Abstract
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.
Highlights
Developing a bioink to produce mechanically stable materials with embedded bacteria and with customized porous structure by 3D bioprinting could lead to innovative bioreactor concepts
We develop a chitosan/ alginate/alumina nanocomposite gel for bacteria encapsulation and demonstrate its suitability for 3D printing. The stability of this gel is reinforced by two different crosslinking methods: during bioink preparation, alginate electrostatically interacts with chitosan, and after shaping the gel is interconnected by covalent crosslinking of the chitosan chains with genipin
Electrostatic crosslinking of chitosan/alumina and alginate considerably increased shape fidelity after printing, allowing to further reinforce the material by covalent crosslinking between chitosan and genipin
Summary
Developing a bioink to produce mechanically stable materials with embedded bacteria and with customized porous structure by 3D bioprinting could lead to innovative bioreactor concepts. In such applications, both the support material and the embedded cells have to survive the rigors of longterm continuous flow processing or in related conditions. Bioinks must fulfill two primary criteria: high cell viability and high printability, the latter being the ability to form 3D structures with good fidelity and integrity [2]. Printable materials should exhibit a solid-like behavior of the printed filaments, which should be strong enough to support the deposition of further layers.
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