1. The Effects of UVC Light on C2C12 Stem Cells Cory Soltys Pittsburgh Central Catholic High School Grade 12 PJAS 2015

2. Ultraviolet Rays  Light waves that have shorter wavelengths, thus greater energy, than visible light  Range from 400nm to 100nm  Given off by the sun but most are absorbed by the ozone layer

3. Ultraviolet-C Light  Shorter wave than UVA or UVB light  Typically range around 280nm-100nm  Completely absorbed by the ozone layer and atmosphere  Commonly used for Ultraviolet Germination Irradiation (UVGI)  Disinfection method involving the use of UVC light to kill microorganisms

4. Damage Caused by UV Light  Damage includes skin burn, sun poisoning, skin irritation, redness, photo- aging, nausea, and possibly skin cancer  FDA Protection methods include sun screen, hats, and radiation-blocking clothing  Can cause DNA to form dimers, leading to replication errors, mutations

5. C2C12 Stem Cell Line  Derived from the mus musculus (mouse) myoblast cell line.  A model type of stem cell line that was discovered in 1977 through experimentation of murinae thigh muscle growth after a crush injury.  Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

6. Purpose  The purpose of this experiment was to test the effects of Ultraviolet-C light radiation on C2C12 Stem Cells

7. Hypotheses  Null Hypothesis- Ultraviolet-C light radiation will NOT have a significant effect on C2C12 cell proliferation and differentiation  Alternative Hypothesis- Ultraviolet-C light radiation WILL have a significant effect on C2C12 cell proliferation and differentiation

8. Materials  Cryotank  Four 75mm 2 tissue culture treated flasks  Twelve 25 mm 2 tissue culture treated flasks  Fetal bovine serum (FBS)  C2C12 Myoblastic Stem Cell Line  Trypsin-EDTA  Pen/Strep  Power macropipette  Sterile macropipette tips (5mL, 10mL)  Incubator

9. Materials (continued)  Inverted microscope  Laminar flow hood  Laminar flow hood UV Sterilizing Lamp  Hemocytometer  Ethanol (70%)  Nitrile gloves  DMEM Media - 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])  Micropipettes and sterile tips

10. Procedure  The culture of cells was passed into four 75mm 2 flasks in preparation for experiment and incubated for 2 days at 37° C.  After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL).  0.1mL of the cell suspension pool was added to each of the eight proliferation 25mm 2 tissue culture treated flasks containing 5mL of DMEM media, creating a lower concentration for cell counting  0.2mL of the cell suspension pool was added to each of the four differentiation 25mm 2 tissue culture treated flasks containing 5mL of DMEM media, creating a higher cell density for more accurate and effective imaging

11. Procedure (continued)  Cell cultures (Flasks) were exposed to UVC radiation (laminar flow tissue culture hood) for the following time intervals: control (0 minutes), 2 minutes, 5 minutes, 10 minutes with three replicates for each duration  Cells were trypsinized prior to counting to release them from the bottom of the flask. 0.5mL of trypsin was inserted into each flask and allowed to incubate for five minutes and 2mL of media was added to stop the reaction.  Twenty μL of trypsinized cells were loaded into hemocytometers. Eight cell counts were performed per group.

12. Hemocytometer Picture

13. P-Value=2.0759E-12

14. Day 1 Dunnett’s Test ComparisonSignificant?T-Value Control vs. 2 MinutesNo2.107 Control vs. 5 MinutesYes8.181 Control vs. 10 MinutesYes14.842 T-Crit= 2.88

15. P-Value=4.86278E-14

16. Day 3 Dunnett’s Test ComparisonSignificant?T-Value Control vs. 2 MinutesYes10.519 Control vs. 5 MinutesYes17.887 Control vs. 10 MinutesYes23.446 T-Crit=2.88

17. Day 1 Differentiation Control 2 Minutes

18. Day 1 Differentiation (continued) 5 Minutes 10 Minutes

19. Conclusion  Proliferation  Based on ANOVA and Dunnett's statistical analysis, the null hypothesis has been rejected. The UVC light radiation had a significant negative effect on the proliferation of the C2C12 stem cell line  Differentiation  It appears that the addition of extreme amounts (10 minutes) of Ultraviolet-C radiation has a significant negative effect on the myotube differentiation of C2C12 stem cells.

20. Limitations  Use of plastic flasks (block radiation)  Hemocytometer counts  Differentiation test is qualitative 20

21. Future Extensions  Use well plates in place of plastic flasks  Multiple cell lines  More time durations 21

22. Day 1 Counting Results Control2 Minutes5 Minutes10 Minutes 594572381335 564557397362 580562451408 564548403339 584521290283 651572319311 607560346323 596512344327

23. Day 3 Counting Results Control2 Minutes5 Minutes10 Minutes 1392634250174 1033602252178 1566656235165 1233627261171 1440698266162 1406502195159 961531210177 1021576237154

24. Day 1 ANOVA

25. Day 3 ANOVA

26. References  Mark Krotec, PTEI  http://www2.centralcatholichs.com/biology/ http://www2.centralcatholichs.com/biology/ 26