Page 139 - IJB-9-2

International Journal of Bioprinting Hybrid biofabrication of neurosecretory structures

also convenient for in vivo transplantation and the secretory the Z-axis thermal insulation fixture was controlled at 37°C.
function of organoids. Biological manufacturing organs have The syringe loaded with bioinks was installed, the tail end was
been widely recognized as disease models [31,32] . Therefore, linked to the air pump, and the head end was linked to the
the hybrid biofabricated 3D neuroendocrine organs are 0.26 mm print sprinkler, and the structure of the hydrogel
expected to become disease models for neuroendocrine scaffold was bioprinted according to the previous methods
research and be applied in clinical transformation research. and parameters . After completing a layer of printing,
[33]
In this study, we aimed to develop an engineering the electrospun nanofibers were used to cover the cell-
strategy for hybrid biofabrication using 3D bioprinting laden hydrogel scaffold again, and after forming a complete
and nanofiber electrospinning technology to produce package, the spinning was suspended, and the bioprinting of
neurosecretory tissue structures. The 3D structure of the next layer of cell-laden hydrogel scaffold was continued.
hybrid biofabrication was characterized in vitro, and the The neurosecretory tissue-like sandwich structure was made
biocompatibility, cell viability, secretory function, and tissue through superimposing and depositing layer-by-layer.
remodeling potential of hybrid biofabricated neurosecretory 2.3. CCK-8 cytotoxicity analysis
tissues were confirmed in vitro and in vivo. Finally, we
successfully manufactured hybrid biofabricated structures To select the solvent with low and residual toxicity for
with neuroendocrine function, and aimed to explore more electrospinning, hexafluoroisopropanol, acetone, and
feasible new avenues for translational clinical applications. dichloromethane were used to dissolve PLLA and gelatin.
After electrospinning, the nanofiber membrane was
2. Materials and methods immediately immersed in DMEM medium for 24 h, and
three kinds of extracts were obtained. PC-12 cells in the
2.1. Preparation of bioinks logarithmic growth phase were inoculated into a 96-well
The neuroendocrine cell line PC-12 was purchased from plate at 100 μL/well from a 4 × 10 /mL cell suspension, and
4
the Shanghai Cell Bank of the Typical Culture Preservation incubated overnight at 37°C. The three extracts were replaced
Committee of the Chinese Academy of Sciences. and cultured for 24 h and each extract was set with three
PC-12 cells were subcultured in DMEM containing 10% gradients, that is, extract at concentrations of 100%, 50%,
of fetal bovine serum. Sodium alginate, gelatin, and and 25%, with fresh medium as control. Next, 100 μL CCK-8
hyaluronic acid (all purchased from Sigma-Aldrich, St. reaction solution (90 μL medium + 10 μL CCK-8) was added
Louis, Missouri, USA) were sterilized by gamma rays and to each well and incubated at 37°C for 2 h. The optical density
were mixed and dissolved in phosphate-buffered saline (OD) of each well at 450 nm wavelength was measured. The
(PBS) without calcium and magnesium ions at a mass ratio relative survival rate (RGR) was calculated using the formula
of 4%, 20%, and 2% by volume, called AGH hydrogel. The below:
PC-12 cells in the logarithmic growth phase were digested Relative survival rate )
into cell suspension, and mixed with an equal volume of (RGR, %
AGH hydrogel at a concentration of 1 × 10 /mL to prepare Average value (OD value of
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bioinks, which was then loaded into an injection syringe experimental group − OD value of blank group)
for cryogenic extrusion bioprinting. = Average value (OD value of control
2.2. Three-dimensional bioprinting of cell-loaded group − OD value of blank group)
structure and electrospinning
The electrospinning blend was prepared by dissolving poly- 2.4. Live/dead assay
L-lactic acid (PLLA) and gelatin in a hexafluoroisopropanol The PC12 cell viability in hybrid biofabricated 3D models
solvent in a mass ratio of 8:3 with thorough stirring (all was assessed using live/dead fluorescence assays according
purchased from Sigma-Aldrich, USA). The blend was then to the manufacturer’s instructions. Briefly, 2 μM calcein
loaded into the syringe, slowly pushed from the micropump AM and 8 μM PI-working solution was obtained by
to the homemade electrospinning device, connected to the diluting live/dead staining solution with PBS, then added
nozzle with a 10 kV high-voltage power supply to instantly to each hole with 1 mL and incubated at room temperature
spray out nanofibers before spraying for approximately for 15 min. The fluorescence was observed under a
10–20 s at approximately 15–20 cm above the 3D bioprinting fluorescence microscope. Five visual fields were randomly
platform. A layer of nanofibers was deposited and bioprinting selected to take pictures. The number of green (living cells)
was initiated. While using cryogenic molding extrusion-based and red (dead cells) cells in each visual field was recorded
bioprinting, the temperature in the molding room where the and the cell survival rate (living cells/living and dead cells
XY forming platform is located was maintained at 4°C, while × 100%) was calculated.

Volume 9 Issue 2 (2023) 131 https://doi.org/10.18063/ijb.v9i2.659