Page 457 - IJB-10-5

International Journal of Bioprinting 3D-printed plasma devices for decontamination

spotted on other materials, including parafilm and P3 R further diluted 1:5. MDCK cells (1.8 × 10 per well, plated a
5
filter tissue, for comparison with measurements performed day before the experiment) in a 24-well plate were washed
in our earlier studies. 18,19 The samples were processed and once with PBS; 300 µL of IGM-containing serially diluted
exposed to CAP generated by device 3, similar to that for P. sample (1.5 µL of influenza A H1N1 virus in the first well)
aeruginosa (described in Section 2.4.1.). was added to the cells; the cells were then incubated for
1 h at 37°C and 5% CO . After 1 h, the cells were washed
2.5. Virologic assessment once with PBS; 300 µL of IGM was added to the cells; the
2
The following virus strains and cell lines were used: (i) SARS- medium was overlaid with 300 µL of 1.2% carboxymethyl
CoV-2 (hCoV-19/Czech Republic/NRL_6632_2/2020) cellulose; and the cells were incubated for 2 days at 37°C
was propagated in Vero E6 cells (ATCC CRL-1586) and 5% CO . For both SARS-CoV-2 and IAV, the infection
2
cultured in Dulbecco’s modified Eagle medium (DMEM) was terminated by aspirating the medium. The cells were
supplemented with 2% fetal bovine serum (FBS); (ii) washed once with PBS, fixed, and stained with naphthalene
IAV (influenza A H1N1/California/07/2009; Diagnostic black. After 45 min of incubation, the naphthalene black
Hybrids, United States of America [USA]) was propagated solution was aspirated, the cells were washed with water,
in MDCK (ATCC CCL-34) cells cultured in influenza and the resulting plaque-forming units (PFUs) were
growth medium (DMEM supplemented with penicillin/ counted. The viral titer was expressed as PFU/mL. The 50%
streptomycin [Pen/Strep], 0.2% bovine serum albumin tissue culture infectious dose (TCID ) of HAdV and HRV
[BSA], 1 mM HEPES, 13.6 mM l-glutamine, 42 mg/L was determined by inoculating serially diluted (10-fold)
50
DEAE-Dextran, and 1 mg/L TPCK-Trypsin); (iii) HAdV samples (10 μL/well) in 96-well microtiter plates, in which
(species C/type 2, ATCC VR-846) was propagated in A-549 A549 and HeLa Ohio cells were seeded, respectively. The
cells (DSMZ ACC107) cultured in DMEM with 10% FBS infected cells were incubated at 37°C in a 5% CO incubator
2
and Pen/Strep; and (iv) HRV (species A/type 2, ATCC VR- for 5 (HRV) or 7 days (HAdV). The cytopathic effect
482) was propagated in HeLa Ohio cells (both a kind gift of (CPE) was visualized via a crystal violet assay as previously
Heinrich Kowalski, Medical University of Vienna, Austria) described. The Spearman–Kärber method 21,22 was used to
20
cultured in DMEM with 10% FBS and Pen/Strep. calculate the respective infectious titers (IU/mL).
Parafilm squares (1 × 1 cm) inoculated with 30 µL of
Quantitative polymerase chain reaction (qPCR) was
virus suspension (infectious dose: 10 –10 IU/mL) were performed to determine the number of recovered virus
3
6
placed in a Petri dish surrounded by two pieces of wet genome copies. For SARS-CoV-2, a commercial kit (gb
cotton wool to increase the humidity. The virus-containing Sarbeco E, Cat. no. 3227--500; Generi Biotech, Czech
suspension was distributed across the parafilm surface Republic) was used in accordance with the manufacturer’s
in six droplets, air-dried for 15 min, and subsequently instructions. IAV genome copies were determined
exposed to CAP for 10, 30, 90, and 120 min. Thereafter, via the Luna Universal Probe One-Step RT‒qPCR Kit
the residual virus was recovered from the parafilm surface (New England Biolabs, USA), as previously described.
55
using 200 µL of phosphate-buffered saline (PBS), and the Recovered HAdV and HRV genome copies were measured
parafilm was thoroughly washed. To reduce the risk of via previously described protocols: HAdV (type C) 23,24 and
bias, we implemented a control protocol for each sample, HRV (multiplex). 28
involving pipetting, 15 min of air-drying, mock exposure,
and subsequent recovery. The recovered suspension was 3. Results
directly used for infecting permissive cells (specified
above) to determine the infectious titer of each virus. All 3.1 CAP composition produced by the
the data were generated from three biological replicates. 3D-printed devices
The concentrations of NO and NO in the gaseous
The SARS-CoV-2 titer was determined by diluting 20 2
µL of each sample in a 24-well plate containing 200 µL environment of devices 1, 2, and 3 were measured during
of cultivation medium in each well. The viral suspension the generation of CAP over 120 min. While NO levels
was further diluted 1:10. Afterward, 300 µL of Vero E6 remained undetectable in all cases (data not disclosed),
NO levels displayed an obvious difference between
cells (2.5 × 10 per well) was added to the suspension and optimized devices 2/3 and the original device 1, suggesting
2
5
incubated for 4 h at 37°C and 5% CO . The mixture was that the optimized devices are more efficient in producing
2
subsequently overlaid with 500 µL of 3% carboxymethyl NO (Figure 2).
cellulose and incubated for 5 days at 37°C and 5% CO . The 2
2
IAV titer was determined by pipetting 50 µL of each sample The concentrations of O and N O obtained from the
2
3
into a 24-well plate containing 300 µL of influenza growth FTIR spectra are depicted in Figure 3 as a function of
medium (IGM) in each well. The viral suspension was the source operating time. The gradual increase in the
Volume 10 Issue 5 (2024) 449 doi: 10.36922/ijb.3679