Genipin-crosslinked chitosan/alginate/alumina nanocomposite gels for 3D bioprinting

Immobilizing microorganisms inside 3D printed semi-permeable substrates can be desirable for biotechnological processes since it simplifies product separation and purification, reducing costs, and processing time. To this end, we developed a strategy for synthesizing a feedstock suitable for 3D bioprinting of mechanically rigid and insoluble materials with embedded living bacteria. The processing route is based on a highly particle-filled alumina/chitosan nanocomposite gel which is reinforced by (a) electrostatic interactions with alginate and (b) covalent binding between the chitosan molecules with the mild gelation agent genipin. To analyze network formation and material properties, we characterized the rheological properties and printability of the feedstock gel. Stability measurements showed that the genipin-crosslinked chitosan/alginate/alumina gels did not dissolve in PBS, NaOH, or HCl after 60 days of incubation. Alginate-containing gels also showed less swelling in water than gels without alginate. Furthermore, E. coli bacteria were embedded in the nanocomposites and we analyzed the influence of the individual bioink components as well as of the printing process on bacterial viability. Here, the addition of alginate was necessary to maintain the effective viability of the embedded bacteria, while samples without alginate showed no bacterial viability. The experimental results demonstrate the potential of this approach for producing macroscopic bioactive materials with complex 3D geometries as a platform for novel applications in bioprocessing.