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International Journal of Bioprinting Microfluidic spinning for neural models

2.4. Microfiber assembly and diffusion experiment was used to label HUVECs. The cells were cultured for 7
In this study, we used a self-made 3D printing device to days, during which fluorescence images of the cells were
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print CaA/GelMA hollow composite microfibers onto a obtained using a laser scanning confocal microscope
microfiber assembly microchip. Afterward, 100 μL NaA/ (FV3000, Olympus, Tokyo, Japan). For cell viability testing,
GelMA mixed solution was added into the rectangular middle the cell-loaded microfibers prepared using the above
chamber of the microfiber assembly microchip to cover the method were stained with propidium iodide on the 1st and
composite microfiber. Subsequently, by adding CaCl solution 7th days. According to the instructions, Alexa Fluor 488
2
and applying UV light at a wavelength of 365 nm for 60 s, phalloidin was used to characterize the F-actin of HUVECs
the assembly of composite microfibers in the microfiber in microfibers and plates. In addition, immunofluorescence
assembly microchip was achieved. During the preparation of staining was used to detect the expression of vWF in
microfibers, Alexa Fluor 568 Dextran (10 kDa) was added to HUVECs in hollow composite microfibers and plates. Cells
the core flow with a final concentration of 100 μg/L. After the were fixed at room temperature in 4% paraformaldehyde
microfibers were generated, they were immediately printed for 10 min. The cells were then soaked in 0.1% Triton
onto the microfiber assembly microchip, and the diffusion of X100 for 3–5 min and then in a blocking buffer for 1 h.
the fluorescent dyes at different times in the assembly system Afterward, rabbit anti-vWF was added (diluted 1:100) and
was imaged using an inverted fluorescence microscope (IX-73, incubated overnight at 4°C. Finally, the secondary antibody
Olympus, Tokyo, Japan). In addition, to characterize the pores (diluted 1:100) was added, the cells were incubated for 1
in the microfibers in the assembly system, blue fluorescent h, and the cell nuclei were stained with DAPI for 10 min.
PS microspheres (0.1 mg/mL) were added to the inlet of Fluorescent images of the cells were captured using a laser
the assembled fibers, and their tracks were tracked using an scanning confocal microscope.
inverted fluorescence microscope.
2.8. Neural model construction and differentiation
2.5. Cell culture evaluation
HUVECs used in this study were donated by Prof. Wang The CaA/GelMA hollow composite microfibers loaded
Li (Fudan University). PC12 cells were purchased from with HUVECs were 3D-printed onto a sterilized microfiber
the Cell Bank/Stem Cell Bank of the Chinese Academy of assembly microchip. The PC12 cells were resuspended in a
Sciences (ATCC Source). The HUVECs were cultured in NaA/GelMA mixture solution. The resultant cell suspension
endothelial cell culture medium supplemented with 1% was added to the middle chamber of the microfiber
v/v double antibiotic solution containing penicillin and assembly microchip, and 100 μL of the cell suspension
streptomycin, 1% v/v endothelial cell growth additive, was added to completely cover the composite microfibers,
and 5% v/v FBS. PC12 cells were cultured in RPMI 1640 which were cured by the addition of CaCl solution and
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medium supplemented with 1% v/v double antibiotic UV light for 60 s to complete the assembly. In a previous
solution containing penicillin and streptomycin, 5% v/v study, neural differentiation induction experiments were
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horse serum, and 10% v/v FBS. Cells were cultured in an performed on PC12 cells, which were stimulated using a
incubator maintained at 37°C with a concentration of 5% complete medium with 100 ng/mL NGF. There were three
CO , and passaged every 2 days.
2 groups in this experiment: NGF- HUVEC- group, NGF+
2.6. Preparation of cell-loaded microfibers HUVEC- group, and NGF+ HUVEC+ group. Among
The HUVECs were digested using 0.25% trypsin-EDTA them, the NGF- HUVEC- group was the negative control
and resuspended in a mixed NaA/GelMA solution to group in which the assembled composite microfibers were
obtain a density of 2 × 10 /mL. To encapsulate cells, a not loaded with HUVECs, and NGF was not added to the
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mixture of PF-127 solution and NaA/GelMA was filtered system. In the NGF+ HUVEC- group, the microfibers
using a 0.22 µm sterile membrane before the operation. were not loaded with HUVECs, and NGF was added to
The CaCl solution was high-pressure sterilized, and the the hollow microfiber in PF-127 solution and continuously
2
spinning device was pre-disinfected with UV light for applied to the system after microfiber assembly. In the
30 min. After production was completed, the cell-loaded NGF+ HUVEC+ group, the microfibers were loaded with
microfibers were transferred to a complete cell culture HUVEC cells, and NGF was added to hollow microfiber
medium containing a 0.1% w/v chitosan solution (filtered in PF-127 solution and continuously applied to the system
for sterilization) for further cultivation, and the medium after microfiber assembly. After 7 days of culture, PC12
was changed every 2 days. cells were stained for F-actin, and the differentiation of
PC12 cells was assessed by measuring the axon length
2.7. Cell tracing and staining on day 7 under a microscope. Tubulin III expression in
To track the HUVECs and their position in the microfibers, PC12 cells was detected by means of immunofluorescence
according to a previous study, Cell Tracker Green CMFDA staining, and all images were acquired using laser confocal
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Volume 10 Issue 2 (2024) 267 doi: 10.36922/ijb.1797