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Hyeong-jin Lee, Young Won Koo, Miji Yeo, et al.

ing process using a core/shell nozzle. They extruded temperature was controlled to enhance printability in
the crosslinked solution through the core and the bio- the 3D structure while the damage to cells was mini-
ink through the shell to create a hollow tube-shaped mized. The rheological property of dECM (decellula-
3D structure. By applying the drop-on-demand print- rized extracellular matrix) bioink was controlled by
ing method, they were also able to fabricate various increasing the temperature to construct a 3D structure
3D cell-laden structures. (Figure 3a) [48] . As the temperature increased beyond
For applications in extrusion-based cell printing, a 15°C, the storage modulus was increased, and a cros-
dual or core/shell nozzle is occasionally used as an slinked gel was observed at 37°C. In this process, in-
alternative crosslinking method, as in the study de- creasing temperature is prerequisite to retain 3D
scribed above [43–46] . The core/shell fibrous collagen– structure, which subsequently makes storage modulus
alginate hydrogel was proposed by Perez et al. [44] greater than loss modulus at the certain temperature. A
(Figure 2c). They placed mesenchymal stem cells high cell viability (> 90%) was maintained over 14
(MSCs) into the inner cell-collagen encapsulated with days of culture for the in vitro and in vivo tests. Fur-
a 2 ~ 5 wt% alginate (the outer portion). The colla- thermore, Yoon et al. [49] varied temperature for opti-
gen–alginate hydrogel was extruded into a bath filled mizing the fabrication of a collagen scaffold. In this
with a 50-mM CaCl 2 solution, and the outer alginate study, the stage containing a circulating pump, water
contacted the CaCl 2 solution for 5 min and crosslinked. chamber, and temperature controller was used to
Using this process, sufficient stability of the colla- maintain the cell-printing plate from 25°C to 60°C.
gen–alginate hydrogel was maintained and repress- The collagen struts were adequately fabricated be-
ented by storage moduli as 30 kPa, 40 kPa, and 50 kPa tween 36°C to 39°C with a ce ll viability of 85%.
at 2 wt%, 3 wt%, and 5 wt% alginate, respectively. Conversely, the strut formation was rather amorphous
The cell viability was approximately 70 to 80% for the and not applicable below 35°C or over 42°C, with a
collagen–alginate (3 wt%) sample and pure collagen significant decrease in cell viability. This phenomenon
sample. In addition, Ahn et al. [46] developed a simple suggests that the temperature and collagen gela-
and innovative cell printing method using a core/shell tion/crosslinking are correlated, and controlling the
nozzle and an absorbing printing stage (Figure 2d). In temperature allows the 3D structure to be formed by
their process, the alginate-based bioink was extruded rapid gelation of the bioink. However, the printed col-
through the core nozzle, and the CaCl 2 solution was lagen scaffold lacks sufficient strength and stiffness
extruded through the shell nozzle to crosslink the
printed bioink simultaneously. The crosslinking solu- (0.01 ± 0.001 kPa of Young’s modulus); therefore,
tion then immediately absorbed into the absorbing further exploration of a non-toxic chemical reagent or
crosslinking process is required.
stage to prevent the crosslinked solution from ruining
the 3D shape of the alginate struts. On a non-absorb- Low-temperature cell printing is a printing method
ing stage, the crosslinked structure can collapse during that plots struts by instantly freezing the bioink ex-
printing owing to the weakened coherence between truded from the nozzle (Figure 3b). The conventional
struts by the remaining crosslinked material. The sur- 3D cell printing has revealed the conversion of dis-
faces of the struts can be constantly indurated, and the pensed nearby struts, which eventually disturbs the
stability of the scaffold increases through the conti- layer-by-layer stacking process. To overcome this
[50]
nuous crosslinking of the previously printed layers. problem, Ahn et al. applied a low temperature from
This method formed a 3D structure easy and more −2°C to −40 °C to fabricate the biaxially porous 3D
consistently than the submerged crosslinking tech- lattice scaffold in solid structure. Throughout the cell
nique, since the submerged process contained a high printing process, the alginate bioink with cells was
possibility of the bioink floating in the crosslinking maintained at 4°C to minimize the cell damage by a
solution during the printing process and required addi- rapid decrease in temperature. As the temperature
tional treatment, such as polyethylenimine (PEI) sur- was close to 0°C, the cell viability increased up to
face coating [42,47] or a layer-by-layer interactively mo- 84%, but the shaping ability decreased. Conversely,
ving stage [39–41,43] . as the temperature decreased to −40°C, the cell via-
bility dropped below 10%, but the shaping ability
3.2 Temperature-controlled 3D Cell Printing was enhanced with high fabricating efficiency of
Process
85%. The scaffolds were printed at -10°C with the
For the scaffold printed with formless materials, the reasonable initial cell v iability (70~84%) and high

International Journal of Bioprinting (2017)–Volume 3, Issue 1 33