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Intravital three-dimensional bioprinting

Nature Biomedical Engineering volume 4, pages 901–915 (2020)Cite this article

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Abstract

Fabrication of three-dimensional (3D) structures and functional tissues directly in live animals would enable minimally invasive surgical techniques for organ repair or reconstruction. Here, we show that 3D cell-laden photosensitive polymer hydrogels can be bioprinted across and within tissues of live mice, using bio-orthogonal two-photon cycloaddition and crosslinking of the polymers at wavelengths longer than 850 nm. Such intravital 3D bioprinting—which does not create by-products and takes advantage of commonly available multiphoton microscopes for the accurate positioning and orientation of the bioprinted structures into specific anatomical sites—enables the fabrication of complex structures inside tissues of live mice, including the dermis, skeletal muscle and brain. We also show that intravital 3D bioprinting of donor-muscle-derived stem cells under the epimysium of hindlimb muscle in mice leads to the de novo formation of myofibres in the mice. Intravital 3D bioprinting could serve as an in vivo alternative to conventional bioprinting.

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Fig. 1: Intravital 3D bioprinting setup and in vivo application.
Fig. 2: Development of injectable HCC-conjugated polymers for i3D bioprinting application.
Fig. 3: Characterization of the photosensitive polymers.
Fig. 4: Three-dimensional objects of photosensitive gelatin hydrogels can be used for in vitro cell culture.
Fig. 5: HCC–gelatin hydrogels can be fabricated into pre-existing 3D environments and are suitable for 3D in vitro hSIO culture.
Fig. 6: Intravital 3D bioprinting.
Fig. 7: Cell-laden i3D bioprinting.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw image data and the analysed data generated in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by 2017 STARS-WiC grant of University of Padova, Progetti di Eccellenza CaRiPaRo, TWINING of University of Padova, Oak Foundation Award (grant no. W1095/OCAY-14-191), ‘Consorzio per la Ricerca Sanitaria’ (CORIS) of the Veneto Region, Italy (LifeLab Program) to N.E. and the STARS Starting Grant 2017 of University of Padova (grant code LS3-19613) to A.U. P.D.C. is supported by the National Institute for Health Research (NIHR; grant no. NIHR-RP-2014-04-046). G.G.G. was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre Catalyst Fellowship. G.G.G., P.D.C. and N.E. were supported by the Oak award W1095/OCAY-14-191. All research at Great Ormond Street Hospital NHS Foundation Trust and University College London Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the National Health Service, the NIHR or the Department of Health. We thank D. Moulding for technical support and S. Schiaffino for scientific advice and discussion.

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Authors and Affiliations

  1. Department of Industrial Engineering, University of Padova, Padova, Italy

    Anna Urciuolo, Luca Brandolino, Cecilia Laterza, Monica Giomo & Nicola Elvassore

  2. Veneto Institute of Molecular Medicine, Padova, Italy

    Anna Urciuolo, Luca Brandolino, Paolo Raffa, Valentina Scattolini, Cecilia Laterza, Laura Brigo & Nicola Elvassore

  3. Department of Women’s and Children’s Health, University of Padova, Padova, Italy

    Anna Urciuolo, Paolo Raffa, Valentina Scattolini & Elisa Zambaiti

  4. ONYEL Biotech, Padova, Italy

    Ilaria Poli

  5. University College London Great Ormond Street Institute of Child Health, London, UK

    Giovanni G. Giobbe, Giulia Selmin, Michael Magnussen, Paolo De Coppi & Nicola Elvassore

  6. Department of Specialist Paediatric and Neonatal Surgery, Great Ormond Street Hospital for Children, London, UK

    Paolo De Coppi

  7. Department of Pharmaceutical and Pharmacological Science, University of Padova, Padova, Italy

    Stefano Salmaso

  8. Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China

    Nicola Elvassore

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  1. Anna Urciuolo
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Contributions

A.U. and N.E. designed the experiments. N.E. designed the photochemistry, I.P. synthesized and chemically characterized the coumarin polymers and S.S. contributed to the chemical characterization of coumarin polymers. A.U. performed and analysed in vitro and in vivo experiments. L.Brandolino. and P.R. contributed to in vitro experiments. V.S. contributed to the analysis of in vivo experiments. C.L. performed hydrogel injection into the brain and derived reporter cells and human ES cell-derived NSCs. G.G.G., E.Z., G.S. and M.M. contributed to organoid experiments. G.G. and P.D.C. characterized human intestinal organoid cultures. L.Brigo. contributed to the design and interpretation of in vitro two-photon crosslinking experiments. M.G. performed AFM analysis. A.U. and N.E. analysed the data and wrote the manuscript. N.E. supervised the project.

Corresponding author

Correspondence to Nicola Elvassore.

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Competing interests

N.E. has an equity stake in ONYEL Biotech s.r.l. A.U. and N.E. are submitting a patent for the intravital 3D bioprinting (provisional patent number 102020000008779).

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Supplementary information

Supplementary Information

Supplementary methods, figures, tables and video captions.

Reporting Summary

Supplementary Video 1

3D projection of z-stack images showing a ‘LIFE’-shaped HCC–4-arm PEG 3D structure related to Supplementary Fig. 12c.

Supplementary Video 2

3D reconstruction of z-stack images showing an empty cuboidal-shaped HCC–8-arm PEG 3D structure related to Supplementary Fig. 12e.

Supplementary Video 3

3D reconstruction of z-stack images showing a flower-shaped HCC–gel 3D structure related to Fig. 3d.

Supplementary Video 4

3D reconstruction of z-stack images showing the maximum fabrication depth related to Fig. 3e.

Supplementary Video 5

Orthogonal 3D reconstruction of z-stack images showing a cell-laden bioprinted structure related to Supplementary Fig. 18a.

Supplementary Video 6

Long-term 3D culture of MuSCs related to Fig. 4g.

Supplementary Video 7

3D reconstruction of z-stack images showing object fabricated into a drop of HCC–gel/Matrigel related to Fig. 5a.

Supplementary Video 8

3D reconstruction of z-stack images showing objects fabricated into a drop of HCC–gel/Matrigel in respect to hSIOs related to Supplementary Fig. 20a.

Supplementary Video 9

Intravital imaging related to Fig. 6b.

Supplementary Video 10

Intravital imaging related to Fig. 6e.

Supplementary Video 11

3D reconstruction related to Fig. 6e.

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Urciuolo, A., Poli, I., Brandolino, L. et al. Intravital three-dimensional bioprinting. Nat Biomed Eng 4, 901–915 (2020). https://doi.org/10.1038/s41551-020-0568-z

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