**3. 3D bioprinting**

The studies previously reported hypothesize that iPSC-derived neurons from PD patients can be very useful in the research of PD pathophysiology and to find new therapeutic targets for innovative drugs. Moreover, the possibility to differentiate iPSCs into nonneuronal cells, such as microglial and intestinal cells, will help to unravel the role of immunity response and the

Amyotrophic lateral sclerosis (ALS) is the most prevalent motor neuron disease and is characterized by the progressive loss of upper and lower motor neurons (MNs), leading to muscle atrophy, paralysis, and finally death usually after 2–5 years from the first diagnosis [21]. Also for ALS the cause is still unknown, but in about 5–10% of cases, several genes are found mutated, among which are SOD1, TARDPB, and FUS [46]. Moreover, in 2013 the GGGGCC-hexanucleotide repeat expansion in C9orf72 locus was found in many familial and sporadic cases of ALS [47].

MNs derived from iPSCs are the most common neural cell type used in ALS involving the use of stem cell differentiation. For example, an increase in oxidative stress and in DNA damage was found in iPSC-derived C9ORF72 MNs, confirming that the reduction of oxidative stress could help to delay patients' death [48]. Moreover, MNs derived from iPSCs with induced mutation in FUS (P525L) were used to investigate the transcriptome and microRNA, finding an alteration of both in pathways with implications for ALS pathogenesis [49]. The role of astrocytes was also investigated in both sporadic and VCP mutant patients, suggesting that in ALS patients, the co-culture between MNs and astrocytes causes alterations in both cell types [50, 51]. Moreover, the genetic correction allowed to study pathways implicated in ALS, like Bhinge and colleagues that found that the activation of AP1 drives neurodegeneration in genetic corrected SOD1 mutant MNs [52]. Small-molecule compounds that regulate IGF-2 expression were found to increase MN resilience, screening the compounds in iPSC-derived MNs [53]. Another example is given by Egawa and colleagues that firstly generated and characterized MNs from iPSCs of patients carrying TDP-43 mutations. They found some pathological hallmark, such as short neurites and abnormal-insoluble TDP-43. Then, they tested trichostatin A, spliceostatin A, garcinol, and anacardic acid and found that the last one, an inhibitor of histone deacetylase, rescued the pathogenic abnormalities like TDP-43 mRNA [54]. All these researches suggest the increasing importance of iPSCs as model for drug screening. These works suggest that MNs derived from iPSCs of mutated and sporadic ALS patients can be a helpful tool to study both disease mechanisms and drug screening. Several investigations can be done in iPSC-derived MN cellular models, e.g., oxidative stress, DNA damage, and transcriptome. The co-culture between astrocytes and MNs can give information about how they interact with each other and whether this interaction could have a pathophysiologic role in ALS.

Huntington's disease (HD) is characterized by loss of neurons mainly in the caudate nucleus, the putamen, and the cerebral cortex with affection in a later stage of other areas, e.g., the hippocampus and hypothalamus [55]. Despite other neurodegenerative diseases, the cause of HD is well known; in fact it is an autosomal dominant genetic disorder caused by an

gastrointestinal disorder that affect PD patients.

*2.2.3. iPSCs in amyotrophic lateral sclerosis*

30 Recent Advances in Neurodegeneration

*2.2.4. iPSCs in Huntington's disease*

The term bioprinting was used for the first time in 2009 by Mironov with the release of the first issue of the journal *Biofabrication*, a magazine that took its name from the eponymous term biofabrication. While the term biofabrication is intended to indicate natural processes such as biomineralization, the term bioprinting is defined by Guillemot in 2010 as [63, 64].

The use of computer-aided transfer processes for patterning and assembling living and nonliving materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies.

3D bioprinting is an emerging technology, used for the manufacture and the generation of artificial tissues and organs [65], adding new approaches to tissue engineering (TE) and regenerative medicine, such as the manufacture of scaffold to support cells, as well as in situ deposition of cell suspensions [63]. Bioprinting technology has allowed to overcome several limits, such as the control of in vitro 3D biological structures and cellular distribution [66]. Bioprinting, through the use of hardware and software, has been used in particular for the design of three-dimensional structures, allowing the creation of "organoids" for biological and pharmacological studies, and to repair and replace human tissues.

First, bioplotters that appeared on the market were intended for a purely industrial use, since they had a high price. The costs limited their development and related researches. With the advent of technology and knowledge of bioprinting, we have witnessed the birth of multiple models of bioplotters, each with characteristics that reflect the needs of the individual creator [12, 13], e.g., increasing the number of nozzles for simultaneous extrusion of several materials

From Neuronal Differentiation of iPSCs to 3D Neural Organoids: Modeling…

http://dx.doi.org/10.5772/intechopen.80055

33

Inkjet bioplotter was the first technique used in the 1980s in offices and then for domestic use. It was readapted around the year 2000 to be used as a biological printer, replacing the normal ink with a bioink, containing cells and biocompatible materials [69]. Droplets of the biomaterial are extruded from very small orifices, deposited on a substrate, maintaining good cell dispersion, viability, and functionality, even with different cellular types [70]. The stream can be continuous, command-driven (drop on demand), and electrodynamic. Both piezoelectric and thermal inkjet printers have been readapted for biological printing, offering many advantages in terms of simplicity, versatility, and material control, both in terms of quantity

Laser-based direct writing was introduced in 1999 and is one of the most used laser-based bioprinting techniques [72]. The technique involves a layer with biological material (donor layer) and a layer that collect cells (acceptor layer) that are pushed by the laser through the first layer. The pulsation of the laser creates bubbles which in turn generate a shockwave, forcing the cells to pass from the donor layer to the acceptor layer. This technique allows to have a good resolution but has some disadvantages, such as irreversible damage to the cells

The advent of TE has allowed extrusion technology to be thoroughly studied and applied to the field of bioprinting, for the generation of living tissues. Extrusion technique includes a combination of different delivery systems combined with an automatic robotic system for extrusion and 3D printing [73]. Deposition of the material takes place through extrusion to form a cylindrical filament made of a biocompatible gelatinous material, in which the cells are encapsulated, maintaining the desired three-dimensional structure [74]. This type of extrusion can be pneumatic or mechanical. Mechanical extrusion involves the use of a piston, which guides the deposit of the material allowing a good flow control through the nozzle, or a screw, which allows the extrusion of more viscous substances, but which can cause leakage of pressure, causing damage to cells. This technique also has some limits, particularly during the development of the biomaterial which must have the right viscosity to be extruded and the ability to be easily homogenized with cell suspension and to maintain three-dimensional structure [65].

One of the fundamental elements that characterize the bioprinting process is the development of biomaterial, which must have specific characteristics: biocompatibility, printability, and

[68] and changing the type of technology that controls the nozzle.

*3.1.1. Inkjet bioplotter*

*3.1.2. Laser technology*

*3.1.3. Extrusion-based bioprinter*

**3.2. Hydrogel and bioink**

and speed in the printing process [71].

because of the heat and light generated by the laser [65].

#### **3.1. Bioprinting and bioplotter techniques**

Bioprinting can be distinguished on the basis of the bioink printing technique, allowing to change the printing processes according to the needs that the different cell types require: inkjet, laser, and extrusion (**Figure 2**) [65]. In addition to the specific printer characteristic, each bioplotter must have common functionalities. The most important is the presence of a robotic displacement system that can move along the three Cartesian axes, x, y, and, for the 3D characteristics, z. Usually, the bioink is extruded from a dispenser, but it is possible to have more dispensers, permitting to have different bioinks in the same scaffold. One of the most recent techniques allows a coaxial extrusion, with a bioink that is surrounded by a second bioink. The sterility of the printout is usually guaranteed by the presence of sterile chamber with laminar flow system. If the bioplotter is quite small, this problem could be overcome by simply placing the bioprinter under a classic cellular hood. Finally, the presence of a dedicated software for the supply of bioink and for the high-resolution control of the design of the construct to be printed is essential [65].

**Figure 2.** On the top the most used printing processes are extrusion-based (performed by piston, pneumatic method, or screw), inkjet-based operated by a piezoelectric actuator, and laser-assisted (composed of an energy-absorbing slide and a donor slide that collect the discharged bioink droplets). On the bottom the complexity of the 3D bioprinted construct, composed of a natural biocompatible material (e.G., alginate), cells, functional peptides, and other biocompatible materials. (the image was taken from Axpe and Oyen [67]).

First, bioplotters that appeared on the market were intended for a purely industrial use, since they had a high price. The costs limited their development and related researches. With the advent of technology and knowledge of bioprinting, we have witnessed the birth of multiple models of bioplotters, each with characteristics that reflect the needs of the individual creator [12, 13], e.g., increasing the number of nozzles for simultaneous extrusion of several materials [68] and changing the type of technology that controls the nozzle.

## *3.1.1. Inkjet bioplotter*

Inkjet bioplotter was the first technique used in the 1980s in offices and then for domestic use. It was readapted around the year 2000 to be used as a biological printer, replacing the normal ink with a bioink, containing cells and biocompatible materials [69]. Droplets of the biomaterial are extruded from very small orifices, deposited on a substrate, maintaining good cell dispersion, viability, and functionality, even with different cellular types [70]. The stream can be continuous, command-driven (drop on demand), and electrodynamic. Both piezoelectric and thermal inkjet printers have been readapted for biological printing, offering many advantages in terms of simplicity, versatility, and material control, both in terms of quantity and speed in the printing process [71].

#### *3.1.2. Laser technology*

Laser-based direct writing was introduced in 1999 and is one of the most used laser-based bioprinting techniques [72]. The technique involves a layer with biological material (donor layer) and a layer that collect cells (acceptor layer) that are pushed by the laser through the first layer. The pulsation of the laser creates bubbles which in turn generate a shockwave, forcing the cells to pass from the donor layer to the acceptor layer. This technique allows to have a good resolution but has some disadvantages, such as irreversible damage to the cells because of the heat and light generated by the laser [65].

#### *3.1.3. Extrusion-based bioprinter*

The advent of TE has allowed extrusion technology to be thoroughly studied and applied to the field of bioprinting, for the generation of living tissues. Extrusion technique includes a combination of different delivery systems combined with an automatic robotic system for extrusion and 3D printing [73]. Deposition of the material takes place through extrusion to form a cylindrical filament made of a biocompatible gelatinous material, in which the cells are encapsulated, maintaining the desired three-dimensional structure [74]. This type of extrusion can be pneumatic or mechanical. Mechanical extrusion involves the use of a piston, which guides the deposit of the material allowing a good flow control through the nozzle, or a screw, which allows the extrusion of more viscous substances, but which can cause leakage of pressure, causing damage to cells. This technique also has some limits, particularly during the development of the biomaterial which must have the right viscosity to be extruded and the ability to be easily homogenized with cell suspension and to maintain three-dimensional structure [65].

### **3.2. Hydrogel and bioink**

**Figure 2.** On the top the most used printing processes are extrusion-based (performed by piston, pneumatic method, or screw), inkjet-based operated by a piezoelectric actuator, and laser-assisted (composed of an energy-absorbing slide and a donor slide that collect the discharged bioink droplets). On the bottom the complexity of the 3D bioprinted construct, composed of a natural biocompatible material (e.G., alginate), cells, functional peptides, and other biocompatible

Bioprinting, through the use of hardware and software, has been used in particular for the design of three-dimensional structures, allowing the creation of "organoids" for biological

Bioprinting can be distinguished on the basis of the bioink printing technique, allowing to change the printing processes according to the needs that the different cell types require: inkjet, laser, and extrusion (**Figure 2**) [65]. In addition to the specific printer characteristic, each bioplotter must have common functionalities. The most important is the presence of a robotic displacement system that can move along the three Cartesian axes, x, y, and, for the 3D characteristics, z. Usually, the bioink is extruded from a dispenser, but it is possible to have more dispensers, permitting to have different bioinks in the same scaffold. One of the most recent techniques allows a coaxial extrusion, with a bioink that is surrounded by a second bioink. The sterility of the printout is usually guaranteed by the presence of sterile chamber with laminar flow system. If the bioplotter is quite small, this problem could be overcome by simply placing the bioprinter under a classic cellular hood. Finally, the presence of a dedicated software for the supply of bioink and for the high-resolution control of the design of the construct to be printed

and pharmacological studies, and to repair and replace human tissues.

**3.1. Bioprinting and bioplotter techniques**

32 Recent Advances in Neurodegeneration

is essential [65].

materials. (the image was taken from Axpe and Oyen [67]).

One of the fundamental elements that characterize the bioprinting process is the development of biomaterial, which must have specific characteristics: biocompatibility, printability, and ability to maintain a three-dimensional structure once printed and maintained in culture [65]. The main feature of the hydrogel, biocompatible material used as a three-dimensional support for cell growth, is the ability to be extremely hydrophilic, making it an excellent candidate in terms of biocompatibility for its use in bioprinting. It was initially used in TE because it was able to simulate the extracellular matrix, guarantying cell growth and communication [75]. Biomaterials are divided, According to their derivation, biomaterials are divided in natural or synthetic compunds. There are naturally derived polymers such as sodium alginate, gelatin, collagen, chitosan, fibrin, and GelMA [76–78] and synthetic polymers such as Pluronic®, polyethylene glycol (PEG), and polyurethanes [79, 80]. Over time, it has been seen how the natural compounds are more performing when placed in contact with the cells than the synthetic ones. Several cell types associated with different biomaterials to compose the bioink have already been used in several research areas, where cellular viability and motility have been demonstrated, as well as a spatial organization similar to in vivo tissue [81]. To create a new biomaterial, we must consider different physical, mechanical, and biological characteristics that are close to the tissue we want to recreate. Thus, researchers tend to create a combination of biomaterials for each cell type, and with well-defined printability specifications, so as to make the process as standardized and reproducible as possible, despite being a very open field and full of new developments. New-generation bioinks are now able to maintain each of these characteristics, thus improving the success in terms of bioprinting. All this is possible if particular attention is paid to the following chemical, physical, and biological properties: rheological studies (viscosity, thinning, viscoelasticity), biofunctional analysis, biodegradation, and polymerization (cytocompatibility, cell adhesion, migration, proliferation, and differentiation) [82]. One of the most important features that has different biomaterials is the ability to cross-link once the bioink has been printed, reticulating the bioink in which cells are encapsulated, without affecting the viability, the differentiation, and the capacity of migration [83]. The ability to polymerize depends on the material used; for example, collagen needs chemical cross-link, through covalent bonds that bind free amines or carboxylic groups of collagen that is able to reticulate, also through a biological process, and through the interaction with transglutaminase. Other compounds, such as sodium alginate, use an ionic cross-link: divalent cations such as Ca2+ bind to two sodium alginate residues, cross-linking the structure. UV radiation is a very promising cross-link technique given its reaction speed but with many questions regarding the possible damage induced to the cells included in the material. Other materials, such as gelatin and agarose, are heat-sensitive, so they are used during the printing process at the melting temperature and then stiffened with cooling [84–87]. One of the most common types of bioink used in bioprinting techniques is the so-called cell-laden hydrogel, which includes natural hydrogels such as agarose, sodium alginate, chitosan, collagen, gelatin, fibrin, and hyaluronic acid and synthetic hydrogels such as Pluronic® and PEG. Hydrogels can be used with the most common bioplotter that mount different printing techniques, allowing the creation of bioinks that combine the advantages of the natural material with the advantages of synthetic materials [88]. Recent findings have shown also the possibility to transfect cells with target DNA or plasmid, directly during the bioprinting process [80]. A new promising method to develop bioinks is the base on decellularized extracellular matrix. This kind of bioink consists in eliminating cells from a tissue of interest, keeping intact

the extracellular matrix that is then pulverized and subsequently used as bioink once dissolved in a saline buffer. Finally, the cell suspension bioinks, characterized by a print that does not have a support material, like a scaffold, are also very common. It uses aggregates of cells in culture medium, placed in mono- or multicell spheres [73]. This technique is based on the liquidity of the tissue and its fusion, such as to allow cells to assemble, merge, and create cell-to-cell interconnections [89, 90]. Organovo was the first medical research company that used this technique to create functional human tissues. They developed a liver model using

From Neuronal Differentiation of iPSCs to 3D Neural Organoids: Modeling…

http://dx.doi.org/10.5772/intechopen.80055

35

In the last decade, the possibility of replacing dead cells in degenerative processes affecting the central nervous system opened the way for a more intense and accurate study of stem cells and their possibility of replacing damaged tissue [92]. It was also thought to exploit the ability of stem cells to secrete cytokines and growth factors, offering benefits such as anti-inflammatory effects, protection of neural cells, and endogenous recovery systems. Transplanting these cells into damaged sites presents various problems such as low cell survival and limited engraftment [93]. To minimize these problems, it was decided to use three-dimensional scaffold printing that mimics the complexity both from the biological and functional points of view of the

The manufacture of three-dimensional prefabricated scaffolds has already given positive results in the treatment and repair of spinal and nerve damage but with a great limitation in terms of control of the external shape of the scaffold and of its internal architecture [95, 96]. These problems have been overcome with the 3D bioprinting, which leaves the operator complete freedom regarding the shape, the material, and its internal architecture. The recent developments in the field of 3D bioprinting are mostly aimed to the field of regenerative medicine, to respond to the growing demand for tissues and organs for transplants, arriving only later for this technology to be applied to basic scientific research. Until now only few studies have focused on using 3D printing applied to the creation of neural tissue compared to other widely studied tissues such as the skin, bones, heart tissue, and cartilaginous structures [97]. The few studies published so far, in which they use nerve cells in 3D printing processes [98, 99], show a poor characterization of bioinks to be used for nerve cells, due to the delicacy of the tissue to be recreated and of the characteristics necessary for the optimal growth of the nervous tissue [94]. Recently, researchers also think that the nervous tissue printed in 3D may be used for the neural regeneration, a huge possibility in the field of neurodegeneration to replace degenerated neural tissue [78, 80].

The creation of nerve tissue by bioprinting is also used for pharmacological studies, for toxicological screening, and for basic research. It is necessary to underline how this field is still in its infancy and how it is necessary to validate this model for the applications described up to now, to be sure that the model completely recapitulates the pathophysiology that we want to

investigate with this tool [94] in particular with regard to neurodegenerative diseases.

parenchyma cells and an extrusion printer of their own creation [91].

**4. 3D bioprinting and neurodegeneration**

tissue to be replaced [94].

the extracellular matrix that is then pulverized and subsequently used as bioink once dissolved in a saline buffer. Finally, the cell suspension bioinks, characterized by a print that does not have a support material, like a scaffold, are also very common. It uses aggregates of cells in culture medium, placed in mono- or multicell spheres [73]. This technique is based on the liquidity of the tissue and its fusion, such as to allow cells to assemble, merge, and create cell-to-cell interconnections [89, 90]. Organovo was the first medical research company that used this technique to create functional human tissues. They developed a liver model using parenchyma cells and an extrusion printer of their own creation [91].
