Three-Dimensional Microvascular Networks Made with Thermoresponsive Microfiber Patterning

Introduction:: A critical hurdle in the development of thick artificial tissues is the fabrication and inclusion of functional vasculature. As cell-dense artificial tissues grow to dimensions which exceed the diffusion limits of essential gases and nutrients, convective pathways in the tissue are required or else the tissue core will necrose and die. The inclusion of microvessels in tissue engineered systems would also enable the creation of more complex tissue models that better mimic natural tissue. Essential to this process is the creation of different kinds of blood vessels, including the capillaries responsible for exchange between the tissue and blood stream. Modern top-down techniques have had difficulty reproducing the microvasculature’s smallest features and complex, branching architecture: additive methods remain limited by feature size¹, and subtractive methods are limited by impractical scalability, expense, and optical working distance². Here we demonstrate dense, complex networks of artificial microvasculature fabricated via sacrificial thermoresponsive fibers embedded in a hydrogel matrix. These patterned microchannels are lined with an endothelial monolayer and are demonstrated to have capillary-like architecture, barrier properties, and the capacity to support co-cultures.

Materials and Methods:: Polyvinyl-caprolactam copolymer was dissolved and solvent spun into microfiber meshes, which were then packed into PDMS frames, and fused to an inlet. The frames with fibers were then plasma treated. A 10% w/v gelatin-microbial transglutaminase solution was cast over the fibers, then allowed to crosslink in a 37 °C incubator. Following crosslinking, devices were flushed with room temperature cell media to trigger gel-to-sol phase transition of the polymer fibers and subsequent removal, forming microchannels. Microchannel architecture was quantitatively evaluated through skeletonization of three-dimensional image stacks collected with light-sheet microscopy. Channels were then seeded with endothelial cells (ECs) at 10⁷ cells/mL and incubated to allow cell attachment. The seeded microchannels were perfused with a peristaltic pump at 100-200 μl/min for their lifetime. To verify long-term cell viability, cells were periodically stained with 5μM Calcein AM and imaged via confocal microscopy. EC-lined channels were also fixed and stained in order to assess channel coverage and the presence of intercellular junctions. A diffusion assay was performed with fluorescently-tagged dextran to quantify the permeability of endothelialized channel walls. Co-cultures with ECs and embedded Human Normal Dermal Fibroblasts (HDNFs) were prepared and assessed for viability with Calcein AM, Propidium Iodide (PI), and Hoechst.

Results, Conclusions, and Discussions:: We produced EC-lined, three-dimensional channel networks with an average diameter of less than 35 μm. These channels exhibit high cell viability for at least 30 days when perfused. Staining of EC-lined channels demonstrated the presence of both PECAM-1 and VE-Cadherin, indicating the presence of a complete monolayer and endothelial barrier. Intensity data from the dextran diffusion assay was used to numerically solve for the permeability of the channel walls: initial results show that channels with an endothelial lining are 5x less permeable to the dextran than channels without. Live-dead assays of HDNF-EC co-cultures currently show a cell viability rate of 88±2% around vascularized regions at 21 days post-seeding.
By embedding solvent-spun, thermoresponsive sacrificial microfibers in a thick enzymatically-crosslinked hydrogel, we have fabricated artificial microvascular channel networks capable of supporting a monolayer of ECs for at least 30 days. These networks exhibit complex, three-dimensional architectures, and are capable of hosting EC monolayers in channels less than 20 μm in diameter. Additionally, the EC monolayers exhibit barrier properties indicative of a healthy endothelium. These networks may also be integrated with parenchymal cells (e.g. NDHFs) in co-culture.


Acknowledgements: I would like to acknowledge and thank the foundations who funded this research: NSF grant 1506717, NIH grants R00EB013630 and R01NS110665, and AHA grant 15GRNT25710148

References: (1) Tomasina, C.; Bodet, T.; Mota, C.; Moroni, L.; Camarero-Espinosa, S. Bioprinting Vasculature: Materials, Cells and Emergent Techniques. Mater. Basel Switz. 2019, 12 (17). https://doi.org/10.3390/ma12172701.

(2) Fleischer, S.; Tavakol, D. N.; Vunjak‐Novakovic, G. From Arteries to Capillaries: Approaches to Engineering Human Vasculature. Adv. Funct. Mater. 2020. https://doi.org/10.1002/adfm.201910811.