# Biological and Technical Challenges Related to Cultured Meat Production
## 2 Cultured Meat Production Process
Moreover, considering the food engineering purposes, scaffolds must be biodegradable or edible or both, and their structure is also involved in the final organoleptic properties of the final product [20]. The traditional approach, based on tissue engineering, involves scaffold fabrication and subsequent cell seeding in a 3D culture. Scaffolds can be obtained through tissue/organ decellularization or can be fabricated. There are several different techniques for scaffold production. The most used are solvent casting and porogen leaching (SCPL), phase separation, gas foaming, sintering, electrospinning, self-assembly, and 3D printing (3 DP) techniques (e.g., powder-bed 3D printing, fused deposition modelling, and stereolithography) [18]. Biofabrication. Recently, another approach is gaining importance: the biofabrication [19]. This process enables the production of cell-laden constructs using materials containing mixed cells and biological molecules. The main advantage is the possibility to fabricate thicker structures and to control the spatial arrangement of cells, even of multiple types, which are the major limitations of the conventional scaffolding methods [21]. 3D bioprinting (3 DBF) is biofabrication technology based on additive manufacturing (AM) allows precise cell positioning, density deposition, and precise control of the structure, and the ratio amongst different populations in case of multicellular constructs. Cell-materials mixtures used in the process are called bioinks [22]. 3 DBP represent a possible game-changer in the field of the in vitro meat manufacturing, assuring high scalability, complexity free process, minimal energy expenditure, and lower N20 emissions [22]. The major bioprinting methods are extrusion-based, inkjet, stereolithography, and laser- and light-based methods [23]. Tissue differentiation and bioreactors: Whatever the construct fabrication, cells inside the 3D scaffold must be placed in a bioreactor for maturation. Mechanical, chemical, and eventually electrical stimuli are required to complete this stage through dedicated culturing systems [24; 25]. bioreactors are closed, automated systems in which cells can proliferate, differentiate, and maturate within the construct forming the tissue. bioreactors provide precise control over relevant variables such as temperature, oxygen concentrations, pH, and cell density [18]. Several types of bioreactors are used, the most common being static culture, spinner flask, and perfusion bioreactors (e.g., stirred tank or rotating wall) [25]. Inside the bioreactors, cells go through proliferation and differentiation. The last process is triggered by changing the scaffold and culture media and through the addition of elements such as proteins, amino acids, and minerals [24]. The lack of cell adhesion and the creation of structures suitable for cell growth are the critical points of this complex procedure. From laboratory to industrial scale-up: The number of cells required to produce 1 kg of protein from muscle cells is in the range of 2.9 \(\times\) 10\({}^{11}\) to 8 \(\times\) 10\({}^{12}\)[25]. To achieve these huge numbers, it is necessary for a cell proliferation stage to occur in large-scale bioreactors [26], such that cells can grow and replicate in the order of 5000 L [27]. According to [PERSON] et al., bioreactors will be essential in the industrial production process of cultured meat [26]. Tissue development would require at least two different stages: (i) the cell proliferation phase to provide a sufficient number of cells for construct fabrication (e.g., using a stirred tank bioreactor), and (ii) the tissue differentiation to a stage (e.g., a porous scaffold placed inside a perfusion bioreactor) which will lead to the final cut of meat providing the proper chemical, mechanical, electrical stimuli [26; 27]. Existing products and processes in the chemical and biomedical industry do not meet requirements for large-scale cell and tissue cultures, mainly in terms of cost and sustainability.