Engineering microvasculature by 3D bioprinting of prevascularized spheroids in photo-crosslinkable gelatin

To engineer tissues with clinically relevant dimensions by three-dimensional bioprinting, an extended vascular network with diameters ranging from the macro- to micro-scale needs to be integrated. Extrusion-based bioprinting is the most commonly applied bioprinting technique but due to the limited resolution of conventional bioprinters, the establishment of a microvascular network for the transfer of oxygen, nutrients and metabolic waste products remains challenging. To answer this need, this study assessed the potential and processability of spheroids, containing a capillary-like network, to be used as micron-sized prevascularized units for incorporation throughout the bioprinted construct. Prevascularized spheroids were generated by combining endothelial cells with fibroblasts and adipose tissue-derived mesenchymal stem cells as supporting cells. To serve as a viscous medium for the bioink-based deposition by extrusion printing, spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) and Irgacure 2959. The influence of gelMA encapsulation, the printing process and photo-crosslinking conditions on spheroid viability, proliferation and vascularization were analyzed by live/dead staining, immunohistochemistry, gene expression analysis and sprouting analysis. Stable spheroid-laden constructs, allowing spheroid outgrowth, were achieved by applying 10 min UV-A photo-curing (365 nm, 4 mW cm^-2), while the construct was incubated in an additional Irgacure 2959 immersion solution. Following implantationin ovoonto a chick chorioallantoic membrane, the prevascular engineered constructs showed anastomosis with the host vasculature. This study demonstrated (a) the potential of triculture prevascularized spheroids for application as multicellular building blocks, (b) the processability of the spheroid-laden gelMA bioink by extrusion bioprinting and (c) the importance of photo-crosslinking parameters post printing, as prolonged photo-curing intervals showed to be detrimental for the angiogenic potential and complete vascularization of the construct post printing.