Biofabrication under fluorocarbon: a novel freeform fabrication technique to generate high aspect ratio tissue-engineered constructs

Bioprinting is a recent development in tissue engineering, which applies rapid prototyping techniques to generate complex living tissues. Typically, cell-containing hydrogels are dispensed layer-by-layer according to a computer-generated three-dimensional model. The lack of mechanical stability of printed hydrogels hinders the fabrication of high aspect ratio constructs. Here we present submerged bioprinting, a novel technique for freeform fabrication of hydrogels in liquid fluorocarbon. The high buoyant density of fluorocarbons supports soft hydrogels by floating. Hydrogel constructs of up to 30-mm height were generated. Using 3% (w/v) agarose as the hydrogel and disposable syringe needles as nozzles, the printer produced features down to 570-μm diameter with a lateral dispensing accuracy of 89 μm. We printed thin-walled hydrogel cylinders measuring 4.8 mm in height, with an inner diameter of ∼2.9 mm and a minimal wall thickness of ∼650 μm. The technique was successfully applied in printing a model of an arterial bifurcation. We extruded under fluorocarbon, cellularized alginate tubes with 5-mm outer diameter and 3-cm length. Cells grew vigorously and formed clonal colonies within the 7-day culture period. Submerged bioprinting thus seems particularly suited to fabricate hollow structures with a high aspect ratio like vascular grafts for cardiovascular tissue engineering as well as branching or cantilever-like structures, obviating the need for a solid support beneath the overhanging protrusions.