# Biological and Technical Challenges Related to Cultured Meat Production
## 4 Technical Challenges in Cultured Meat Production
### Biofabrication and 3D Bioprinting
#### 4.3.1 3D Bioprinting Strategies  
There are different bioprinting strategies, each with its own pros and cons (provided in Table 3. Figure 4 provides a graphical representation of the most common methods or those of higher research). According to [PERSON] et al., no one method could be excursively used to achieve the goal of biofabrication of complex tissue, the current trend being research in the development of hybrid methods [94].  
Extrusion bioprinting: The most common 3 DBP method is the extrusion-based one, mainly because it is versatile and affordable, and several entry-level bioprinters are available on the market [95]. [PERSON] et al., for example, successfully used it to bioprint meat-like constructs [96]. Extrusion bioprinting usually relies on a dispensing system, such as a syringe with an appropriate nozzle or needle, installed on a print-head used to precisely deposit bioinks on a print bed. The dispensing system can be pressure, piston, or screw driven: the first allows better cell viability, but with low control on material flow rate and shape fidelity [91; 93]. The main advantages of the extrusion-based method are scalability, printability of a wide range of materials of high viscosity, and high cell concentration [94]. Nevertheless, the resolution is the lowest if compared with the other methods and it is related to the nozzle diameter, in that reduction is limited by the consequent cell viability drop [94]. Moreover, the post-printing cell viability depends on the bioink viscosity and cell concentration [97]. Further drawbacks are nozzle-clogging and the limitation due to material rheology: only bioinks with shear-thinning property can be used [94].  
Inkjet bioprinting: A liquid droplet dispensing system, based on temperature or piezoelectric driving technology is used. Due to the nature of the process, inkjet bioprinting is characterized by high speed and resolution, and associated to lower costs. However, it can be used only with low viscosity materials, and it is strongly limited by the nozzle-clogging mechanism. Moreover, to enhance and facilitate droplet formation, low cell concentration is allowed [91; 94].  
Stereolithography and two-photon polymerization-based bioprinting. Stereolithography is based on the polymerization of light-sensitive polymers; UV or visible lights can be used to photo-cure the material in a layer-by-layer manner [23]. It is a nozzle-free method, and so does not face the problem of nozzle clogging, which represents a great limitation in the previously explained methods. Moreover, it allows obtaining very high resolution and high printing velocity. However, only light-sensitive polymers can be used, and the UV lights, as well as UV-activated photo-initiators, can damage cells and consistently reduce post-printing viability [23; 94; 97; 98]. On the basis of this technology, digital  
Figure 4: Different 3D bioprinting techniques, adapted from [PERSON] et al. [95].  
light processing (DLP) has been developed to photo-cure polymers. The method uses a projector emitting visible light to overcome problems due to UV irradiation [98]. With the advent of two-photon polymerization-based stereolithography nanoscale resolution is achieved. The highest spatial resolution is among biofabrication techniques but with costly systems [94,99].  
Laser-assisted bioprintingThe method is based on the laser-induced transfer principle [100]: it uses a pulsed laser beam that acts, through a focusing system, on a ribbon composed by a donor transport support material covered by a laser-energy-absorbing and a biological material layer. The focused laser pulses on the absorbing layer generates high-pressure bubbles that propel the material against a collector substrate facing the ribbon. LAB is a nozzle-free technology capable of high speed and high cell density bioprinting. However, it is less common than inkjet and extrusion, mainly due to the costs of the technology and the difficulty to produce ribbons. Moreover, it is difficult to scale-up for high-volume production [91].