Page 276 - IJB-10-2

International Journal of Bioprinting Microfluidic spinning for neural models

microscopy. Quantitative polymerase chain reaction used; these 7 microchannels converge at the J position, at
1
(qPCR) assays for growth cone marker growth-associated a similar height as M and M . The design height of M Ⅰ
Ⅱ
Ⅳ
protein 43 (GAP-43) and sympathetic marker tyrosine and the outlet is 0.63 mm, with the actual measurement
hydroxylase (TH) of neural-associated mRNAs were height being 0.62 ± 0.01 mm, M with confluence point J
1
Ⅰ
performed. The primer sequences are as follows: converging again at the J position, and J position being
2
2
connected to the outlet with a height similar to that of M and
• GAP-43: 5’-CGACAGGATGAGGGTAAAGAA-3’ the outlet. Resin templates with different channel heights
Ⅰ
(forward)
can be prepared by one-step 3D printing. The generated
5’-GACAGGAGAGGAAACTTCAGAG-3’ template exhibits exceptional attributes, such as smooth
(reverse) surface, clear microchannels, minimum difference between
• TH: 5’-GTGAACCAATTCCCCATGTG-3’ the actual printing height and the design height, and high
(forward) fineness, which are the most sought-after characteristics in
the preparation of in microfluidic spinning chips.
5’-CAGTACCGTTCCAGAAGCTG-3’ After the resin template was cleaned and silanized, the
(reverse)
PDMS layer was obtained by the PDMS molding method.
• GAPDH: 5’-TCCAGTATGACTCTACCCACG-3’ One PDMS layer was perforated and plasma-sealed with
(forward) another identical PDMS layer to obtain a microfluidic
5’-CACGACATACTCAGCACCAG-3’ spinning microchip, as shown in the inset of Figure 1A . The
2
(reverse) transparent PDMS material of the microfluidic spinning
chip also provided favorable conditions for observing the
2.9. Statistical analysis fluid flow in the microchannel under a microscope. To
In this work, all experiments were performed at least three prepare CaA/GelMA microfibers with hollow structures, as
times. All data are presented as mean ± standard deviation shown in Figure 1A , we injected CaCl solution as a sheath
2
2
(SD), unless otherwise stated. Results are considered flow in channel M , sample solutions containing NaA,
Ⅰ
statistically significant when p <0.05, as determined by GelMA, and the photoinitiator in channels M and M , and
Ⅱ
Ⅳ
Student’s t-test. Data processing of the FTIR spectra was core flow solution in M . The transient ionic crosslinking of
Ⅴ
performed using Origin software. The cross-sectional area NaA and CaCl was used to form hollow microfibers, and
2
of the microfibers, number of cells in the microfibers, and CaA/GelMA microfibers were obtained after crosslinking
axon length in the fluorescence images and 3D videos were was induced by a second UV exposure (Figure S1 in
analyzed using ImageJ software, an inverted fluorescence Supplementary File). Procedurally, the spinning process
microscope, and confocal microscope self-contained involves two crosslinking reactions: ionic crosslinking
software. Analysis of qPCR outputs was performed using and UV crosslinking. The first step of ionic crosslinking
the Lightcycle 96 (Roche) self-contained software. was achieved by the rapid formation of a calcium-alginate
hydrogel via an ion-exchange reaction of NaA and calcium
3. Results and discussion ions. This step is crucial as it encapsulates the prepolymer
26
of GelMA and the photoinitiator I2959, enabling the second
3.1. Design and preparation of microfluidic spinning step of photocrosslinking. Under UV light irradiation at a
microchip wavelength of 365 nm, the photoinitiator I2959 underwent
In this study, we employed a microfluidic spinning a cleavage reaction to form reactive radicals, and the
strategy to fabricate CaA/GelMA microfibers with a GelMA-containing photosensitive groups underwent a
hollow structure, and the microfluidic spinning microchip polymerization reaction to form GelMA hydrogels. To
45
was designed according to a previous report. The resin facilitate the observation of the morphology and structure of
41
templates were prepared using 3D printing technology, as the hollow fibers, we added PS microspheres with a diameter
shown in Figure 1A . The microchip resin template was fixed of 5 μm each to the sample solution, and the particles in the
1
on glass using an AB adhesive to ensure that the template walls of the hollow microfiber tubes could be viewed under
was not bent or deformed. The resin template consists of the microscope, as shown in the inset of Figure 1A .
9 microchannels distributed in a symmetric structure, 2
named M (2 microchannels), M (2 microchannels), M 3.2. Manufacturing and characterization of
Ⅱ
Ⅰ
Ⅲ
(2 microchannels), M (2 microchannels), and M (1 composite hollow microfibers
Ⅴ
Ⅳ
microchannel) in order, where the design height of M Using this fast and efficient microfluidic spinning method,
Ⅲ
and M is 0.21 mm, and the actual measured height is 0.16 the material selection, concentration, and flow rates of the
Ⅴ
± 0.02 mm; for M and M , a design height of 0.45 mm sample, sheath, and core flow solutions were optimized. The
Ⅱ
Ⅳ
and an actual measurement height of 0.41 ± 0.02 mm were effects of the different sheath and sample flow rates on the
Volume 10 Issue 2 (2024) 268 doi: 10.36922/ijb.1797