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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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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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.
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).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary methods, figures, tables and video captions.
3D projection of z-stack images showing a ‘LIFE’-shaped HCC–4-arm PEG 3D structure related to Supplementary Fig. 12c.
3D reconstruction of z-stack images showing an empty cuboidal-shaped HCC–8-arm PEG 3D structure related to Supplementary Fig. 12e.
3D reconstruction of z-stack images showing a flower-shaped HCC–gel 3D structure related to Fig. 3d.
3D reconstruction of z-stack images showing the maximum fabrication depth related to Fig. 3e.
Orthogonal 3D reconstruction of z-stack images showing a cell-laden bioprinted structure related to Supplementary Fig. 18a.
3D reconstruction of z-stack images showing object fabricated into a drop of HCC–gel/Matrigel related to Fig. 5a.
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.
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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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