*3.1. Alginate*

Alginate, the salt of alginic acid, is an anionic polysaccharide derived from brown seaweed algae. It consists of β-D-mannuronic acid (M block) and α-L-guluronic acid (G block) monomers in its molecule. Alginate itself can dissolve in water and be crosslinked by divalent cations, such as calcium (Ca<sup>2</sup>+), barium (Ba<sup>2</sup>+), and strontium (Sr<sup>2</sup>+) ions, due to ion exchange reactions. This characteristic is particularly attractive in many biomedical fields, such as nanoparticle preparation, drug delivery, wound healing, tissue engineering, and regenerative medicine [75–78]. An obvious characteristic of alginate solution is that its physical sol–gel transition point is below 0 ◦C. Under ambient temperature, it is hard for pure alginate solution to be printed in layers without chemical crosslinking. The in vivo biocompatibility of alginate is not as good as those of animal- or human-derived natural polymers, such as gelatin and fibrinogen [41].

Generally, the physiochemical properties of alginate hydrogels depend on the ratio of M/G blocks. The higher the M/G ratio, the higher the activity of the polymers. The first pertinent alginate 3D bioprinting technology was reported in 2005, in which alginate was used as an additive in gelatin-based cell-laden bioinks (Figure 6) [25–33]. The blending of alginate with gelatin molecules can improve the printing resolution and increase the shape fidelity. Only a certain range of the alginate/gelatin concentrations can be printed in layers. Optimal concentration of alginate in gelatin-based bioinks varies from 0.5% (w/v) to 3% (w/v) depending on the polymer resources. After 3D printing, calcium ion crosslinks (i.e., ion bonds) can significantly improve the structural stability to a certain degree. The 3D

printed constructs can be crosslinked through various liquid exposures, such as spraying, soaking, and filtering of calcium solutions [79–81]. This exposure leads to the exchange of sodium ions in the alginate molecules with Ca2+ ions whereby the divalent Ca2+ ions form chemical crosslinks or chelates between two carboxyl groups in the same or different polymer chains.

**Figure 6.** 3D bioprinting of chondrocytes, cardiomyocytes, hepatocytes, and adipose-derived stem cells (ASCs) into living tissues/organs using a pioneering 3D bioprinter made in Prof. Wang's laboratory at Tsinghua University: (**a**) the pioneering 3D bioprinter; (**b**) schematic description of a cell-laden gelatin-based hydrogel being printed into an one-layer grid lattice using the 3D bioprinter; (**c**) schematic description of the cell-laden gelatin-based hydrogel being printed into large-scale 3D construct using the 3D bioprinter; (**d**) 3D printing process of a chondrocyte-laden gelatin-based construct; (**e**) a grid 3D construct made from a cardiomyocyte-laden gelatin-based hydrogel; (**f**) hepatocytes encapsulated in a gelatin-based hydrogel after 3D printing; (**g**) hepatocytes in a gelatin-based hydrogel after 3D printing; (**h**) a gelatin-based hydrogel after 3D printing; (**i**) hepatocytes in a 3D printed construct after a certain period of in vitro culture; (**j**) a magnified photo of (i); (**k**) the shape of the hepatocyte-laden grid construct maintained well after certain period of in vitro culture; (**l**) a magnified photo of (k), showing hepatocytes formed vortex in the hydrogel; (**m**) hepatocytes in a 3D printed construct after a longer period of in vitro culture; (**n**) a magnified photo of (m), showing the vortex structure was still there; (**o**) immunostaining of the hepatocyte-laden 3D construct after certain period of in vitro culture, showing new hepatic tissue formed in the gelatin-based hydrogel with close cell-cell connection or tight junction; (**p**) a dark-field microscopy of (o). Images reproduced with permission from [7,22].

It is very interesting that the chemical crosslinking of alginate molecules using calcium ions is reversible. When the 3D printed constructs are placed in a liquid containing no or less Ca2+, the crosslinked Ca2+ dissolves gradually within about one week. Further reinforcement is necessary when long-term in vitro culture is required. This means that the 3D constructs need to be further stabilized on and o ff using calcium ions during long-term in vitro cultures.

Alginate-based 3D bioprinting processes can be completed through di fferent working mechanisms, such as extrusion-based cell-laden fiber deposition on a platform [82], coaxial nozzle-assisted crosslinking deposition, bioplotting in a plotting medium (i.e., crosslinker pool) [83], and precrosslinked alginate hydrogel coextruded with cells [84]. Each of these 3D bioprinting technologies has some merits in 3D printing of bioartificial organs.

In extrusion-based 3D bioprinting, grea<sup>t</sup> e ffort has been made to improve printing resolution and shape fidelity of the cell-laden alginate-containing 3D constructs by optimizing the processing parameters, such as nozzle size, dispensing pressure, and printing speed [85–89]. For example, Markstedt and coworkers blended alginate hydrogel with collagen and nanofibrillated cellulose to effectively enhance extrusion-based 3D printing resolution from 1000 to 400–600 μm using a proper diameter nozzle [90]. Kundu and coworkers printed cell-laden alginate with synthetic polycaprolactone (PCL) using a double-nozzle 3D bioprinter [14].
