## 4 Discussion  
The development of carriers and bioinks for 3D printing have been widely investigated, along with the conditions for printing with these substrates [18]. However, the cell mixing step, which is critical for highly reproducible bioprinting, has not been considered in detail [4]. The gelling properties of hydrogels are typically used to control viscosity during cell suspension [4; 19]. If the carrier is too viscous, mixing is inefficient and air bubbles are more likely to form, resulting in a heterogenous cell distribution [4]. On the other hand, if the carrier is not viscous enough then cells begin to settle, not only resulting in a heterogeneous distribution but also causing cells to accumulate and block the nozzle [4]. To address this issue, we developed and optimized a cell mixing procedure based on a defined requirements profile, followed by the analysis of rheology and homogeneity. Static mixers achieve efficient mixing in a single step [20], whereas a pair of coupled syringes allow the mixing process to be repeated easily [21]. However, more than eight repetitions are normally required to achieve satisfactory homogeneity, which also reduces the strength of the gel [21]. In our study, we tested three devices with a 1:10 mixing ratio,  
Figure 9: Representative non-processed images of a cell-laden strand over time. Left panels show transmitted light images. Center panels show the green fluorescence channel (FDA), indicating the viable cells. Right panels show the red fluorescence channel, indicating dead cells (PI). Scale bar = 100 \(\upmu\)m. For high quality images, the reader is referred to the web version. revealing a lack of homogeneity in the mixing sections or rheological properties that were incompatible with instant printing using our existing bioprinting protocol, thus causing too much shear. Accordingly, we chose a 1:1 mixing ratio in the static mixer system with a long mixing section, which had less impact on the rheology of the carrier and thus a lower shear intensity on the cells. We adapted the volume flow and rheological properties based on temperature-dependent measurements and applied a defined pressure using a syringe pump, resulting in homogenous mixing sections and a low shear impact. Adaption of the volume flow by separating the gelling agents also reduced cell sedimentation compared with a pure aqueous solution. Cell sedimentation is dependent on incubation time and the polymer content of the carrier [22; 23]. For example, lower alginate concentrations in a bioink (0.5-4% \(w/v\)) accelerated cell sedimentation [22]. In the absence of shear, the cell sedimentation rate was 0.33 \(\upmu\)m\(\cdot\)s\({}^{-1}\) in 1.5% alginate hydrogels and 0.05 \(\upmu\)m\(\cdot\)s\({}^{-1}\) when the alginate concentration was 4% [22]. In contrast, our sedimentation rate was \(\sim\)6.8 \(\times\) 10\({}^{-4}\)\(\upmu\)m\(\cdot\)s\({}^{-1}\) for bioink with zero shear (according to Carreau-Yasuda) at 37 \({}^{\circ}\)C, and 1.7 \(\times\) 10\({}^{-4}\)\(\upmu\)m\(\cdot\)s\({}^{-1}\) for a gelatin solution at 28 \({}^{\circ}\)C (0.1 s\({}^{-1}\)). We therefore do not anticipate any complications caused by cell sedimentation in our bioink. In contrast, cells suspended in water sedimented with a velocity of 1.91 \(\upmu\)m\(\cdot\)s\({}^{-1}\), which will result in heterogeneous mixing results. An optimized mixing procedure based on a specific requirements profile was therefore adapted to achieve highly reproducible homogenous distributions, which is the prerequisite for successful bioprinting. The syringe pump and the selected mixing system are also easy to scale for different applications. The volume flow of the bioink through the nozzle during bioprinting determines the intensity and duration of shear stress for the carrier and the cells. Using an established printing process, we were able to vary the volume flow to predict the behavior of the carrier and the cells within the nozzle. The Reynolds number can predict the flow profile of fluids in a defined geometry [16].