Our project heavily integrated wet-lab, modeling, and hardware by demonstrating the functionality of our 3D bioprinter to print two bioinks, weighing in predictions from modeling regarding the diffusive, biocompatible, and shape fidelity properties of our hydrogels while concurrently determining optimal print parameters through multiple DBTL cycles. As our project extensively characterized considerations that factored into optimizing both the 3D bioprinter and the bioinks, we recognize that this array of parameters can be overwhelming for iGEM teams starting out. Teams embarking on building on our dual-channel bioprinting system likely will have different goals — they may be using different organisms, synthesizing different compounds, printing different structures, or more.

Therefore, we sought to provide a starting point for conducting testing for future iGEM teams interested in replicating our 3D bioprinting system. We designed, performed, and analyzed a simple, all-in-one test to assess both cell viability and print precision, providing a model for future teams on how to cross-examine and optimize two bioink properties simultaneously.

The two factors we chose to cross-analyze were cell viability and fidelity to the intended print structure. Regardless of the application, cell viability and structural fidelity hold as key parameters in microbial printing, as the biocompatibility and physical properties of the bioink and print media determine whether we can effectively print and harness the living system for biomanufacturing. For future teams unsure of which bioprinter and bioink optimization factors to test first, our experimental design template is an excellent starting point. Teams that do have a grasp on which properties they intend to test can still benefit from our template for optimizing two bioprinting factors in parallel, customizing it to their own needs.

We conducted our printing tests with alginate, due to its relatively low cost, high biocompatibility, and relatively simple protocol to prepare. Alginate requires crosslinking with CaCl₂ to form its hydrogel state; however, CaCl₂ has been shown to be toxic to bacteria in concentrations above 0.1 M [1]. Optimizing the solid agar media on which alginate is printed requires lower concentrations of CaCl₂.

On the other hand, print fidelity favors higher concentrations of CaCl₂ in the solid media plate. Plates with higher concentrations of CaCl₂ structures have cleaner, higher resolution prints. This is because a greater crosslinker concentration gellifies the bioink quicker before the structure has time to spread out and pool on the plate, which causes low resolution prints or for more complicated shapes, collapsed structures.

The challenge thus lies in finding an ideal CaCl₂ plate concentration that maximizes print resolution while preserving cell viability. During this experiment, all other printing parameters, such as print speed or extrusion height, were kept constant to avoid confounding variables. We tested 10 different CaCl₂ agar plate concentrations, from 0.1 M to 1.0 M. For each concentration, we performed three replicates. On each plate, three prints of 4% alginate bioink containing our engineered bacteria were performed: 3D-printed (left), hand-extruded (middle), and streaked (right).

As we increased the concentration of CaCl₂ in our plates, the print fidelity generally improved for both manual and 3D printed lines. At higher CaCl₂ concentrations, the sol-gel transition time was shorter, and the bioink lines were visibly thinner as they did not spread out as much on the plate. This verified our hypothesis that increasing CaCl₂ concentrations boosted print fidelity to the intended line shape. Concentrations from 0.6 M CaCl₂ and greater were visibly up to our print fidelity standards.

Next, we integrated our print fidelity optimization into the context of customizing cell viability. We compared our 3D-printed, hand-printed, and streaked hydrogels across CaCl₂ to a control plate made with LB agar.

Through visual analysis, it was evident that bacterial growth in the hydrogel was most robust on the control plate without CaCl₂, and the least on the 1.0 M plate.

To balance optimizing both printability and cell viability in our alginate bioink, we selected the concentration of 0.6 M CaCl₂ as our ideal solid media print surface, as it meets our print fidelity threshold and maintains an intermediate level of cell viability necessary for our parallel culture system.