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AJ P of Bioelectrical Devices
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Parylene-C-coated NTE substrates (3 replicas). Matrigel ink. Biocompatibility was estimated by a direct rATP
(Corning) was drop casted on the substrates and left for assay (CellTiter-Glo 3D Cell Viability, Promega cat.
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1 h at 37°C. A line of human (hiPSCs) was reprogrammed, no. G9681) and performed at 24, 48, and 96 h (three
characterized, and differentiated in NSCs, by following replicas each), including plastic as positive control. In
the procedure presented by Ferraro et al. . A 100 µm details, this assay measures the quantity of viable cells
[30]
strainer (Fisher Scientific) was used to filter the NSCs to by quantifying the adenosine triphosphate (ATP) values
achieve a single cell suspension. Subsequently, the cells present in each well. ATP is recognized as a marker for
were plated at a concentration of 1 × 10 cells cm on metabolic cellular activity. A preliminary experiment
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the specimens, including the control. After 30 min of was conducted to identify the suitable cell concentration
incubation, a proper volume of Neural Expansion Medium to be cultured in order to reach a desirable amount of
(PSC Neural Induction Medium diluted 1:1 with Advanced confluence (70 – 80%). From this analysis, a concentrated
DMEM/F-12 Thermo-Fisher Scientific) was added to each NSCs suspension of 6 × 10 cells cm was selected.
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specimen. The well-plate was then incubated at 37°C in an Cells, previously kept at 37°C in an incubator, were
atmosphere at 5% CO . After 24 h and 5 days of culture, detached, counted, seeded onto each sample, and further
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cells were fixed and further analyzed following the same incubated for a period of 30 min. Later, each well was
procedure adopted for HFs, as previously described. filled with a proper amount of neural expansion medium
and incubated at 37°C and in a 5% CO atmosphere. The
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(2) Immunofluorescence of PEDOT: PSS samples ATP assay was performed according to supplier protocols
An additional immunofluorescence assay with hiPSC- after 24, 48, and 96 h of culture on triplicated samples.
derived NSCs was performed on two samples each of Briefly, a volume of CellTiter-Glo 3D Reagent equal to
Matrigel-coated PEDOT: PSS ink and plastic, as control. the volume of cell culture medium present in each well
The ink was printed in a square shape of 8 × 8 mm, with the was added to induce cell lysis. After incubation at room
combination of parameters: Ø nozzle = 300 μm, s = 25 mm/min, temperature for 25 min to stabilize the luminescent
A = 40 sccm, S = 80 sccm, T = 40°C, and for 20 layers. Post- signal, the total volume was transferred into a 96-well
printing curing was performed in a thermal oven (Heraeus) opaque-walled multiwell plate and the luminescence
at T = 150°C for 8 min. Later, Matrigel (Thermo-Fisher was recorded using Microplate Reader Infinite 200
Scientific) was drop casted on each sample for 1 h at 37°C. (Tecan). Afterward, an ATP standard curve was plotted
NSCs were filtered with a 100 μm strainer (FisherScientific) in the range of 10 μM to 10 nM, using the ribonucleoside
in order to collect a single cell suspension. Cells were then triphosphate rATP (Promega cat. no. P1132) as positive
plated at a concentration of 5 × 10 cells cm and later control to compare the luminescence values acquired
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deposited on the substrates. The samples were incubated from the standard wells and the samples. Hence, the
for 30 min and eventually filled with an adequate volume of ATP concentration was calculated and plotted. To assess
Neural Expansion Medium (Thermo-Fisher Scientific). The the material biocompatibility at multiple conditions
well-plate was finally incubated at 37°C in an atmosphere of use, indirect rATP assays were also performed. In
at 5% CO , over a period of 4 days. The same procedure for particular, PEDOT: PSS samples on Parylene-C-coated
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the NTE substrates was then applied to these specimens. NTE substrates of 1 cm were placed in a 24-well plate
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and let to release any potential cytotoxic components
(3) Cell viability assay on NSCs into 1 mL of Neural Expansion Medium for 5 days in a
Cell viability was executed to evaluate the cellular humidified incubator at 37°C and in 5% CO atmosphere.
2
viability and proliferation of NSCs on PEDOT: PSS As reference, the same volume of medium was poured
printed samples. The presence of the DEG co-solvent in an empty dish and placed in the incubator at the same
might indeed affect the biocompatibility of the final conditions. The day before the test, NSCs were seeded
printed pattern, despite the post-curing process that should either in flat bottom 48-well plates Matrigel-coated (5
4
allow its complete evaporation. In the literature, a couple × 10 cell/well). Three replicates were set up for each
of studies have demonstrated that DEG and its primary condition. The day after, cell supernatant was removed
metabolite diglycolic acid are toxicants . Reed et al. and replaced by either ink sample conditioned medium
[32]
[31]
stated DEG at high concentrations (100 mmol/L) induces or control medium. As experimental positive control
the necrosis of SH-SY5Y neuroblastoma cells at 120 h NSCs were also seeded in fresh medium to verify that the
already. However, to the best of the authors’ knowledge, neural medium, maintained at 37°C for 5 days, could not
no research has ever been performed on PEDOT: PSS- affect the cell healthy. Finally, the cells seeded in the 48-
based inks containing DEG in percentages of 12 – 20 wt%. well plates were subjected to an ATP cell viability assay,
The samples used were AJ printed, cured, and coated as previously described for the direct rATP. Tests were
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with Matrigel, following the same procedure applied conducted on both printed and spin coated PEDOT: PSS
for the immunofluorescence assay on the PEDOT: PSS samples, and at different conditions of temperature and

56 International Journal of Bioprinting (2022)–Volume 8, Issue 1