PubMed Google ScholarMengfei Yu (俞梦飞)View author publicationsSearch author on:PubMed Google ScholarYong He (贺永)View author publicationsSearch author on:PubMed Google ScholarContributionsThese 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 authorsCorrespondence to Yiyu Cheng (程翼宇), Mengfei Yu (俞梦飞) or Yong He (贺永).Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationNature Protocols thanks Hyungseok Lee, Yeong-Jin Choi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Key referencesHe, C. F. et al. Research 8, 0613 (2025): https://doi.org/10.34133/research.0613He, C. F. et al. Adv. Funct. Mater. 33, 2301209 (2023): https://doi.org/10.1002/adfm.202301209Yu, K. et al. Bioact. Mater. 11, 254–267 (2022): https://doi.org/10.1016/j.bioactmat.2021.09.021Sun, Y. et al. Biofabrication 13, 035032 (2021): https://doi.org/10.1088/1758-5090/aba413Extended dataExtended 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. 2 Photographs of the vat after printing TPMS structure (18mm×18mm×18mm).a) With utilizing oil-seal process. b) Without utilizing oil-seal process.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. 4Immunofluorescence 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. 5High-magnification Immunofluorescence images showing staining of the cells after 7 days of culture.Extended Data Fig. 6Immunofluorescence images showing HUVEC (red), USMC (green), and RS1 (blue) within the printed structures after 0, 2, 4, 6 days of culture.Extended Data Fig. 7 Immunofluorescence images (cross section) showing staining for DAPI (blue), α-SMA (green), Phalloidin (red) of the cells encapsulated in the printed structures after 7 days of culture.The encapsulated cells spread on the surface of structures and also exhibit a degree of proliferation within the hydrogels.Extended Data Fig. 8Low-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.Supplementary informationReporting SummarySupplementary Video 1Preparation of the hydrogel precursor solution.Supplementary Video 2Adjustment of the pH of bioink.Supplementary Video 3Sterilize the bioink by filtration and add AEBSF.Supplementary Video4Oil-seal printing process (use PEGDA bioink without encapsulating cells to demonstrate the process).Supplementary Video 5Organ-scale bioprinting process.Supplementary Video 6Post-printing processing of organ-scale 3D structures.Supplementary Video 7Dynamic cultivation of organ-scale 3D structures.Supplementary Data 1TPMS model for organ-scale bioprinting (10 mm × 10 mm × 10 mm.Supplementary Data 2Curing depth test model serving as the base.Supplementary Data 3TPMS model for demonstrating the effectiveness of oil-seal process (18 mm × 18 mm × 18 mm).Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article