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.
Direct 3D bioprinting of bioreactors containing microorganisms embedded inside hydrogel structures is a promising strategy for biotechnological applications. Nevertheless, microporous hydrogel networks hinder the supply of nutrients and oxygen to the cell and limit cell migration and proliferation. To overcome this drawback, we developed a feedstock for 3D bioprinting structures with hierarchical porosity. The feedstock is based on a highly particle-filled alumina/alginate nanocomposite gel with immobilized E. coli bacteria with the protein ovalbumin acting as foaming agent. The foamed nanocomposite is shaped into a porous mesh structure by 3D printing. The pore radius diameters inside the non-printed, non-foamed nanocomposite structure are below 10 µm, between 10 and 500 µm in the albumin-stabilized foam and with additional pores in the range of 0.5 and 1 mm in the printed mesh structure. The influence of albumin on the bubbles and hence pore formation was analyzed by means of interfacial shear rheology and porosity measurements with X-ray microtomography (µCT). Furthermore, averaged diffusion coefficients of water in printed and non-printed samples with different albumin concentrations were recorded using nuclear magnetic resonance (NMR) tomography to assess the water content in the porous structure. Moreover, the effective viability and accessibility of embedded E. coli cells were analyzed for various material compositions. Here, the addition of albumin induced bacterial growth and the porosity increased the effective viability of the embedded bacteria, most likely because of enhanced accessibility of the cells. 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.
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