# 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  
Bioink formulation is one of the most important branches in bioprinting research, with about 25% of the overall publications in the field [95]. These materials are typically based on cytocompatible hydrogels, and they must have several key properties, both from a mechanical (e.g., viscosity, printability, and stiffness) and biological (e.g., cytocompatibility and cell-material interaction) point of view [101]. Hydrogels are a class of hydrophilic polymers that can be cross-linked, forming a 3D network capable of absorbing and holding a large quantity of water (even upward of 100 times their dry weight). This property allows the polymers to reach the hydration levels found in most tissues. Moreover, the porous network allows for a great degree of \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Properties** & **Extrusion** & **Inkjet** & **Laser-Assisted** & **Stereolithography** & **Two-Photon** \\ \hline Speed & Slow & Fast & Medium & Fast & Very fast \\ Cost & Moderate & Low & High & Low & Very high \\ Cell viability & 85–95\% & 80–95\% & \textless{}85\% & 25–90\% & \textgreater{}80\% \\ Cell density & High & Low & Medium & Medium & Medium \\ Scalability & High & High & Low & Medium-high & - \\ Resolution & 100–500 \(\upmu\)m & 100–500 \(\upmu\)m & 20–100 \(\upmu\)m & 20–100 \(\upmu\)m & 0.1–10 \(\upmu\)m \\ Bioink viscosity & 6–30 \(\times\) 10\({}^{7}\) mPa-s & \textless{}10 mPa-s & 1–300 mPa-s & No limitation & No limitation \\ \hline \multirow{4}{*}{**Advantages**} & Is Simple, is capable of printing & Has the ability to print low viscosity & Has a high resolution, is nozzle-free, and biomaterials, fast can deposit biomaterials in solid or liquid phase & Is nozzle-free, has high complexity, and has a high height resolution, and has high cell viability \\  & & & & & \\ \cline{1-1}  & biomaterials, high & & & & \\ \cline{1-1}  & cell densities & & & & \\ \cline{1-1}  & & & & & \\ \hline \multirow{4}{*}{**Drawbacks**} & \multirow{4}{*}{Only for viscous liquids, resolution} & \multirow{4}{*}{Limited to low viscous fluids, resolution, cell density} & High cost, thermal damage due to nanosecond/femtosecond & Lack of printing during & \\ \cline{1-1}  & & & & & \\ \cline{1-1}  & & & & & \\ \cline{1-1}  & & & & & \\ \cline{1-1}  & Tissue models for cell research, drug & & & & \\ \cline{1-1}  & testing and regenerative & & & & \\ \cline{1-1}  & medicine, & & & & \\ \cline{1-1}  & meat-analogue & & & & \\ \cline{1-1}  & constructs [101] & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Comparison of major bioprinting methods considering several properties, advantages and drawbacks, and main applications. Table adapted from [23,94,95]. diffusion of nutrients and wastes out of the material. These hydrogels can be chemically stable, or they may degrade and dissolve [102]. Hence, they offer the opportunity to recreate engineered microenvironments suitable for cells, mimicking the natural ECM properties and native cellular niche important for tissue regeneration [86]. Hydrogels can be formed using natural biopolymers, synthetic biopolymers, or combinations of the two. The most common have been formed using proteins (such as collagen, elastin, and fibrin), polysaccharides (alginate, agarose, chitin/chitosan, etc. ), and synthetic polymers (such as polyethylene glycol, polyvinyl alcohol, polyacrylamide, and polylactic acid) [86]. A more complete framework of materials used for hydrogel fabrication is provided in Table 4.  
The main advantage of natural polymers is the higher cytocompatibility and presence of recognizable biological moieties (typically only from animal sources) that can act as signals and modulate cellular responses such as attachment, proliferation, and differentiation. However, they are affected by batch-to-batch variability, they often require stringent extraction and purification protocols, and they face procurement problems related to sustainability and availability [27, 86].