Page 70 - IJB-9-6

International Journal of Bioprinting 3D Aerosol Jet® printing for microstructuring

in the presence of higher AgNPs concentrations and Later, HFs were seeded at a concentration of 2 × 10 cells/
4
smaller AgNPs sizes (at an equal mass) , we can deduce cm onto the samples, incubated for 20 min and before
2
[38]
that the cytotoxicity level of the AgNPs-based ink used filling the well with DMEM. Cells were then fixed at day 5
for the printing 3D microstructure printing will be even using the Fix&Perm Sample Kit® (SIC) for 30 min (fixation
greater than the ink analyzed. The samples used were three and permeabilization), and later incubated for 45 min with
replicas of 10 printed lines (line width ~ 400 µm, line length a blocking solution (iBind 5× Buffer, Invitrogen). Finally,
TM
1 cm, n = 40 layers), thermally sintered at 200°C for 1 h samples were stained with phalloidin (Sigma Aldrich) and
on a biocompatible electrospun porous polyacrylonitrile counterstained with Hoechst 33342 to mark cell cytoskeletal
(PAN) substrate used as a neural scaffold . Samples and nuclei, respectively. Samples mounted on a glass
[37]
were first washed for 3 h in 1× PBS (Sigma Aldrich, IT), slide were then detected under a fluorescence inverted
which was sterilized by autoclaving process, and disposed microscope (Olympus IX70) and analyzed with the Image-
in a 24-well with a sterile tweezers. Cell culture on plastic Pro Plus software v7.0 (Media Cybernetics).
dish was used as experimental positive control. A control
h-iPSC line derived from commercial human fibroblast 2.4.2. PEDOT:PSS-based ink
(HFs) cell line (BJ cell line ATCC® CRL-2522™) previously Similarly to the AgNPs-based ink, cell biocompatibility
reprogrammed to differentiate into NSCs . Before cell assays were also carried out on the own-formulated
[40]
[39]
seeding, Matrigel was coated on the substrates at 37°C for PEDOT:PSS ink, being the one with the best results
1 h. Afterward, NSCs were enzymatically detached with obtained in each print strategy. A direct rATP assay was
accutase (Thermo Fisher Scientific) and passed through a performed on h-iPSCs-derived NSCs at 72 h as the protocol
100-μm strainer in order to obtain a single-cell suspension previously described for the AgNPs-based ink. Differently,
that was collected and centrifuged at 1300 rpm for 4 min. the samples (three replicas) were printed squares (6 ×
The supernatant was removed and h-iPSCs-derived NSCs 6 mm, n = 20 layers), thermally sintered at 150°C for
were seeded at a cellular density of 6 × 10 cells/cm on the 1 h on a biocompatible Au/Ti substrate, as a potential
2
4
samples. A concentrated cell suspension was deposited onto bioelectronic interface for cell monitoring. A concentrated
4
2
each support and incubated for 20 min before filling each suspension of 6 × 10 cells/cm was cultured on the samples,
well with a proper volume of neural expansion medium. and plastic was again used as positive control. In addition,
Cells were maintained at 37°C in a saturated humidity immunofluorescence assays on the same printed squares
atmosphere containing 95% air and 5% CO . After 48 h, a were performed on HFs with the same procedure of the
2
direct rATP assay was performed to measure the quantity AgNPs-based ink.
of viable cells by quantifying the adenosine triphosphate 2.4.3. Collagen-based ink
(ATP), typical marker for metabolic cellular activity. Cells Cell viability assays on collagen-based inks were performed
were lysed on the samples using the CellTiter-Glo 3D to evaluate their biocompatibility in the vision of their use
Reagent and incubated at room temperature for 25 min in for bone tissue engineering applications according to the
order to stabilize the luminescent signal. The total volume protocol reported in Degryse et al. A mouse calvaria
[41]
was transferred into a 96-well opaque-walled multiwell preosteoblast cell line (MC3T3-E1) was expanded in T175
plate and the luminescence was recorded using a Tecan culture flasks in MEM alpha medium (Bioconcept), with
Infinite® M200 microplate reader (Tecan, Männedorf, the addition of 10% FBS (Gibco), 100 U mL penicillin,
−1
Switzerland). Parallelly, an ATP standard curve (range and 100 µg mL streptomycin at 37°C, 90% rH and 5%
−1
of 10 µM–10 nM) was created using rATP disodium salt CO . Every 2 days, the medium was refreshed until a
(Promega cat. P1132) to compare the samples luminescence confluency of 80% was reached, then cells were trypsinized
2
to the standard ones in order to determine the correct ATP (TrypLE; Gibco) and subcultured. Collagen films for each
concentration. Hence, the ATP concentration recorded ink composition (collagen, collagen-HAp) were prepared
from each sample was calculated and plotted. in triplicate by casting 250 µL in a 24-well plate, and
Immunofluorescence assays were also conducted on drying the inks for 24 h under a chemical hood. A UV-
the AgNPs-based SI-AJ20X ink in order to detect cellular light exposition (254 nm) for 1 h was used to sterilize the
morphology (cellular cytoskeleton and nuclei). After being thin films, later washed in ethanol and 1× PBS solution.
drop-casted on glass coverslips, the ink was thermally Eventually, MC3T3-E1 cells were seeded on the top of
sintered, washed, and sterilized as previously described. collagen films at a concentration of 1 × 10 cells per cm
4
2
HFs (BJ cell line) were cultured in a Dulbecco’s Modified (24-well plate). MC3T3-E1 were also seeded on 24-well
Eagle Medium (DMEM), supplemented with 10% fetal plates at the same concentration (positive control). A Live-
bovine serum (FBS), 1% penicillin/streptomycin, and 1% Dead staining (ThermoFisher Scientific) was performed at
L-glutamine (all Euroclone), and maintained in an incubator. two time points (1 and 7 days) following the manufacturer’s

Volume 9 Issue 6 (2023) 62 https://doi.org/10.36922/ijb.0257