(M-507) Stacking thick perfusable human microvasculature to create densely vascularized biomimetic tissues

Introduction:: Tissue engineering holds great promise in generating biomimetic tissue for regenerative medicine; however, creating thick, large-scale tissues with sufficient vascularization remains a challenge. This vascularization is particularly important for organs like the heart, which have a high metabolic demand. While there have been recent advances in creating perfusable vasculature within engineered tissues, current methods lack the necessary vascular density, complexity, or microvascular size to support large-scale fabrication of highly metabolic tissues. Therefore, further studies are needed to develop new approaches to create complex, perfusable microvascular networks that are compatible with different tissues and organs. These advances could revolutionize tissue engineering and lead to novel therapies for tissue regeneration and repair.


Materials and Methods:: We have developed a modular fabrication approach to generate thick, perfusable, highly vascularized tissues, by stacking multiple layers of vasculature, patterned using soft lithography, within a remodelable collagen hydrogel (Figure 1A). These constructs contain endothelial cells within the bulk collagen, and additional endothelial cells within the perfusable vasculature, to create endothelial-lined lumens. We compared these perfusable vascularized tissues to non-perfusable, control, self-assembled tissues which contain endothelial cells only within the bulk collagen using various techniques, including confocal microscopy, scanning electron microscopy (SEM), optical coherence tomography, transcriptional profiling, and graft implantation onto infarcted rat hearts. We evaluated the structure, perfusability, and host vascular integration of these thick vascularized networks. To assess the vascular structure and perfusion dynamics within the grafts after in vivo implantation, we used lectin perfusion, tissue clearing, histological analysis and optical microangiography (OMAG).


Results, Conclusions, and Discussions:: Results

Our layered thick vascular networks showed robust cellular junctions, in vivo-like vascularity and angiogenic sprouting, and high perfusability, when compared to non-perfusable, self-assembled tissues (Figure 1B,C). RNAseq analysis demonstrated that these thick vascular networks have transcriptional profiles towards vascular development, and adding additional layers of vasculature did not significantly change the gene expression of vascular tissues. We further showed that these vessels could support highly metabolic cells, such as C2C12s (mouse skeletal muscle cells) within these constructs, with a healthy C2C12 phenotype, which is not observed within the self-assembled constructs. Lastly, we demonstrated that these multilayer microvessels improved host vascular integration in vivo. Implantation of microvessel constructs onto infarcted rat hearts showed that these grafts had a significantly greater depth and density of microvasculature perfused by the host heart and greater engraftment of implanted cells, as compared to self-assembled grafts. Furthermore, OMAG and OMAG-based velocimetry confirmed that multilayer microvessel grafts had greater overall vascular density, perfusion, and velocity of perfusion. All together, these results suggest that incorporation of perfusable, multilayer microvessels promotes host vascular integration and graft perfusion throughout the full thickness of the implanted tissue.

Conclusions/Discussion

We have developed a modular approach to generate thick ( >mm), highly vascularized tissues capable of supporting highly metabolically active cells. This modular assembly has the potential to overcome previous hurdles in the creation of large-scale complex tissues and allows for immediate tissue perfusion after assembly. This modular approach also provides precision in creating small diameter vessels and makes it possible to recapitulate tissue heterogeneity as different layers can have different cellular or matrix compositions and patterns. Our work lays the groundwork for using modular assembly to create highly metabolic thick engineered tissues with extensive vascularization for applications in both tissue repair and in vitro modeling.


Acknowledgements (Optional): : We acknowledge the Lynn and Mike Garvey Imaging Laboratory in the Institute of Stem Cell and Regenerative Medicine, the Nanotech User Facility, and Flow Cytometry Facility, all at the University of Washington. Electron microscopy data were generated using the Fred Hutchinson Cancer Research Center Electron Microscopy Shared Resource (EMSR). The EMSR is supported in part by the Cancer Center Support Grant P30 CA015704-40. We thank Dr. Kelly Stevens and Susana Simmonds Bohorquez for protocol sharing and assistance in tissue clearing. We acknowledge the financial support of National Institute of Health grants R01HL141570 (to Y.Z. and C.E.M.), R01HL146868 (to C.E.M.), R01HL148081 (to C.E.M.), T32EB1650 (to N.Z., training grant), F31HL158061 (to N.Z.), and T32EB032787-01 (to A.L.F, training grant).


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