1. Brandon Perrotte Grade 11 Pittsburgh Central Catholic High School Effects of UV Radiation on C2C12 Cells

2. A Brief History of UV Radiation Ultra Violet light is the invisible light that exists just beyond the shortest light rays (violet) in the visible light spectrum. In 1801, Johann Wilhelm Ritter, a German physicist, discovered that these invisible lights rays were very effective at darkening silver chloride soaked paper, and named them, "deoxidizing rays“. The interesting paradox about the former term for ultraviolet light, "chemical rays", is that Ultraviolet C, light wave technology actually offers a chemical-free way in which to sanitize and safely de-germ a variety of industrial and household surfaces.

3. Background Information and Household Applications Natural ultraviolet light is emitted as UVA, UBC and UVC light. 98.7% of that light is absorbed by the earth's atmosphere. UVC light waves, which are the shortest and most powerful in the UV range, have germicidal properties, and are capable of breaking down the DNA of microorganisms such as viruses, bacteria, dust mites and other allergens and contaminants. UVC light is used to sanitize surfaces and tools in medical labs and to sanitize water in water treatment plants. Many spring water bottlers now utilize UVC light to disinfect bottles and sterilize equipment and increasingly, public water treatment facilities are utilizing UVC technology to upgrade safety standards for drinking water.

4. Radiation Therapy in Production The Halo UVX Sanitizing Vaccuum and the Verilux UVC Sanitizing Wand are 2 products currently available to consumers that can safely eliminate invisible allergens and contaminants in areas that chemical cleaners would be completely unwelcome.

5. Clinical Applications of UV Radiation Conditions Hypertension hypertensive crises angina pectoris chronic arthritis erectile dysfunction cerebral ischemia chronic skin ulcers Treatment: Administering to the patient a therapeutically effective amount or dose, or series of amounts or doses, of ultraviolet light.

6. Stressed Cells Stressing a cell Interfering with the cell’s ability to carry out basic functions. Stresses include: prolonged exposure to heat deprivation of food pollutants various chemicals infection UV radiation Apoptosis Programmed cell death Cell’s response UV light exposure to cells was used to stress cells in this experiment.

7. C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. Mouse stem cell line is used as a model in many tissue engineering experiments.

8. C2C12 Cell Line (contd.) Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells. Expresses muscle proteins and the androgen receptor (AR). AR- DNA binding transcription factor which regulates gene expression.

9. Purpose To examine the effects of ultraviolet radiation on the survivorship, proliferation, and differentiation of C2C12 Cells.

10. Hypotheses The increasing amounts of UV Radiation will have a significant effect on the proliferation, differentiation, and survivorship of C2C12 cells. The null hypothesis is that the UV radiation will not have any significant effect on the proliferation, differentiation, or survivorship of the C2C12 cells.

11. Materials Cryotank Three 75mm 2 tissue culture treated flasks Twenty-four 25 mm 2 tissue culture treated flasks 10% fetal bovine serum C2C12 Myoblastic Stem Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM Serum -1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) 75 mL culture flask Incubator Zeis Inverted Compound Optical Scope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Sterile 60x100 mm Petri dish Labeling Tape Hemocytometer Ethanol (70% and 100%) Distilled water

12. Procedure (Stem Cell Line Culture) A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached. The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2. Procedure (Proliferation Experiment) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 7 mL of cell suspension was transferred into a sterile 60x100 mm Petri dish within a laminar flow hood. 1 mL aliquots of cell suspension were transferred to 24 25mm 2 culture flasks containing 4 mL of fresh media. 1 mL aliquots from the initial cell suspension (non UV-stressed) were transferred to the 24 25mm 2 culture flasks. The lid was removed and the cells were subjected to 0, 20, 40, and 60 seconds of UV light respectively.

13. Experimental Groups Vitamin C [ ]UV Light Exposure 03060 0.5 ml Sterile water, 4.5 ml cells.5 ml Sterile water, 4.5 ml cells.5 ml Sterile water, 4.5 ml cells 0.05 4.5ml cells,.o5 Vitamin C,.45 ml Sterile Water 0.5 4.5 ml cells,.5 Vitamin C Procedure (Proliferation Experiment contd.) Different concentrations of 10% Vitamin C were then added to determine if the antioxidant would relieve the oxidative stress. The cells were incubated (37°C, 5% CO 2 ), and cell densities were determined at Days 1,2,4 and 6 as follows: The cells were trypsinized and collected into cell suspension. 50 µl aliquots were transferred to a hemacytometer for quantification.

14. Procedure Serum Starvation (Differentiation) The differentiation experiment was identical to the proliferation experiment with the following exceptions: After the first day of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

15. Proliferation Stressed(20 sec. Exposure) Vs. Unstressed Days 2, 4, 6 All Pictures were taken at 10x Magnification. P-value: 0.107074 P-value: 0.001071 P-value: 0.000495

16. Proliferation Stressed(40 sec. Exposure) Vs. Unstressed

17. Proliferation Stressed(60 sec. Exposure) Vs. Unstressed

18. C2C12 Cell Proliferation 0 sec. 20 sec. 40 sec. 60 sec. Exposure to UV Light Y-axis= Number of cells x 10^3 Series 1= Day 0; Series 2= Day 2; Series 3= Day 4; Series 4=Day 6

19. Summary of Proliferation Data ANOVA Statistical Analysis Stressed (20 sec.) Stressed (40 sec.) Stressed (60 sec.) Day 2 0.1070740.0008518.2E-08 Day 4 0.0010718.47E-063.29E-06 Day 6 0.0004952.6E-085.53E-09

20. Differentiation UV Stressed(20 second exposure) vs. Unstressed Days 2, 4, 6

21. Differentiation UV Stressed(40 second exposure) vs. Unstressed

22. Differentiation UV Stressed(60 second exposure) vs. Unstressed

23. Qualitative Summary of Differentiation Stressed(20 sec.) vs. Unstressed Appearance- Insignificant Stressed-Low density; Unstressed-high density+ myotube formation Stressed(40 sec.) vs. Unstressed Appearance -Significant Stressed-Low density; Unstressed-high density+ myotube formation. The survivorship of the C2C12 cells seems to sharply decrease. Stressed(60) vs. Unstressed Appearance -Significant Apparent myotube formation was difficult to assess because the density was so low due to the stress. Stressed + Vitamin C vs. Unstressed +Vitamin C: Appearance -ability of Vitamin C to help cells survive the stressor- Significant Apparent myotube formation was again difficult to assess because the density was so low due to the stress. The Vitamin C, even in the smallest quantities, had an adverse or toxic effect on the C2C12 cell populations.

24. Conclusion The evidence (p-value F- critical) strongly supports the hypothesis. The null hypothesis is rejected. The increasing amounts of UV Radiation had a significant effect on the proliferation, differentiation, and survivorship of C2C12 cells.

25. Limitations and Extensions Limitations Amount (length) of UV light stress No clear quantitative measure of differentiation Extensions Quantitative measure of differentiation (ex. MyoD expression) Different types of stressors C2C12 Migration on TE Scaffolds CyQUANT™ Cell Proliferation Assay More quantitative than counting cells on a Hemocytometer Fluorescent dye binds to nucleic acid in the cell

26. Sources Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University John Wilson, Biostatistician for the University of Pittsburgh Mark Krotec, PTEI http://lpi.oregonstate.edu/infocenter/vitamins/vitaminC / http://earthobservatory.nasa.gov/Features/UVB/ www.stanford.edu/group/blau/protocols/c2lineproto col.html http://www.physoc.org/publications/proceedings/archiv e/article.asp?ID=Proc%20Physiol%20Soc%203PC125