# Biological and Technical Challenges Related to Cultured Meat Production
## 2 Cultured Meat Production Process  
Cultured meat production follows a precise workflow, which is briefly summarized in Figure 1.  
Cell harvesting. The fundamental step in the production of cultured meat is cell procurement, for which three methods are used. (1) A cell or tissue biopsy is acquired from a living animal or recovered postmortem. The cells acquired in this way are called primary lines. (2) Pluripotent cells, such as embryonic stem cells or induced pluripotent stem cells, are used [14]. (3) Immortal cell lines. Although primary cell cultures make it possible to study the mechanisms of cultured meat production on short time scales, they undergo a finite number of divisions before reaching senescence. Immortal or continuous cell lines, on the other hand, do not undergo senescence and can therefore undergo infinite divisions, allowing more consistent cultured meat to be obtained without the need for continuous animal biopsies. However, there are currently no immortalized cell lines suitable for cultured meat available to researchers and developers. The most similar existing cell lines are the myoblasts of model species commonly used in research, such as mice, rats, hamsters, and Japanese quails. These cell lines are characterized by the absence of taste, nutrition, and texture of traditional meat. Therefore, the continuous lines used for the production of cultured meat should be developed from cell types and species that are familiar to consumers and that are tasty, nutritious, and safe for food [15]. Working with tissue biopsies involves an additional step. It is necessary to isolate individual muscle stem cells from the rest of the fibers, achieved using proteases including trypsin, pronase, dyspnea, and collagenase. After enzymatic dissociation it is crucial to remove fiber fragments, tissue debris, and connective tissues in order to ensure an efficient selection process [16]. The next step is to maintain the harvested cells in culture in order to first achieve cell proliferation and then cell differentiation and/or maturation with the ultimate goal of increasing biomass. ProliferationThe cells are initially allowed to proliferate small flasks in which they can grow in two-dimensional sheets, anchored to a surface. This method represents the standard for laboratory cell culturing methods, mainly devoted to research activities. However, for large-scale industrial cell production, suspension cultures in bioreactors are required to increase the efficiency of the process: these systems require generating a great number of cells with minimal energy and resource consumption (i.e., culture medium, consumables materials), time, and handling steps [17]. The current cells culturing method involves the use of specific growth media, which contain all those substances necessary to ensure cell proliferation such as glucose, inorganic salts, amino acids, vitamins, growth factors, antibiotics, and antifungals. Other substances can be added to confer organoleptic properties, such as proteins or pigments that give a meat-like color [18]. However, to date, viable alternatives to classical culture media are  
Figure 1: Cultured meat production process (Source Biorender, Toronto, ON, Canada). being sought, where the goal is to introduce into the medium edible by-products to ensure rapid cell growth. The same principle should be applied to fetal bovine serum (FBS), which is essential for cell proliferation, but not at all ethical and sustainable. Scaffolding. Tissue fabrication and maturation processes can be accomplished only if the cells are provided with the correct environment in which they can adhere, proliferate, and differentiate [19; 20]. Scaffolds are three-dimensional structures characterized by correct porosity, texture architecture, and mechanical and chemical properties suitable for specific cell type maturation.