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Aspiration-assisted bioprinting of spheroids

Nature Protocols (2025)Cite this article

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Abstract

Aspiration-assisted bioprinting (AAB) is a versatile biofabrication technique that enables the precise and selective patterning of biologics, such as tissue spheroids and organoids, addressing limitations of conventional bioprinting techniques. AAB facilitates the fabrication of (1) tissues with physiologically relevant cell densities using spheroids and (2) advanced tissue models that replicate three-dimensional microenvironments essential for studying cellular responses, disease development and drug testing. Here we provide reliable and reproducible guidelines for the precise positioning of abovementioned biologics, incorporating two operational modes: (1) a single-nozzle mode for precise, one-by-one bioprinting and (2) a high-throughput mode using a digitally controllable nozzle array, enabling the rapid and simultaneous placement of multiple spheroids for scalable tissue fabrication. Comprehensive instructions are included for setting up the AAB platform, operating software and key operational procedures, including optimization of bioprinting conditions. This Protocol enables users to build and operate their own AAB platform depending on target applications, achieving fine control over spheroid positioning through successful aspiration and their precise placement under optimized conditions. This Protocol enables the setup of the AAB platform within 1–2 d. Bioprinting time varies depending on the number of spheroids to bioprint: the single-nozzle mode requires ~30 s per spheroid, while the high-throughput mode can print 64 spheroids in 3–4 min. Designed for accessibility and adaptability, this Protocol is suitable for users from a variety of backgrounds, including engineering, biology, pharmacy and medical sciences, who require bioprinting of spheroids for creating microphysiological systems for drug testing and disease modeling and implantable grafts for regenerative medicine.

Key points

  • Aspiration-assisted bioprinting (AAB) is a versatile biofabrication technique that enables the precise and selective patterning of biologics, such as tissue spheroids and organoids. This Protocol provides comprehensive instructions for setting up the AAB platform, operating software and key operational procedures, including the optimization of bioprinting conditions.

  • AAB overcomes the limitations of conventional bioprinting techniques, enabling flexible, precise, heterogeneous and scalable spheroid bioprinting across both two-dimensional and three-dimensional configurations.

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Fig. 1: The fabrication and assembly process for the AAB platform.
Fig. 2: The pneumatic system in AAB.
Fig. 3: Key steps in the assembly of the DCNA and full setup of the AAB platform.
Fig. 4: The key workflow of the AAB process.
Fig. 5: The AAB software interface.
Fig. 6: Detailed control diagram for AAB operations.
Fig. 7: Detailed schematic description of key steps for optimal AAB procedure preparation.
Fig. 8: Applications of single-nozzle AAB.
Fig. 9: Applications of high-throughput AAB.

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Data availability

The main data discussed in this Protocol are available in supporting primary research publications. STL files used in this Protocol are accessible via figureshare.com at https://doi.org/10.6084/m9.figshare.28405133.v1 (ref. 35). Example datasets to demonstrate the software for prepositioning (Steps 145–151) and automated motion movement (Steps 117–118) are provided in the Supplementary data.

Code availability

The AAB software can be accessed in the Supplementary software. Details of the software interface and instructions were included in the manuscript. We will post updated versions of the software via our GitHub repository at https://github.com/MHKim-software/HITS-Bio.git, if necessary.

References

  1. Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C. & De Boer, J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 31, 108–115 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Dababneh, A. B. & Ozbolat, I. T. Bioprinting technology: a current state-of-the-art review. J. Manuf. Sci. Eng. 136, 061016 (2014).

    Article  Google Scholar 

  3. Dey, M. et al. Chemotherapeutics and CAR‐T cell‐based immunotherapeutics screening on a 3D bioprinted vascularized breast tumor model. Adv. Funct. Mater. 32, 2203966 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ayan, B. et al. Aspiration-assisted freeform bioprinting of pre-fabricated tissue spheroids in a yield-stress gel. Commun. Phys. 3, 183 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kim, M. H., Banerjee, D., Celik, N. & Ozbolat, I. T. Aspiration-assisted freeform bioprinting of mesenchymal stem cell spheroids within alginate microgels. Biofabrication 14, 024103 (2022).

    Article  Google Scholar 

  6. Wu, Y. et al. Hybrid bioprinting of zonally stratified human articular cartilage using scaffold‐free tissue strands as building blocks. Adv. Healthc. Mater. 9, 2001657 (2020).

    Article  CAS  Google Scholar 

  7. Ayan, B. et al. Aspiration-assisted bioprinting for precise positioning of biologics. Sci. Adv. 6, eaaw5111 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kim, M. H. et al. High-throughput bioprinting of spheroids for scalable tissue fabrication. Nat. Commun. 15, 10083 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Heo, D. N. et al. Aspiration-assisted bioprinting of co-cultured osteogenic spheroids for bone tissue engineering. Biofabrication 13, 015013 (2020).

    Article  Google Scholar 

  10. Dey, M. et al. Biofabrication of 3D breast cancer models for dissecting the cytotoxic response of human T cells expressing engineered MAIT cell receptors. Biofabrication 14, 044105 (2022).

    Article  CAS  Google Scholar 

  11. Celik, N. et al. miRNA induced 3D bioprinted-heterotypic osteochondral interface. Biofabrication 14, 044104 (2022).

    Article  CAS  Google Scholar 

  12. Celik, N., Kim, M. H., Hayes, D. J. & Ozbolat, I. T. miRNA induced co-differentiation and cross-talk of adipose tissue-derived progenitor cells for 3D heterotypic pre-vascularized bone formation. Biofabrication 13, 044107 (2021).

    Article  CAS  Google Scholar 

  13. Banerjee, D. et al. Biofabrication of an in-vitro bone model for Gaucher disease. Biofabrication 15, 045023 (2023).

    Article  CAS  PubMed Central  Google Scholar 

  14. Ayan, B., Wu, Y., Karuppagounder, V., Kamal, F. & Ozbolat, I. T. Aspiration-assisted bioprinting of the osteochondral interface. Sci. Rep. 10, 13148 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Alioglu, M. A. et al. Nested biofabrication: matryoshka‐inspired intra‐embedded bioprinting. Small Methods 8, 2301325 (2024).

    Article  CAS  Google Scholar 

  16. Dey, M., Ayan, B., Yurieva, M., Unutmaz, D. & Ozbolat, I. T. Studying tumor angiogenesis and cancer invasion in a three‐dimensional vascularized breast cancer micro‐environment. Adv. Biol. 5, 2100090 (2021).

    Article  CAS  Google Scholar 

  17. Daly, A. C., Davidson, M. D. & Burdick, J. A. 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels. Nat. Commun. 12, 753 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Han, G. et al. 3D printed organisms enabled by aspiration‐assisted adaptive strategies. Adv. Sci. 11, 2404617 (2024).

    Article  Google Scholar 

  19. Roth, J. G. et al. Spatially controlled construction of assembloids using bioprinting. Nat. Commun. 14, 4346 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ellison, S. T. et al. Cellular micromasonry: biofabrication with single cell precision. Soft Matter 18, 8554–8560 (2022).

    Article  CAS  PubMed  Google Scholar 

  21. Liu, Y. et al. hESCs‐derived early vascular cell spheroids for cardiac tissue vascular engineering and myocardial infarction treatment. Adv. Sci. 9, 2104299 (2022).

    Article  Google Scholar 

  22. Gutzweiler, L. et al. Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications. Biofabrication 9, 025027 (2017).

    Article  PubMed  Google Scholar 

  23. Zieger, V. et al. Towards automation in 3D cell culture: selective and gentle high‐throughput handling of spheroids and organoids via novel pick‐flow‐drop principle. Adv. Healthc. Mater. 13, 2303350 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Murata, D., Arai, K. & Nakayama, K. Scaffold‐free bio‐3D printing using spheroids as ‘bio‐inks’ for tissue (re‐)construction and drug response tests. Adv. Healthc. Mater. 9, 1901831 (2020).

    Article  CAS  Google Scholar 

  25. Aguilar, I. N. et al. Scaffold-free bioprinting of mesenchymal stem cells with the regenova printer: optimization of printing parameters. Bioprinting 15, e00048 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Moldovan, N. I., Hibino, N. & Nakayama, K. Principles of the Kenzan method for robotic cell spheroid-based three-dimensional bioprinting. Tissue Eng. Part B 23, 237–244 (2017).

    Article  CAS  Google Scholar 

  27. Ip, B. C., Cui, F., Tripathi, A. & Morgan, J. R. The bio-gripper: a fluid-driven micro-manipulator of living tissue constructs for additive bio-manufacturing. Biofabrication 8, 025015 (2016).

    Article  PubMed  Google Scholar 

  28. Miao, T. et al. High-throughput fabrication of cell spheroids with 3D acoustic assembly devices. Int. J. Bioprinting 9, 733 (2023).

    Article  Google Scholar 

  29. Theret, D. P., Levesque, M., Sato, M., Nerem, R. & Wheeler, L. The application of a homogeneous half-space model in the analysis of endothelial cell micropipette measurements. J. Biomech. Eng. 110, 190–199 (1988).

    Article  CAS  PubMed  Google Scholar 

  30. Sato, M., Theret, D. P., Wheeler, L. T., Ohshima, N. & Nerem, R. M. Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties. J. Biomech. Eng. 112, 263–268 (1990).

    Article  CAS  PubMed  Google Scholar 

  31. Rocca, M., Fragasso, A., Liu, W., Heinrich, M. A. & Zhang, Y. S. Embedded multimaterial extrusion bioprinting. SLAS Technol. 23, 154–163 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Spencer, A. R. et al. Bioprinting of a cell-laden conductive hydrogel composite. ACS Appl. Mater. Interfaces 11, 30518–30533 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Madadian, E. et al. In‐foam bioprinting: an embedded bioprinting technique with self‐removable support bath. Small Sci. 2300280 (2024).

  34. Uzel, S. G., Weeks, R. D., Eriksson, M., Kokkinis, D. & Lewis, J. A. Multimaterial multinozzle adaptive 3D printing of soft materials. Adv. Mater. Technol. 7, 2101710 (2022).

    Article  Google Scholar 

  35. Kim, M. H. & Ozbolat, I. Digital files for customized parts in AAB system. Figshare https://doi.org/10.6084/m9.figshare.28405133.v1 (2025).

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Acknowledgements

This work has been supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) award no. R01EB034566 (I.T.O.) and National Institute of Dental and Craniofacial Research (NIDCR) award no. R01DE028614 (I.T.O.). We thank J. C. Moses, S. Liu, M. Yeo and D. Gupta at Penn State University for proofreading the Protocol.

Author information

Authors and Affiliations

  1. The Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA

    Myoung Hwan Kim & Ibrahim T. Ozbolat

  2. Engineering Science and Mechanics Department, Penn State University, University Park, PA, USA

    Myoung Hwan Kim & Ibrahim T. Ozbolat

  3. Department of Biomedical Engineering, Penn State University, University Park, PA, USA

    Ibrahim T. Ozbolat

  4. Department of Neurosurgery, Penn State Milton S. Hershey Medical Center, Hershey, PA, USA

    Ibrahim T. Ozbolat

  5. Materials Research Institute, Penn State University, University Park, PA, USA

    Ibrahim T. Ozbolat

  6. Biotechnology Research and Application Center, Cukurova University, Adana, Turkey

    Ibrahim T. Ozbolat

Authors
  1. Myoung Hwan Kim
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  2. Ibrahim T. Ozbolat
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Contributions

M.H.K. developed the hardware and software for the AAB, designed the detailed Protocol and wrote the manuscript. I.T.O. supervised the study, provided funding and revised the manuscript. Both authors approved the final version of the manuscript.

Corresponding author

Correspondence to Ibrahim T. Ozbolat.

Ethics declarations

Competing interests

I.T.O. has an equity stake in Biolife4D and is a member of the scientific advisory board for Biolife4D and Healshape. The remaining author declares no competing interests.

Peer review

Peer review information

Nature Protocols thanks Subha Narayan Rath and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key references

Ayan, B. et al. Sci. Adv. 6, eaaw5111 (2020): https://doi.org/10.1126/sciadv.aaw5111

Kim, M. H. et al. Nat. Commun. 15, 10083 (2024): https://doi.org/10.1038/s41467-024-54504-7

Supplementary information

Reporting Summary

Supplementary Data 1

An example .csv file for saved position for panel 4A in Fig. 4.

Supplementary Data 2

An example .csv file for the automated motion for panel 9A in Fig. 4.

Supplementary software

A software program to perform AAB.

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Kim, M.H., Ozbolat, I.T. Aspiration-assisted bioprinting of spheroids. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01240-x

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