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Cell Culture & Plating: Human neuroblastoma cells of the IMR-32 cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and were cultured routinely in coated 75 cm² tissue culture flasks using Eagle’s Minimum Essential Medium (EMEM) with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (complete medium). Flasks were coated with 5mL of 10 μg/ml Fibronectin (MilliporeSigma) in phosphate-buffered saline (PBS) solution and incubated at 37°C at 5% CO₂ for 30 minutes. When confluent, cells were passaged using 2-3 mL 0.25% Trypsin-0.53 mM ethylenediamine tetraacetic acid (EDTA) solution. The cells were then added to the coated flask with 8 mL of culture medium. The culture medium was changed every 2-3 days and continuously incubated at 37°C and 5% CO₂. Optimal experimental conditions were determined in a 12-well plate with sterile glass coverslips coated with 600 μL/well of either 10 μg/mL Fibronectin or 50 μg/mL Laminin (Thermo Fisher Scientific, MA, USA) in PBS. Cell cultures from the 75 cm² flask were detached with the Trypsin EDTA solution and centrifuged at 130 xg for 5-7 min. A serial dilution was then performed with the complete medium to attain the desired densities. For our study, cells were diluted to ~8.0x10⁴ cells/well for experimental groups and 2.0x10⁴ cells/well for control groups (counted by hemocytometer). 1 mL of the diluted cell cultures were added to each well and incubated with medium changes every 2-3 days. After 48-hour incubation, the complete medium was aspirated, and the various treatments of our study were performed.

Plasma-Activated Liquid Treatment: A surface micro-discharge (SMD) air plasma device was constructed on printed circuit board (PCB, Express PCB LLC, Mulino, OR). The electrode was rubbed with 70% ethanol prior to use. A 5-mL aliquot of PBS solution was transferred to a 6-cm glass petri dish (6.3 cm i.d. x 0.85 cm H) and placed directly under the electrode. The plasma discharge time was 45 seconds at room temperature. Applied voltage was 8.8 to 10 kV and frequency was 21 kHz at 100% duty cycle. Plasma activated solution (PAS) was used immediately after the activation. The PAS was used for evaluation of differentiation and cell proliferation/viability.

Differentiation: Upon seeding & incubation in the 12-well plates, the medium was exchanged after 2-3 days for filtered neurobasal medium (Thermo Fisher Scientific, MA, USA; Gibco, Cat. No. 21103) supplemented with 10μM retinoic acid (MilliporeSigma, MO, USA), 1% L-glutamine, 2% B27 serum, and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, MA, USA), and the cells were incubated at 37°C and 5% CO₂. The cultures were given half volume medium changes of this differentiation medium every 2-3 days and continuously incubated until differentiation was complete (after 5-7 days). The table below summarizes PAS and differentiation conditions:

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Proliferation/Viability: 24 hours following the PAS treatments, proliferation was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). In brief, after aspirating media, cells in 12-well plates were washed with PBS, followed by the addition of 880 µL of complete medium containing 5 mg/mL MTT, and incubated for up to 4 hours. At the end of the incubation, an 850-µL aliquot of the liquid from each well was aspirated and 1 mL of dimethyl sulfoxide (DMSO) was added to each well. After incubation for 5 minutes, samples were mixed, and a 100μL aliquot of each sample was transferred into a 96-well plate. The absorbance was read at 540 nm using a plate reader (FLUOstar Omega, MBG Labtech, Germany). Net absorbance was calculated and viability was determined using the following formula: (Net absorbance treated sample/Net absorbance control sample).

Fluorescent Staining & Imaging: Desired coverslips were fixed in 2% paraformaldehyde (PFA) in PBS for 50 minutes at room temperature, and were then washed with PBS three times. A 10% donkey serum containing 0.3% Triton-X 100 in PBS was used to permeabilize and block the cells for 1 hour at room temperature. Cells were then incubated in a petri dish with primary antibody BIII-tubulin (1:800) diluted in 5% donkey serum in PBS overnight at 4°C. After washing in PBS-0.2% Tween-20 three times and then once with PBS, the cells were incubated in the secondary antibody Alexa Fluor 488 (Life technologies) diluted with 5% donkey serum in PBS for 1 hour at room temperature in the dark. After washing in PBS+0.2% Tween-20 three times and then once with PBS, cells were counterstained for nuclei with 2μg/mL Hoechst diluted in PBS+0.1% Tween-20 for 15 min at room temperature. Another wash (3x PBS+0.2% Tween-20, 1x PBS) was performed followed by a deionized water rinse, and then the cells were mounted on coverslips using ProLong Gold Antifade Mounting medium. The immunofluorescence was observed under a fluorescent microscope (Olympus BX53), and fluorescent images were obtained using CellSens software (Olympus). For unstained cultures, images were taken with a compound microscope (Olympus) equipped with an Infinity camera and Lumenera software. All measurements were performed with ImageJ software.

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Effects of Neurodifferentiation and Viability by Plasma-Activated Solution (PAS) Treatment on IMR-32 Cells

Madison Lieurance, Neuroscience Department

Frank Reidy Research Center for Bioelectrics at Old Dominion University

Methods

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Discussion

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Results

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References

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Background Information

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Future Directions

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Acknowledgements 

A big medical problem of our time is that after injury or disease, the central nervous system’s ability to regenerate is limited. Damage to the central nervous system can lead to lifetime impairment and disability, and there are currently no completely effective treatments in the clinical world. (Wang et al, 2018). Recent studies show that some treatments can lead to a certain degree of regeneration, such as cold atmospheric plasma (CAP) treatment. Cold atmospheric plasma (CAP) research has greatly advanced in recent years, and findings show that it has many biomedical applications. Recent studies show that CAP can eliminate pathogens, help with wound healing and regeneration, and selectively treat and kill cancer cells (Laroussi et al, 2012). Conventional treatment can lead to healthy cell death that can have permanent damage, but with CAP, cell removal of targeted harmful cells in addition to regeneration can be performed (Keidar et al, 2013). Similar to direct cold atmospheric plasma, plasma-activated solutions (PAS) are a potential cancer and regeneration treatment that can be performed without dependence on a CAP device, and this is the form of plasma treatment we used for our study on IMR-32 cells.

IMR-32 cells are a form of human neuroblastoma cells that originated from an abdominal mass that has neuronal projections similar to that of the cerebral cortex (Rao & Kisaalita, 2002). This cell line is typically associated with in vitro studies and is a good model for differentiation. One study for example, used IMR-32 cells to determine that fatty acids could inhibit proliferation and induced differentiation suggesting their role in cell development (Burdge et al, 2000). Differentiation allows for the specialization of functions and determines the neural connections within our nervous system. Differentiation with retinoic acid has positive clinical results and is shown to induce structural changes in cells to allow for neurite outgrowth (Kunzler et al, 2016), which is why we chose this treatment as a positive control. A successful treatment also results in viability which is the ability of something to survive, grow and develop properly. Viability for cells like the IMR-32 cell line it can be determined after a staining agent assay (i.e. MTT or trypan blue) and simple absorbance measurements (Puty et al, 2021). Both of these characteristics can be telling signs of successful treatment.

Overall, the main objective of our study was to determine if IMR-32 cells treated with PAS could differentiate and be viable in order to support the evidence of PAS as a potential clinical treatment.

Due to the time constraint of this study there was not enough time to perfect all conditions, so in the future one could continue this study with more repetition to allow for even more optimal results. One method of perfecting our treatment to enhance neurodifferentiation and viability could be to look at the reactive oxygen species (ROS) present within the treated samples. In order to measure ROS in the cultures one could use test kits for hydrogen peroxide or nitrite, or for short-term ROS a spin trap or electron spin resonance spectrometer could be used (Tian et al, 2021). Low levels of ROS are ideal as this indicates no toxicity and would ensure viability of the samples. Another assay that was started but unsuccessful is looking at how the PAS affects cells with glutamate excitotoxicity. Overactivation of glutamate within the nervous system is associated with neurological disorders and can occur after neural injury, but CAP and PAS have shown promising neuroprotection to glutamate excitotoxicity (Tian et al, 2021). The goal of a study of this nature would be to induce this excitotoxicity to pre-incubated neural cells that have been treated with PAS and compare the viability to those that have not.

Big thanks to Michael Kong, Hai-Lan Chen, and Francis of the Frank Reidy Research Center for Bioelectrics at Old Dominion University for not only providing me with the opportunity to complete a research internship at their facility, but also taking me under their wing in teaching me a multitude of procedures to complete this research. I am truly grateful for the experience and have grown so much this past summer with their guidance!

Burdge, G. C., Rodway, H., Kohler, J. A., & Lillycrop, K. A. (2001). Effect of fatty acid supplementation on growth and differentiation of human

IMR‐32 neuroblastoma cells in vitro. Journal of cellular biochemistry, 80(2), 266-273.

Clementi, M. E., Tringali, G., Triggiani, D., & Giardina, B. (2015). Aloe arborescens extract protects imr-32

cells against alzheimer amyloid beta peptide via inhibition of radical peroxide production. Natural

Product Communications, 10(11). https://doi.org/10.1177/1934578x1501001147

Guo, L., Xu, R., Zhao, Y., Liu, D., Liu, Z., Wang, X.,Chen, H., & Kong, M. G. (2018). Gas plasma pre-treatment

increases antibiotic sensitivity and persister eradication in methicillin-resistant staphylococcus aureus. Frontiers

in Microbiology, 9. https://doi.or.3389/fmicb.2018.00537 g/10

Heymanns, J., & Unsicker, K. (1987). Neuroblastoma cells contain a trophic factor sharing biological and molecular properties with

ciliary neurotrophic factor. Proceedings of the National Academy of Sciences, 84(21), 7758-7762.

Keidar, M., Shashurin, A., Volotskova, O., Ann Stepp, M., Srinivasan, P., Sandler, A., & Trink, B. (2013). Cold

atmospheric plasma in cancer therapy. Physics of Plasmas, 20(5), 057101.

Kunzler, A., Zeidán-Chuliá, F., Gasparotto, J., Girardi, C. S., Klafke, K., Petiz, L. L., ... & Gelain, D. P. (2017).

Changes in cell cycle and up-regulation of neuronal markers during SH-SY5Y neurodifferentiation by retinoic acid are mediated by reactive species production and oxidative stress. Molecular neurobiology, 54(9), 6903-6916.

Laroussi, M., Kong, M. G., Morfill, G., & Stolz, W. (Eds.). (2012). Plasma medicine: applications of

low-temperature gas plasmas in medicine and biology. Cambridge University Press.

Puty, B., Bittencourt, L. O., Nogueira, I. C., Buzalaf, M. A., Oliveira, E. H., & Lima, R. R. (2021). Human cultured imr-32

neuronal-like and u87 glial-like cells have different patterns of toxicity under fluoride exposure. PLOS ONE, 16(6).

https://doi.org/10.1371/journal.pone.0251200

Rao, R. R., & Kisaalita, W. S. (2002). Biochemical and electrophysiological differentiation profile of a human

neuroblastoma (IMR-32) cell line. In Vitro Cellular & Developmental Biology-Animal, 38(8), 450-456.

Tian, M., Qi, M., Liu, Z., Xu, D., Chen, H., & Kong, M. G. (2021). Cold Atmospheric Plasma Elicits

Neuroprotection Against Glutamate Excitotoxicity by Activating Cellular Antioxidant Defense. Plasma

Chemistry and Plasma Processing, 1-10.

Wang, Y., Tan, H., & Hui, X. (2018). Biomaterial scaffolds in regenerative therapy of the central nervous system. BioMed research international, 2018.

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The task at hand was to determine if PAS could allow for adequate neurodifferentiation and neuroprotection in comparison to a known treatment of retinoic acid and a untreated cultures. Figure B’s differentiated images allowed us to narrow down our coating surface to Laminine due to the fact that the Fibronectin-coated cultures were more crowded, had less defined cell bodies, and also had somewhat of an incorrect morphology after the differentiation period. When comparing with undifferentiated cells, differentiation did successfully occur. According to another study, laminin is known to be an agent that promotes neurite outgrowth and survival (Heymanns & Unsicker, 1987). Figure C’s overlaid images depict that the PAS treated cells have comparable neurite outgrowth to the RA treated cells and much more outgrowth than the SHAM group, which is all promising, but to be sure we needed more quantitative data.

After a proliferation assay to determine viability we found that PAS3 seems to allow for the highest percentage of viability (<100%). PAS 1 and PAS 1:5 are a close second also with percentages around 100%, but in a previous experiment we found that diluted PAS treatments do not allow for the best differentiation. Viability percentages above 100% did occur in our study which could be due to cell stress or random experimental fluctuation. Our experimental conditions and focus groups were set, and after analyzing multiple images and 58 neurites for each treatment, Figure D was compiled in order to determine the overall success of neurodifferentiation. Both PAS groups had much more outgrowth than the SHAM group (about 40-50 micrometers on average with a large statistical difference) and PAS3 in particular did not have statistical difference from the RA treated group. Based on this data we can confirm that our PAS treatment did allow for neurodifferentiation and viability. While this treatment is still in its beginning stages and a long way from reaching the approved clinical treatment stage, our results back up the studies in support of this treatment.

Figure E: Average differentiation length in micrometers of neurites after 5 days of incubation (N=56 for each group) were measured with Image J software for various treatment groups. SHAM indicates the negative control, PAS1 indicates cells treated with PAS for 1 minutes, PAS3 indicates cells treated with PAS for 3 minutes, and RA indicates the positive control group of cells treated with retinoic acid. Error bars indicate the standard deviations, and starred bars show statistical significance based on a t-test(one star= p<0.05, two stars= p<0.01, three stars= p<0.001, and N.S.= no significant difference).

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Figure B: Images of two trials of differentiated cells were captured after seven days from the compound microscope using Infinity Lumenera software at 10x magnification. Images A and B are from Trial 1 and Images C and D are from Trial 2. Images A and C depict cells coated with Laminine and Images B and D depict cells coated with Fibronectin. * Image E depicts undifferentiated cells with normal culture medium for comparison.

Figure C: Images of differentiated cells were captured after five days from the fluorescence microscope using DAPI and GFP overlay at 10x magnification. Image A depicts untreated cells for the SHAM group, Image B depicts cells treated with PAS for one minute, Image C depicts cells treated with PAS for three minutes, and Image D depicts cells treated with RA for the positive control group.

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Figure D: Viability percentages were calculated following absorbance readings for four different PAS treated cultures with three samples averaged from each of the two wells per treatment type as indicated by the legend. Four control wells were used to calculate the viability of the cells based on the given formula. PAS 1 indicates cells treated with PAS for 1 minute, PAS 3 indicates cells treated with PAS for 3 minutes, PAS 1:5 indicates cells incubated with medium-diluted PAS at a 1:5 ratio for 1 hour, and PAS 1:10 indicates cells incubated with medium-diluted PAS at a 1:10 ratio for 1 hour.

*E