# 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
Moreover, those from animal-derived source are useful just for research activities, but they are inadequate for large-scale cultured meat production. Due to their source, synthetic polymers potentially can reach higher reproducibility and uniformity in mechanical and rheological behaviors, with high controllability of physical properties. In addition to these advantages, they have the worst biological behavior, lacking in moieties to interact with cells and create an appropriate environment. Thus, several combinations have been formulated to combine the properties of the two classes. Alternatively, functionalization processes are applied to synthetic polymers in order to enhance cell adhesion [86, 102]. As an example, [PERSON] et al. show the beneficial effect of alginate modification with the peptide motif RGD (arginine-glycin-aspartic acid), an integrin-binding ligand [103]. Integrins are transmembrane receptors of cells and turned out to be fundamental in tissue engineering because they activate signal transduction and regulate the cell cycle, including cell spreading, migration, guidance, proliferation, and apoptosis [103, 104]. The network of the hydrogel is formed via cross-linking (fixation or gelation) with hydrogel precursor polymers. This can be carried out before, during, or after the 3D printing and it is fundamental to preserving shape and structural integrity and avoiding collapse. The gelation mechanisms can be divided into two main categories: chemical and physical. Typically, physical cross-linking is a reversible process but associated with poor mechanical stability, while chemical reagents are able to increase the mechanical stability by creating covalent crosslinks [101]. Crosslinking can be stimulated by light (i.e., UV or visible), heat, or crosslinker bath (i.e., ionic crosslinking). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Natural Polymers** & **Synthetic Polymers** & **Combination of Natural and Synthetic Polymers** \\ \hline \multicolumn{3}{c}{Polyesters:} \\ Anionic polymers: & PEG-PLA-PEG, PEG-PLGA-PEG, PEG-PCL-PEG, & \\ HA, alginic acid, pectin, & PLA-PEG-PLA, PHB, & P(PEG-co-peptides), \\ carrageenan, chondro & P(PF-co-EG) \(\pm\) acrylates, & alginate-g-(PEO-PPO-PEO), \\ sulfate, dextran sulfate & P(PEG/PBO \(\mathrm{terephthalate}\)) & P(PLGA-co-serine), \\ \hline \multicolumn{3}{c}{Other polymers:} \\ \multicolumn{3}{c}{Cationic polymers:} & PEG-bis-(PLA-acrylate), PEG \(\pm\) CDs, & alginate-acrylate, \\ chitosan, polylysine & PEG-g-P(AAAm-co-Vamine), & P(HPMA-g-peptide), \\ \hline \multicolumn{3}{c}{Amphipathic polymers:} & PAAm, P(NIPAAAm-co-AAc), & P(HEMA/Matrigel®), \\ collagen (and gelatin), & P(NIPAAm-co-EMA), PVAC/PVA, PNVP, & HA-g-NIPAAm, GelMA \\ carboxymethyl chitin, fibrin & P(MMA-co-HEMA), P(AN-co-allyl sulfo- mate), & \\ \hline \multicolumn{3}{c}{Neutral polymers:} & P(biscarboxy-phenoxy-phosphozene), & \\ dextran, agarose, pullulan & P(GEMA-sulfate) & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Hydrophilic polymers used to synthesize hydrogel matrices, adapted from [102]. A combination of mechanisms or steps may be pursued to improve the process. For example, [PERSON] et al. proposed a bioink blend composed of alginate and gelatin methacroyl (GelMA) at low concentrations (<5% w/v) characterized by low viscosity, which positively impacts on cell viability during the extrusion process. The ink is crosslinked in two steps, CaCl2 during (using a coaxial nozzle) and after bioprinting, and then the construct is further stabilized by UV-crosslinking [105]. The formulation and preparation of a bioink and its bioprinting is complicated because of the presence of cells and their strict requirements for sterility and viability. Concerning printability, the most important physio-chemical parameters of a hydrogel include the rheological behavior, swelling properties, surface tension, gelation properties, and kinetics. These properties must be tuned, taking into consideration the bioprinting technique (e.g., extrusion or inkjet) and the type of cell to be used.