Sacrificial templates for patterning perfusable vascular networks in engineered tissues have been constrained in architectural complexity, owing to the limitations of extrusion-based 3D printing techniques. Here, we show that cell-laden hydrogels can be patterned with algorithmically generated dendritic vessel networks and other complex hierarchical networks by using sacrificial templates made from laser-sintered carbohydrate powders. We quantified and modulated gradients of cell proliferation and cell metabolism emerging in response to fluid convection through these networks and to diffusion of oxygen and metabolites out of them. We also show scalable strategies for the fabrication, perfusion culture and volumetric analysis of large tissue-like constructs with complex and heterogeneous internal vascular architectures. Perfusable dendritic networks in cell-laden hydrogels may help sustain thick and densely cellularized engineered tissues, and assist interrogations of the interplay between mass transport and tissue function.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. Much of the source and analysed data are available in Zenodo (https://doi.org/10.5281/zenodo.3723373). Some source datasets are too large to be shared in public repositories and are available from the corresponding author on reasonable request. Design files and documentation for our open-source selective laser sintering hardware and software are available in the Zenodo repository and at https://github.com/MillerLabFTW/OpenSLS.
A custom Python add-on for Blender to generate bifurcating vascular structures is available in the Zenodo repository and at https://github.com/MillerLabFTW/IntussusceptionAddon. Image-processing and analysis scripts are also available in the Zenodo repository. The mutual tree attraction algorithm for generating dendritic networks is closed source, but the generated architectures are included in the Zenodo repository.
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We thank A. Bastian, A. Ta and T. Schmidt for assistance with OpenSLS hardware and firmware; E. Watson and A. Mikos for assistance with mechanical testing; J. Wagner, P. Desai and C. F. Higgs for assistance with powder rheology; D. De Santos for technical assistance with carbohydrate SLS; D. Kaplan and W. Stoppel for providing silk fibroin; D. L. Gibbons for providing the 344SQ lung adenocarcinoma cell line; and C. Fortin for help with hepatocyte isolations. This work was supported in part by a Medical Research Grant from the Robert J. Kleberg Jr and Helen C. Kleberg Foundation (J.S.M.), National Institutes of Health (grants HL134510 and DK115461 (K.-D.B.)), the Texas Hepatocellular Carcinoma Consortium (THCCC) (CPRIT RP150587 (K.-D.B.)), National Insitutes of Health grant DP2HL137188 (K.R.S.) and National Insitutes of Health NIBIB Cardiovascular Training grant (T32EB001650 (S.H.S.)). I.S.K. acknowledges support by an F31 National Research Service Award (NRSA) from the National Institutes of Health (HL140905). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
J.S.M. is a co-founder and holds an equity stake in Volumetric, Inc. J.E.R. and J.D.L.-R. are co-founders and hold equity stakes in Nervous System, Inc.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Annotated video recording of one layer of carbohydrate SLS.
Mutual-tree-attraction algorithm for the generative design of dendritic networks.
Fluorescent bead perfusion through a whole planar dendritic network.
Magnified view of fluorescent bead perfusion through the centre of a planar dendritic network.
Perfusion of dendritic architectures at a high volumetric flow rate.
Animated volume rendering of a region in an endothelialized planar dendritic network.
Rotating rendering of a volumetric μCT scan of a dendritic carbohydrate template.
Fly-through rendering of a volumetric μCT scan of a dendritic carbohydrate template.
Fly-through sequence of MTT staining in sections from a cell-laden gel with dendritic architecture.
Fly-through sequence of nuclear staining in sections from a cell-laden gel with dendritic architecture.
Fly-through sequence of processed MTT staining images from a cell-laden gel with dendritic architecture.
Rotating rendering of a volumetrically reconstructed MTT signal in a cell-laden gel with dendritic architecture.
Computational fluid-dynamics simulation of perfusion through a 3D dendritic architecture.
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Kinstlinger, I.S., Saxton, S.H., Calderon, G.A. et al. Generation of model tissues with dendritic vascular networks via sacrificial laser-sintered carbohydrate templates. Nat Biomed Eng 4, 916–932 (2020). https://doi.org/10.1038/s41551-020-0566-1
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