## 2 In Vitro Models to Study CAC
### Skeletal Muscle Wasting  
#### 2.1.1 C2C12 Cell Line  
The muscle precursor cell line C2C12, firstly isolated from mouse skeletal muscle myoblasts [32], is a common model to study muscle atrophy [33] but also cellular senescence [34], dystrophy [35], sarcopenia [36], diabetes mellitus [37], obesity [38], and hepatic steatosis [39]. In culture, the cells are mononucleated and spindle-shaped. During differentiation, upon serum withdrawal from the culture medium and low concentration of horse serum cells can fuse forming multinucleated structures turning into elongated fibre-shaped myotubes [40]. As such, the cell line can be induced to undergo stepwise differentiation since a muscle precursor (satellite cells) to a myoblast, followed by a myocyte, and ultimately a multinucleated myotube [41]. These features enable the characterisation of the mechanisms that regulate each step of muscle development and functioning [41; 42].  
Therefore, the C2C12 cell line provides an invaluable tool to study the molecular biology and mechanisms of myogenic regulation [43; 44], muscle differentiation [45], regeneration, and muscle atrophy [41].  
After differentiation, C2C12 myotubes remain stable until 10 days in culture [46]. However, the differentiation potential of satellite cells is limited [47]. Late passages of C2C12 myoblasts present significant impairment of myogenic differentiation potential, caused by the reduced expression of myogenic regulatory factors, being the lifespan of the cell line restricted to 50 passages before senescence (for cell differentiation even lower passages are required) [48].  
Muscle contraction and insulin can stimulate peripheral glucose uptake. Despite the cell line being insulin-responsive and therefore perfectly capable of serving as a model of study insulin- or contraction-stimulated glucose uptake, C2C12 cells are not as generally used because the expression levels of the glucose transporter 4 (GLUT-4) protein are lower when compared to other cell models, as L6 rat cell line [40; 46; 49]. Overall, this insulin-stimulated response is perfectly capable of serving a as model of study insulin resistance and other metabolic conditions [40].  
This cell model's particular features and the resemblance in myosin and glycogen content with the human muscle depict C2C12 as a remarkable model to study exercise and stress [40]. A unique characteristic of this cell line is its capability to develop a contractile apparatus of sarcomere units [49]. C2C12 cells contract when subjected to electrical stimulation, in fact, following an extended period of time after differentiation, cells can spontaneously contract [43]. The cells express high levels of myosin heavy chain genes MYH1 and MYH4, which are associated with glycolytic fibres (type II), indicating contractility properties [40]. The expression level of MHC and sarcomeric organisation are responsible for the capacity of force generation in C2C12 cells, which in part mirrors the most important attribute of skeletal muscles [50]. As a result, murine cells have been often used in pharmaceutical science and biomedical research proving to be useful to successfully identify drug candidates with high specificity resourcing to screening methods based on their phenotypic traits, such as myotube formation [51; 52].  
In opposition to the traditional 2-dimensional (2D) monolayer cultures, 3-dimensional (3D) has been gaining popularity. The most recent method to manufacture complex 3D architecture and alignment of muscle fibres uses bioprinting, which allows the combination of biomimetic materials to resemble the native tissue and generate more faithful models [53; 54; 55; 56]. The microenvironment created consists of bioink (the most usual biomaterial is hydrogel) and biological material (cells) [56]. This technique significantly improves cell differentiation and response to stimuli [53; 56].