Fabrication and patterning of alginate microspheres for 3D bioprinting

Abstract

Alginate microspheres can provide a three-dimensional (3D) space for cell adhesion and proliferation for 3D bioprinting. Monodispersed microspheres can be patterned into complex 3D shapes with controllable features. In this project, the fabrication of monodispersed alginate microspheres through simple extrusion method were presented. The fabrication parameters include the height of droplet release, the concentration of alginate droplets, the concentration of the calcium chloride (CaCl2) bath, and the mechanical rotation speed to agitate the CaCl2 bath. The best combination of the parameters tested was found to be 1 mm height of droplet release, 3% (wt/v) of alginate, 50 mM of CaCl2 solution and 150 rpm mechanical agitation speed. Nevertheless, some of the alginate droplets were floating on the CaCl2 solution, producing deformed alginate beads that are not spherical. A new method of using ethanol in the CaCl2 bath was explored to prevent the alginate droplets from floating on the surface of the bath. Spherical alginate spheres can be fabricated in a solution of 50 mM CaCl2 in 23% ethanol. The size of the spheres were ~2 mm, which can be reduced to 1 mm after post-crosslinking in a 50 mM CaCl2 solution. Following the alginate microspheres fabrication, the microspheres were patterned via two techniques, i.e. direct and indirect extrusion patterning methods. The bioink for these patterning methods was made up of alginate microspheres encapsulated in partially cross-linked alginate hydrogel, which produced a self-supporting structure with immediate gelation upon immersing in a CaCl2 bath. In the direct extrusion patterning method, various shapes were producible. In the indirect extrusion patterning method, a pluronic F127 mold was printed before incorporating bioink. The two patterning techniques were both feasible. Although indirect extrusion patterning produced shapes of lesser printing accuracy due to the diffusion of the alginate gel, they can both be performed depending on the design complexity of the desired biological construct. The elastic modulus of the cross-linked bioink was found to be higher than those of alginate hydrogel without spheres via a compression test. Fabricating and patterning alginate spheres via simple and inexpensive methods open up more possibilities to introducing hydrogel spheres into 3D bioprinting.