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Pei, et al.
of transferring the printed structure to an incubator after Table 1. Parameters of the integrated cell printing/culture
printing. Moreover, we also optimize the printing process equipment
by adjusting the printing parameters of the bio-inks and Environment Temperature 0 – 50±0.8°C
the pre-processing and post-processing conditions of the control module Humidity 50 – 95%
bio-inks so that we can accurately control the compression Oxygen concentration 0 – 5%
modulus of the printed structure. Collagen is the main Material Channels 4
component of natural ECM, accounting for about 30% extrusion Maximum stroke 90 mm
of the total protein of human body , which provides module Linear velocity range 7.49 μm/min –
[14]
structural stability and strength to numerous tissues such 79.4 mm/min
as skin, bone, cartilage, or even teeth. Therefore, in this Stroke resolution 0.165 μm
study, collagen was added to the bio-inks to increase the Motion control Stroke X 150 mm
survival rate of the cells in printed structure. Then, the module Y 150 mm
distribution of pore size in brain tissue was simulated Z 150 mm
by changing the concentration of collagen in bio-inks to Speed X,Y 0 – 50 mm/s
alter the inner pore size of printing structure. Therefore, Reposition X,Y,Z 10 μm
an artificial brain-like tissue model with a gradient accuracy
distribution of pore size was constructed in vitro. Position X,Y,Z 20 μm
Accuracy
2. Materials and methods
2.1. Building an integrated cell printing/culture 2.3. Preparation of the bio-inks
equipment
The bio-inks used for 3D cell printing needed to be both
The integrated cell printing/culture equipment consists printable and biocompatible. Under certain conditions,
of both hardware and software. The hardware consists gelatin and sodium alginate had higher viscosities which
of three parts: Mobile control module, material extrusion were easy to form and had good printing properties, but
module, and environment control module. The main they were poorly biocompatible. Collagen and silk fibroin
components of the mobile control module and the material had good biocompatibility, but they were difficult to form
extrusion module were placed outside of the environment and cannot print. Therefore, it was difficult to use a single
control module to avoid the corrosion of the precision biomaterial to meet the requirements of the printing. In
components caused by humidity and other conditions in this paper, gelatin and sodium alginate were mixed as
the printing environment. The software was based on the the basic components of the bio-ink, and collagen was
VC language which can realize the coordinated control of added as a regulating factor to form the final bio-ink,
the mobile platform and the material extrusion module. which has been proven to have good biocompatibility and
QT5.8 programming software was used to write a set printability.
of graphical control software. We can achieve the goal The sodium alginate (medium viscosity, purity
of printing a brain-like layered structure with multiple ≥98%) and gelatin (from pigskin, medium-strength) used
bio-inks and multiple cells using multi-nozzles. Finally, in this experiment were purchased from Sigma company.
the printed tissue can be cultured in the equipment after Collagen was collagen type I, which was extracted from
the printing process. Moreover, the environment within rat tail by our research group, and its concentration was
the equipment can be controlled to provide a suitable 6 mg/ml. According to the preliminary experiment,
environment for the survival of cells, formation of bio- we selected 6 wt% gelatin and 1 wt% sodium alginate
inks, and the culture of printed tissue. The integrated cell as the basic bio-inks, and the collagen with different
printing/culture equipment is shown in Figure 1, and the concentrations was added as the regulatory factor. By
parameters of each part of this equipment are shown in changing the concentration of collagen in the bio-ink (the
Table 1. concentrations of collagen are 1.5 mg/ml, 1 mg/ml, and
0.5 mg/ml, which are denoted as G6A1C1.5, G6A1C1,
2.2. Printing principle and G6A1C0.5, respectively), we could obtain the

Multiple nozzles were used to print the layered gradient bio-inks that can print the layered gradient brain-like
brain-like structure by the extrusion method. Each nozzle structure.
contained a bio-ink with different compositions and According to the characteristics of collagen
different cells (Figure 2B), and all the nozzles printed the that gelates at high temperature and of gelatin at low
structure layer by layer in turn until the printing process temperature, we developed a configuration process to mix
was completed. the bio-inks (Figure 3).

International Journal of Bioprinting (2021)–Volume 7, Issue 3 73