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Pei, et al.

A B

C D E

Figure 9. (A) The morphology of the layered structure and the distribution of cells inside of the layered structure which was cultured in a
cell incubator for 1 day. (B) The macro morphology of the layered structure when cultured for 7 days. (C-E) The viability of cells in the
layered structure when the structure was cultured for 1 day, 3 days, and 7 days, respectively. Green and red fluorescence denote living cells
and dead cells, respectively.

Table 5. Printing parameters of bio-inks with different collagen concentration
Parameter type Ink composition Needle diameter Extrusion rate of Moving speed of
D (μm) the ink u (μl/min) platform v (mm/s)
Top layer G6A1C0.5 200 (27G) 60 10
Middle layer G6A1C1 200 (27G) 60 10
Bottom layer G6A1C1.5 200 (27G) 30 10

Due to the complexity of the brain structure, a high resemblance to the brain. Lee et al. [44] proposed
humans attempt to construct brain-like tissue models a direct ink-jet 3D printing method with multi-layer
in vitro to study the structure and function of the brain. collagen gel containing rat embryonic astrocytes and
At present, Chwalek et al. [42] have used the molding neurons. Gu et al. [45] proposed a direct printing method
method to produce a donut-shaped silk-collagen protein using a new bio-ink combining human neural stem
scaffold which can form a 3D neural network that has cells to print a 3D porous grid structure. These methods
similar electrophysiological characteristics as normal can be used to construct 3D brain like tissue by 3D
neural tissue. Odawara et al. [43] developed a technique printing, but the brain like tissue constructed by these
to form the directional fibers using collagen gel and also methods does not simulate the hierarchical structure
developed a 3D culture method in a polydimethylsiloxane and gradient characteristics of brain tissue, which
micro-chamber. Finally, they proved that this method verified the feasibility of using the printing method to
could generate a 3D neural network by accurately construct brain-like tissues but lacked the simulation
controlling the location of the cells. These studies of the layered structure and gradient characteristics of
built a 3D neural tissue model in vitro using the brain tissues. Lozano et al. [46] used a peptide-modified
molding method. Although this method was simple to gellan gum to prepare a three-layer brain-like layered
operate, had low requirements for forming, and did not structure by hand-held extrusion printing method. This
require complex external conditions, it was difficult to study preliminarily simulated the layered structure
manufacture complex structures, and at the same time, with or without cells, but it was quite different from
it was impossible to accurately control the density and the continuous layered structure of natural brain tissue.
spatial distribution of the nerve cells. Therefore, it was Gu et al. [47] developed a novel and optically visible
difficult to produce complex brain-like tissues that bear bio-ink for printing 3D neural tissue. The bio-inks can

International Journal of Bioprinting (2021)–Volume 7, Issue 3 81