# Biological and Technical Challenges Related to Cultured Meat Production
## 4 Technical Challenges in Cultured Meat Production
### Scaffold Fabrication
#### 4.1.1 Conventional Porous Scaffold Fabrication Technologies  
* **Solvent casting and porogen leaching (SCPL)**[68]: This process involves mixing a polymer solution dissolved in an organic solvent composed of insoluble particulates (porogen). The mixture is then cast into a mold or a membrane, and the solvent is evaporated. Finally, the structure is immersed in an aqueous solution to leach out particulates in the structure. Porosity, in terms of shape, size, and uniformity, depends on the particulate choice of particulates, typically salt particles. The main drawbacks are the lack of control of internal architecture and uniformity, reduced reproducibility, formation of a skin layer due to polymer thickening that can limit access to internal porous, limited thickness (2-3 mm) [69], weak mechanical properties, and possible cytotoxicity due to residual solvent and porogen [70].
* **Phase separation**[71]: The technique is used to produce a scaffold through the separation of a mixture into two phases: a polymer-rich one and a polymer-poor one. This is achieved under thermodynamically unstable conditions. For example, cooling the solution below the freezing point of the solver induces crystal nucleation inside the solution; after that, the solid material is sublimed, ensuring that the structure is composed of only the polymer-poor region with porosity, because the solvent and the polymer-rich phase are evacuated from the scaffold. This technique leads to highly interconnected porosity which can be used to reproduce channel-like structures by applying a directional temperature gradient. Nevertheless, control and optimization of process parameters (e.g., temperature, polymer concentration, surfactants use, crystal nucleation) are the main problems in managing pore size and distribution [72]. Moreover, the typical pore size achievable is often smaller than the typical dimension in tissue engineering applications (<200 \(\upmu\)m).
* **Gas foaming**[73]: This is a class of techniques for scaffold fabrication exploiting a blowing agent to generate gas inside the material which acts as a porogen agent. The main advantage is the absence of solvents or porogen materials, which can induce  
Figure 2: Conventional porous scaffold fabrication technologies process representation and example of scaffold, respectively **(a)** SCPL, **(b)** phase separation, **(c)** gas foaming, **(d)** sintering.  
cytotoxicity due to possible residuals. The Gas formation can be induced chemically or thermally or by pressure change. The main drawbacks of the technique are low control over pore size and interconnectivity, low reproducibility and structural uniformity, and difficulty in incorporating biological molecules in thermally induced processes [74,75].
* **Sintering**[76]: The technique is used to produce cohesive porous scaffolds through bonding of a polymeric phase and ceramic particles or fibers. The usual procedures involve a bed of randomly packed particles bonded through heating up to a temperature above the glass transition temperature of the base material, but lower than its melting point, creating a local fuse area only in the contact surfaces, leading to a porous microstructure. Alternative sintering modes are mild solvent treatment and pressure. Sintered scaffolds are characterized by lower porosity, small pore size with difficulty in precise control and distribution, and higher mechanical properties, and they are mainly used in dental and bone-repairing applications [77].