# Biological and Technical Challenges Related to Cultured Meat Production
## 4 Technical Challenges in Cultured Meat Production
### Biofabrication and 3D Bioprinting
#### 4.3.2 Bioink Formulation
Thus, the characteristics of the bioink should meet the mechanical requirements from the process point of view and at the same time ensure cell survival after the bioprinting and within the constructs [101]. Most of the recent papers outline the necessity to find the best compromise between printability and specialization for the specific cell type or tissue under analysis [95]. According to [PERSON] et al., the optimal formulation of a bioink must take into account the overall process. The author refers to the extrusion bioprinting, which is to date the most common, and lists the major factors responsible for achieving an optimal bioink design capable of reaching high cell viability and evaluates their impact [106] (Figure 5). Viability is considered the key challenge because it impacts the subsequent cellular events, such as proliferation, differentiation, and tissue formation, even if many factors impacting cell stress and their severity are not yet completely understood. Additionally, cellular density, cellular projections, and network formation retained are of fundamental importance [106]. According to [PERSON] et al., the major factors in bioink design affecting cell viability and network formation are the following. Their effect is schematically displayed in Table 5.  
Figure 5: Schematic representation of major process parameters, grouped by bioink, printing, and post-printing design phases, and the biological behavior of cells on which parameters can act. Adapted from [106]. * There is a circular relationship between cells and bioink rheology: the firsts impact the rheology, and thus process parameters, and vice versa. For example, [PERSON] et al. found a twofold lowering in the viscosity of a GelMA-based bioink when prepared with 0.5 and 1.5 million cells/mL and a fourfold lowering when prepared with 2.5 million cells/mL [107]. Hence, there is the need to predict or test the rheological properties of ink with cells inside. * Mechanical stresses must be minimized, reducing printing pressure and increasing nozzle diameter because cells are mechanical sensing and suffer higher mechanical stresses. * The modulus of the gel-phase highly impacts cell viability, and probably also molecular weight and polydispersity. The mechanical properties of the material surrounding the cells is a crucial aspect that is poorly understood. * Post-printing crosslinking can affect cell viability as well. Frequently, bioprinted constructs are UV-crosslinked and the amount irradiation cells can tolerate is not clear, probably between 30 s and a few minutes. Moreover, the final degree of cross-linking can interfere with cell projections and network formation, an important mechanism to ensure tissue formation. This can become important when polymer concentration is increased to increase printability. According to the author, the concentration of the polymer must be between 5 and 10%, but it is obviously a polymer-dependent quantity. Another important matter of focus these days is the assessment techniques to standardize the evaluation of printability [108; 109]. [PERSON] et al. proposed a printability assessment method to evaluate the rheological properties of bioinks for extrusion processes. It is divided in two steps: (1) a qualitative screening of fiber formation and layer stacking capabilities, and (2) a rheological evaluation focused on flow initiation, shear-thinning, and post-printing recovery properties [108]. Through image analysis during bioprinting, other parameters related to the shape fidelity can be calculated, such as the printability index (Pr) based on the circularity calculation of a grid-like structure [109]. Research for novel solutions in bioink development for 3D cell culture and bioprinting purposes can push emerging technologies in tissue engineering and food bioprinting. The challenge is to accomplish the correct balance between a material's chemical, morphological, and structural features that have a positive effect on cellular cycle processes.