Abstract
Natural tissues are composed of diverse cells and extracellular materials whose arrangements across several length scales—from subcellular lengths1 (micrometre) to the organ scale2 (centimetre)—regulate biological functions. Tissue-fabrication methods have progressed to large constructs, for example, through stereolithography3 and nozzle-based bioprinting4,5, and subcellular resolution through subtractive photoablation6,7,8. However, additive bioprinting struggles with sub-nozzle/voxel features9 and photoablation is restricted to small volumes by prohibitive heat generation and time10. Building across several length scales with temperature-sensitive, water-based soft biological matter has emerged as a critical challenge, leaving large classes of biological motifs—such as multiscalar vascular trees with varying calibres—inaccessible with present technologies11,12. Here we use gallium-based engineered sacrificial capillary pumps for evacuation (ESCAPE) during moulding to generate multiscalar structures in soft natural hydrogels, achieving both cellular-scale (<10 µm) and millimetre-scale features. Decoupling the biomaterial of interest from the process of constructing the geometry allows non-biocompatible tools to create the initial geometry. As an exemplar, we fabricated branched, cell-laden vascular trees in collagen, spanning approximately 300-µm arterioles down to the microvasculature (roughly ten times smaller). The same approach can micropattern the inner surface of vascular walls with topographical cues to orient cells in 3D and engineer fine structures such as vascular malformations. ESCAPE moulding enables the fabrication of multiscalar forms in soft biomaterials, paving the way for a wide range of tissue architectures that were previously inaccessible in vitro.
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Fig. 1: Capillary pumps for sacrificial moulding.
Fig. 2: Endothelialized structures—vessel topologies and fine features.
Fig. 3: Hierarchical vascular trees and epithelial ducts.
Fig. 4: 3D applications of ESCAPE—epithelial ducts, multicellular orthogonal networks and cell-dense structures with proximal vasculature.
Data availability
The main data supporting the findings in this study are available in the main text, methods and Supplementary Information. Other data generated or analysed are available from the corresponding authors on request.
Code availability
Blender/Python code used to computationally generate vascular trees, OpenSCAD code to generate CAD designs and MATLAB code to analyse images are available from the corresponding authors on request.
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Acknowledgements
S.S. thanks H. Shea and R. C. Hayward for their comments and discussions. We gratefully acknowledge support from the NIH National Institute of Biomedical Imaging and Bioengineering (NIH-EB00262, NIH-EB033821), National Science Foundation Engineering Research Center on Cellular Metamaterials (EEC-1647837), National Science Foundation Science and Technology Center for Engineering Mechanobiology (CMMI-1548571), Allen Distinguished Investigator programme, U.S.-Israel Binational Science Foundation (BSF 2017239), American Heart Association Postdoctoral Fellowship (20POST35210045), NIH National Heart, Lung, and Blood Institute (F31HL156517), NIH T32 Quantitative Biology and Physiology training grant and the Portuguese Foundation for Science and Technology (FCT; doctoral grant SFRH/BD/129224/2017).
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Authors and Affiliations
Biological Design Center, Boston University, Boston, MA, USA
Subramanian Sundaram, Joshua H. Lee, Isabel M. Bjørge, Christos Michas, Sudong Kim, Alex Lammers, Jeroen Eyckmans & Christopher S. Chen
Department of Biomedical Engineering, Boston University, Boston, MA, USA
Subramanian Sundaram, Joshua H. Lee, Isabel M. Bjørge, Christos Michas, Sudong Kim, Alex Lammers, Jeroen Eyckmans & Christopher S. Chen
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
Subramanian Sundaram, Sudong Kim, Alex Lammers, Jeroen Eyckmans & Christopher S. Chen
Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
Isabel M. Bjørge & João F. Mano
Department of Mechanical Engineering, Boston University, Boston, MA, USA
Christos Michas & Alice E. White
Department of Physics, Boston University, Boston, MA, USA
Alice E. White
Department of Material Science and Engineering, Boston University, Boston, MA, USA
Alice E. White
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Subramanian Sundaram
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Contributions
S.S. and C.S.C. conceived and designed the research. S.S. designed the process, wrote custom code, analysed data and was involved in all aspects of the work. S.S., J.H.L. and I.M.B. performed experiments. S.S. and C.M. printed two-photon direct writing moulds under the supervision of A.E.W. S.K. advised on cell experiments. A.L. performed multiphoton microscopy optimization. J.F.M. co-supervised the work of I.M.B. S.S., J.E. and C.S.C. wrote the manuscript. All authors discussed and contributed to the manuscript.
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Correspondence to Subramanian Sundaram or Christopher S. Chen.
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Competing interests
A patent application (U.S. application no. 18/422,963) has been filed by Boston University based on this work. C.S.C. is a founder and owns shares in Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of the dimensional stability and architecture of collagen gels on exposure to NaOH.
a, To study the dimensional stability of features in collagen, we polymerized collagen gels around acupuncture needles (160 µm diameter; see Methods). After polymerizing collagen, we withdrew the needle and added different concentrations of NaOH to the reservoirs ensuring flow through the channel and placed devices on a hotplate maintained at 32 °C for 30 min. b, Post NaOH treatment, we imaged these collagen gels using SHG imaging. Until 20 mM NaOH treatment, the dimension of the channel closely matched the size of the acupuncture needles and the quantification of the collagen intensity showed clear boundaries (see arrows). After 30 min exposure to 50 mM NaOH, the gels lost their structural integrity, leading to broadening of the features. Gels treated with 100 mM NaOH did not show clear edges and dimensions of the channels could not be measured (not shown here). c, Quantification of the channel dimensions post-exposure to different concentrations of NaOH for 30 min (mean ± s.d. across five devices per concentration). d, Collagen architecture post 30-min exposure to NaOH as observed through SHG imaging of bulk collagen gels (regions shown are 100 µm × 100 µm). e, Collagen intensity changes in response to different concentration of NaOH post 30-min exposure normalized with respect to the PBS control (mean ± s.d. across 18 measurements from six devices per concentration).
Extended Data Fig. 2 3D cavities in collagen.
a, Printed Stanford bunny. b, PDMS negative. c, Solid Ga cast. d, Cavity in collagen. e, Depth-coded projection of cell nuclei post cell seeding. f, Confocal image projections of DAPI, F-actin and VE-cadherin. g, Comparison of 3D design (sections shown in white) and the fabricated cavity (collagen SHG signal). Scale bars, 200 µm (a,d); 100 µm (e,f).
Extended Data Fig. 3 Capillary pumping (Ga ESCAPE) in different materials.
a, Design of bifurcations for making Ga casts. b, Ga can be evacuated in soft materials such as collagen. On removing the Ga cast, the empty cavity can be filled with cells or other materials; here the branching conduits are filled with coloured beads for visualization. c, Geometries with both flow-accessible conduits and dead ends can be retracted at once. Critically, this differs from using Ga as a sacrificial material with non-porous surrounding materials (for example, PDMS) without interstitial flow, in which dead-ended branches are severed during evacuation. d, Retraction process works in other soft hydrogels, such as fibrin or agarose. e, Photographs of devices with different materials after Ga retraction with coloured beads lining the conduits. f, Culturing cells in direct contact with Ga does not result in cell toxicity. g, Cells grow on top of Ga (probably on the native oxide layer) when cultured on dishes containing Ga droplets. Scale bars, 100 µm. Dead cells could be seen occasionally (marked by white arrowheads on the right) at the contact line between Ga and the culture dish, which—we posit—is from the increased mechanical movement of the interface at the contact line.
Extended Data Fig. 4 Endothelialized vessels.
a, 150-µm-diameter cylindrical vessel seeded with ECs. Scale bar, 200 µm. b, Close-up of the vessel showing F-actin and VE-cadherin (scale bar, 100 µm) and the cross-section (scale bar, 50 µm). c, Tapered vessel with calibre decreasing from 150 µm to 20 µm. Scale bar, 200 µm. Close-up multiphoton images show collagen and the cell nuclei in a single plane. Corresponding confocal maximum projections show that cells line the vessels uniformly until the cavity is comparable in size to the cell nuclei (scale bars: left, 25 µm; right, 10 µm). d, Sinusoidal vessel design and fabricated device. Scale bar, 200 µm. e, Bifurcating vessel with one dead-ended branch and maximum projections of the fabricated device (f; scale bar, 200 µm). The close-up images show the perfused and dead-ended sections. Scale bars, 100 µm. g, Two-level branching Murray design. Ga cast (h) and immunofluorescence images (i). Scale bars, 200 µm (tile scan); 100 µm (close-up image). j, The two-level branching design with narrow constrictions in the smallest branches (j) and the fabricated device (k; scale bar, 200 µm). Close-up images show four constricted sections (scale bar, 50 µm).
Extended Data Fig. 5 Self-intersecting structures.
a, 3D knot design. b, By introducing a narrow continuous wall under the part, self-intersecting structures such as the knot can be moulded. The thickness of the wall is made to be much smaller than the feature. After demoulding the negative structure, the high surface tension of the liquid is used in controlling where Ga flows, that is, the injection pressure is greater than the value required to inject Ga into the knot itself but not high enough for entering the gap left by the narrow wall. c, The modified design of the knot used for printing and the resulting shape of the Ga cast. d, PDMS negative copy of the printed structure (side view in e). f, Photo of the moulded Ga knot. g, Side and top views of the cell nuclei. Scale bar, 100 µm. h, Immunofluorescence images of the EC-lined knot labelling F-actin and VE-cadherin. Scale bar, 100 µm.
Extended Data Fig. 6 Fine control of vascular structures—vascular malformations and microgrooves to control cell alignment.
a, Tilted-angle SEM images of the printed vascular malformation design. Scale bar, 200 µm. Close-up images show spherical blebs with different offsets. Scale bar, 20 µm. b, Phase-contrast image of the collagen device seen from top of the blebs. The fabrication process yields high-resolution smooth spherical blebs (shown by the arrowhead). c, SHG slice of collagen (averaged) through one bleb structure before cell seeding. Scale bar, 50 µm. d, 3D volumetric views of the different spherical blebs. e, Design of a cylindrical vessel with no orientation cues and five regions to align cells progressively from 90° to 0° (along the length of the vessel). f, Orientation histograms of the cells (F-actin) at different regions of the vessel. g, Half vessel maximum projections of DAPI, F-actin and VE-cadherin at different vessel regions. Scale bar, 50 µm.
Extended Data Fig. 7 Hierarchical branching trees with both perfused and dead-ended sections.
a, Branching vessel architecture with a tenfold reduction in vessel calibre. The vessels are dimensioned according to Murray’s law (with exponent 2.96) at each branching hierarchy. b, Tilted-angle SEM images of the printed structure. Scale bar, 200 µm. c, SEM image of the smallest branches of the Ga cast. d, Tile scan of the device showing the cell nuclei and VE-cadherin. The insets show vessel cross-sections at different regions of the device. Scale bar, 200 µm. The close-up images (bottom) show a branch point and cells sprouting from the dead ends at approximately 60-µm-sized vessels. Scale bars, 50 µm. e, Design of a deterministic tree with symmetric bifurcations with each vessel (perfusable). f, Maximum projection of tile scans of the deterministic binary tree. Scale bar, 250 µm.
Extended Data Fig. 8 Rectangular tree.
a, PDMS negative of the 4 × 4-mm2 tree. b, Tile scan of the vascular tree showing the cell nuclei and F-actin. Scale bar, 250 µm. c, Time-course images of the Ga retraction process with 20 mM NaOH (see Supplementary Video 12). d, When high concentrations of NaOH are used, the retraction process is expedited, showing that the retraction rate is determined by how the surface oxide is removed (spatially) and not limited by the evacuation rate of liquid Ga through the central root.
Extended Data Fig. 9 Marginal growth and hierarchical 3D vascular trees.
a, Design generated through marginal growth (in which the boundary is grown iteratively; see Methods), the corresponding PDMS negative and the Ga cast. b, Tile scan of the marginal growth tree showing F-actin and DAPI. Scale bar, 250 µm. c, Hierarchical 3D vascular tree design branching from one inlet to 16 outlets, with the branches sized per Murray’s law. The projected thin support wall (10 µm thick) makes the printed 3D structure mouldable, that is, PDMS can be polymerized around this structure and removed. Subsequently, when liquid Ga is injected into the mould, it preferentially fills the branching vascular structure (all features ≫10 µm) but not the narrow wall yielding a cast of the intended design. d, Photographs of the gallium cast as viewed from the top and when tilted. e, Tilted (volumetric) view of the cell nuclei of the endothelial monolayer through SHG imaging as viewed from the array of outlets; colour denotes the vertical position. Scale bar, 300 µm. Maximum projections of the inlet (f), the first bifurcation (g) and a quadrant of outlets (h) showing DAPI, VE-cadherin and F-actin. Scale bars, 100 µm. The insets in h show the corresponding regions in a titled volumetric view. The regions shown in f, g and h are marked in the full device image (e) as *, ** and ***, respectively.
Extended Data Fig. 10 Applications—epithelial buds and orthogonal (blood and lymphatic) networks.
a, Maximum projection of the 3D epithelial bud geometry (E-cadherin). Scale bar, 100 µm. b, The close-up images show the cell nuclei and F-actin at a slice through a single bud. Scale bar, 20 µm. c, Design of the 3D, enmeshed blood and lymphatic networks and supporting structures. The enmeshed architecture requires thin support walls (20 µm thick) projected onto the substrate to make the design mouldable. The Ga pieces corresponding to the blood and lymphatic network are aligned with respect to each other through a supporting structure (outside the device/gel region). d, Ga cast. e, Maximum projections of the device. Scale bar, 300 µm. Volumetric (f) and close-up projections (g; scale bar, 100 µm) of the enmeshed parts of the device (see Fig. 4b–d).
Extended Data Fig. 11 Cardiac bundles with vasculature.
a, Design consists of helical cardiac bundles maximally packed with iPSC-CMs (90%) and CFs (10%) twisted along with aligned vascular channels separated by 150 µm. Thin supporting walls projected onto the substrate (14 µm) are used to make the design mouldable. Tabs at the right end hold the cardiac and vascular portions of the Ga cast together at the ends of the cast. b, Ga casts of the twisted cardiac and vascular regions. c, To study the effectiveness of maximally packing cells in the cardiac portions, devices were filled with a mixture of iPSC-CMs (90%) and CFs (10%) in the cardiac portions alone and the vascular regions were left empty. Depth-coded projection of the cell nuclei. Scale bar, 200 µm. d, iPSC-CMs are confined to bundles and the cells in the bulk are CFs, as seen in the maximum projection images. Scale bars, 100 µm. e, Voltage waveforms used for 1-Hz electrical stimulation and a close-up of the biphasic pulse. f, Phase image of the device region showing the cardiac bundle and the vascular channel with beads (not in focus). Scale bar, 100 µm. g, On the addition of tracer beads into the vascular conduit, the displacement of particles can be tracked along specific paths in the form of kymographs (see Supplementary Video 15). The beads appear as black spots forming traces that show the baseline flow rate from the pressure head and the impact of cardiac contraction at different stimulation frequencies. h, Maximum projections of the devices containing both cardiac cells (in the cardiac portions) and a confluent layer of ECs in the vascular conduits. Scale bar, 200 µm. i, Close-up images showing the cell nuclei of regions marked in h and corresponding to Fig. 4g. Scale bars, 100 µm.
Supplementary information
Supplementary Information
Supplementary Video 1
Time-lapse showing liquid Ga droplets attached to PDMS surfaces. Different concentrations of NaOH were added to these wells, capped and inverted. When Ga detaches from PDMS, it drops down and rolls (speed 150×). Second part compares Ga droplets attached to PDMS and agarose on the addition of 10 mM NaOH (speed 2×).
Supplementary Video 2
Capillary pumping of a 150-µm gallium filament in collagen using 100 mM NaOH, placed on a hotplate at 32 °C (speed 60×). Several frames of the video show the addition of NaOH to the reservoirs.
Supplementary Video 3
Capillary pumping of a 150-µm gallium filament in collagen using 50 mM NaOH, placed on a hotplate at 32 °C (speed 60×). Several frames of the video show the addition of NaOH to the reservoirs.
Supplementary Video 4
Capillary pumping of a 150-µm gallium filament in collagen using 20 mM NaOH, placed on a hotplate at 32 °C (speed 60×). Several frames of the video show the addition of NaOH to the reservoirs.
Supplementary Video 5
Time-lapse showing the capillary pumping of a 150-µm gallium filament in collagen using 10 mM NaOH (speed 60×). A few frames of the video show the addition of NaOH to the reservoirs. Second part of the video shows the addition of coloured beads to visualize the cavity formed in collagen. Note that the non-uniform pink colour in the gel is because of phenol red in the gel region being washed from right to left.
Supplementary Video 6
Comparison between using a hydrogel as the surrounding material and a non-porous material during evacuation of a bifurcation with one dead-ended branch and one through branch.
Supplementary Video 7
ESCAPE process with a symmetric bifurcation in collagen.
Supplementary Video 8
ESCAPE process with a symmetric bifurcation in fibrin.
Supplementary Video 9
ESCAPE process with a symmetric bifurcation in agarose.
Supplementary Video 10
Diffusion of 70-kDa dextran from ESCAPE-fabricated vessels (speed 50×).
Supplementary Video 11
Growth of the space colonization tree designed to nourish a 4-mm × 4-mm area; see design parameters in Methods.
Supplementary Video 12
ESCAPE process with rectangular space colonization tree design using 20 mM and 100 mM NaOH.
Supplementary Video 13
Rotating volumetric view of the 3D orthogonal networks design showing the cell nuclei imaged by multiphoton imaging. Cell nuclei are colour-coded by z position (see Fig. 4c).
Supplementary Video 14
Regions of the cardiac bundle devices without ECs in the nearby vascular conduits under electrical stimulation.
Supplementary Video 15
Cardiac bundle devices with tracer beads in the vascular conduits (but without ECs) under electrical stimulation. See kymographs in Extended Data Fig. 11g.
Supplementary Video 16
Region of a cardiac bundle device with endothelialized vascular conduits electrically stimulated at 1 Hz. Movement of beads shows the coupling between cardiac contractions and the flow through the vascular conduits (see kymograph in Fig. 4h).
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Sundaram, S., Lee, J.H., Bjørge, I.M. et al. Sacrificial capillary pumps to engineer multiscalar biological forms. Nature (2024). https://doi.org/10.1038/s41586-024-08175-5
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Received:16 October 2023
Accepted:08 October 2024
Published:11 December 2024
DOI:https://doi.org/10.1038/s41586-024-08175-5
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