# Biological and Technical Challenges Related to Cultured Meat Production
## 4 Technical Challenges in Cultured Meat Production
### Bioreactors  
In the context of tissue engineering, bioreactors are used to apply and control environmental parameters and conditions to constructs or cell cultures. The most important parameters in culture are temperature, pH, CO\({}_{2}\), and other biological, biochemical (such \begin{table}
\begin{tabular}{c c c} \hline \hline
**Bioink** & & **Cells** \\ \hline Printing pressure \(\uparrow\) & \(\downarrow\) Viability & \\ Nozzle diameter \(\downarrow\) & \(\downarrow\) Viability & \\ Printing time \(\uparrow\) & \(\downarrow\) Viability & \\ Degree of crosslinking \(\downarrow\) & \(\uparrow\) Density in bioink & \\ Viscosity \(\downarrow\) & \(\uparrow\) Density in bioink & \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Relationship between bioink material properties and cellular viability and behavior, adapted from [106]. as oxygen, nutrients transfer, or waste removal), and physical (mechanical stimuli) conditions [25]. The specific requirements and the architecture of a bioreactor depend on the cell type or tissue culture. Thus, they must be designed and manufactured for tissue-specific purposes [25]. * **Static culture systems:** They are the simplest and provide the required nutrient in a static fluid environment. Thus, the media must be changed often, and it perfuses by passive fluid diffusion [25]. These systems can be easily coupled with load-bearing mechanisms, for example, to provide a compressive load to the engineered tissues [110; 111]. * **Spinner flasks:** Spinner-flask-based systems are used to apply fluid-induced shear stresses to constructs submerged within a re-circulating and nutrient-rich medium solution [25]. Although this system provides a better environment to construct with respect to the static culture, spinner flasks may not be optimal due to turbulent flow and the related higher shear stress generated [112]. * **Perfusion systems:** The poor diffusion condition of static culture can be improved by perfusion bioreactors, especially in the internal parts of porous scaffolds [113]. These systems are characterized by a culture encasing bioreactor, vessels for the medium (nutrient-rich and oxygenated), and a pump generating the flow [27]. Moreover, perfusion systems allow for automatic media circulation, waste removal, and provide shear stress due to the flow, which is beneficial in specific tissue cultures such as for dermis and cartilaginous tissues [23; 112]. * **Rotating wall vessel:** An alternative approach for reducing diffusional limitations of nutrients and waste with limited shear stress is the use of rotating wall vessel bioreactors [24]. Although shear stress is important for cell maturation, an excessive force will lead to damages or to the formation of undesired capsules surrounding the tissue [112]. This method uses a dynamic laminar flow induced by the rotating fluid inside the bioreactor, and it has been proved to be effective for cell cultures, especially chondrocytes and cardiac cells [24]. The main drawback is the non-uniform tissue growth, due to the force field. Moreover, the centrifugal force can cause collision between scaffolds and the walls of the bioreactor [112]. * **Pulsatile flow:** For cardiovascular cell cultures that require a pulsatile stimulation, bioreactors exploiting pulsatile flow are used to mimic in vivo conditions. Typically, vascular cells are cultured into tubular scaffolds [112]. With technological and design tools, increasingly powerful bioreactors designed for specific application can be fabricated, with high specialization and effectiveness [24]. The main assumption in bioreactor design is that the same factors and stimuli that determine phenotypic nature and functionalities to tissue and cells in vivo also determine the progression of cells in vitro [114]. Several tissues are cultured, providing mechanical, electrical, chemical, and mixed systems mimicking the environment of in vivo conditions.