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International Journal of Bioprinting NIR-secretome release for nerve regeneration

Biochrom), and the tissue was mechanically dissociated at Samples were dehydrated and sputter-coated with gold.
room temperature with a fire-polished Pasteur pipette. The They were then imaged with SEM Supra 25 (Zeiss). Images
cell suspension was harvested and centrifuged at 235 × g were acquired at several magnifications. The chemical
for 8 min. The pellet was suspended in 88.8% Minimum analysis of microbeads was carried out using attenuated
Essential Medium (Biochrom), 5% fetal bovine serum (FBS), total reflectance-Fourier transform infrared spectroscopy
5% horse serum, 1% glutamine (2 mM), 1% penicillin– (ATR-FTIR) by Spectrum One spectrometer (Perkin
streptomycin–neomycin antibiotic mixture (Invitrogen), Elmer). The material under investigation was directly laid
and glucose (25 mM). Cells were plated at a density of 105 upon the ATR crystal, and the spectra were recorded in the
cells/mL on a 24-well plate precoated with poly-L-lysine wave number range of 4000–550 cm .
−1
(0.1 mg/mL; Sigma). Twenty-four hours later, the culture
medium was replaced with a mixture of 96.5% neurobasal 2.4. Near-infrared radiation and release rate of the
medium (Invitrogen), 2% B-27 (Invitrogen), 0.5% glutamine microbeads
(2 mM), and 1% penicillin–streptomycin–neomycin Microbeads were transferred on a 96-well plate (Corning)
antibiotic mixture. After 72 h, this medium was replaced with 100 μL of culture medium. The plate was placed
with a glutamine-free version of the same medium, and the under an 808 nm laser (Laser Ever) equipped with a
cells were grown for 10 more days before experiments. All thermal imaging camera (Optris). Different power
cell lines were cultivated in T75 flasks and kept at 37°C in densities were adjusted for all the tested concentrations
5% CO humidity. Mesenchymal stem cells were purchased of GO to induce the same thermal effect, increasing from
2
2
from the American Type Culture Collection (ATCC). Cells room temperature to 39°C: 0.91, 2.40, and 3.30 W/cm for
were maintained in Dulbecco’s modified Eagle’s medium bioinks having 0.5, 0.2, and 0.1 mg/mL of GO, respectively.
(Sigma-Aldrich) supplemented with 10% FBS (EuroClone), Thermal increase was recorded in a time span of 60 s. To
2% penicillin–streptomycin (Sigma-Aldrich), and 2% measure the release rate of the secretome, fluorescein-5-
L-glutamine (Sigma-Aldrich) on T25 flasks. After three isothiocyanate (FITC)–Dextran macromolecules having
passages, supernatant was gathered at 24 h from the change a molecular weight of 10,000 kDa were embedded in the
of culture medium and centrifuged at 1000 rpm for 10 min. bioink. After irradiation, fluorescence of the supernatant
Supernatant was then stored at -80°C for further use. was observed by removing the microbeads at different
timepoints. Fluorescence intensity of the supernatant of
2.2. Bioprinting of alginate-graphene oxide non-irradiated beads was subtracted for each sample,
microbeads to consider the natural swelling of the bioprinted beads.
To bioprint AGO microbeads, a biocompatible bioink Cumulative release was then plotted over time.
composed of the mixture of the two materials was first
prepared. Alginate was kept at a concentration of 1.5% w/v, 2.5. Cell viability and production of reactive
while GO was added at different final concentrations for oxygen species
further tests: 0.5, 0.2, and 0.1 mg/mL. Control microbeads Cells were first treated with hydrogen peroxide (H O ) at
2
2
were prepared without GO. Culture medium, astrocyte- 250 μM for 30 min or with 1× PBS (control group). After
conditioned medium (ACM; ScienCell), and MSCs incubation, to measure viability, cells were put in contact
secretome were added at a ratio 1:1 with respect to AGO. with bioprinted microbeads having concentrations of GO
The mixture was then loaded into a bioprinting cartridge ranging from 0.5 to 0.1 mg/mL. After NIR irradiation,
and transferred on an electromagnetic droplet printhead of cells were incubated for 72 h at 37°C and 5% CO . Then,
2
BIO X bioprinter (Cellink, BICO Company). Bioprinting viability was measured using CellTiter-Glo® (Promega).
was performed at room temperature, extruding microbeads Briefly, an amount of CellTiter-Glo® reagent equal to the
with a speed of 50 mm/s and a pressure of 120 kPa. An volume of culture medium was added to each well. Then,
open time of 100 miliseconds (ms) and a cycle time of the plate was orbitally shaken for 2 m to ensure complete
1000 ms were applied to set the number of extruded cell lysis and incubated in the dark at room temperature
beads. The droplets were extruded on a 6-well (Corning) for 10 min. After incubation, luminescence was recorded
filled with a 2% w/v CaCl solution. The microbeads were using Cytation 3 cell imaging multi-mode reader. Results
2
left crosslinking for 10 min, and then they were carefully were reported as % of control (untreated) cells. To observe
gathered and observed under the optical microscope of viable cells through fluorescence microscopy, cells were
Cytation 3 Cell Imaging Multi-Mode Reader (Biotek). stained with calcein AM (Invitrogen). Culture medium
was removed, and calcein at a final concentration of 10 μM
2.3. Characterization of microbeads in PBS was added for 20 min at 37°C and 5% CO . Then,
2
Scanning electron microscopy (SEM) images were acquired calcein was removed and replaced with culture medium.
to investigate the surface composition of the microbeads. Fluorescence was observed under the fluorescence

Volume 10 Issue 1 (2024) 231 https://doi.org/10.36922/ijb.1045