1. Effect of Microwave Radiation on C2C12 Stem Cells
Anthony Tirone
Grade 11
Central Catholic HS
Second PJAS

2. Overview of Stem Cells
Unspecialized cells that are capable of renewing themselves through cell division
Under certain physiological or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions

3. C2C12 Stem Cells
Subclone of the mus musculus (mouse) myoblast cell line
Immortalized
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins

4. Microwave Radiation Ranges from one meter to one millimeter
Studies suggest that long-term exposure can have a mutagenic effect
Frequency inside a microwave can reach 2.45 billion hertz
Frequency shown to start harming the human body is only 10 hertz

5. Microwave Radiation Exposure
Mobile phones use EMR in the microwave range
Wireless LAN protocols, such as Bluetooth emit microwave radiation
GPS receives signals via microwave radiation
Most objects involving wireless connection or transmission to satellites, other devices, etc.

6. Purpose
The purpose of this study is to observe the effects of varying amounts of microwave radiation on the proliferation, differentiation, and survivorship of C2C12 stem cells

7. Hypothesis
Null: Microwave Radiation will not have a significant effect on the proliferation, differentiation, and survivorship of C2C12 stem cells
Alternative: Microwave Radiation will have a significant effect on the proliferation, differentiation, and survivorship of C2C12 stem cells

8. Materials Cryotank 75mm2 tissue culture treated flasks
Twenty 25 mm2 tissue culture treated flasks
Fetal bovine serum (FBS)
C2C12 Myoblastic Stem Cell Line
Trypsin-EDTA
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])
Incubator
Nikon Inverted Microscope
Aspirating Vacuum Line
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing
Lamp
Labeling Tape
Hemocytometer
Sterile PBS
Ethanol (70% and 100%)
Distilled water
Emerson 1.6 cu ft 1100W Microwave Oven

9. Procedure
A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells
The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/flask was reached
The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2

10. Procedure (Continued)
After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/flask)
2 mL of the cell suspension was added to mm2 tissue culture treated flasks containing 5 mL of DMEM media, creating a cell density of approximately 105 cells per flask
The cells were incubated (37° C, 5% CO2) for one day to allow the cells to adhere to the flask
The cells were exposed to microwave radiation for times of 5, 10, and 15 seconds
The cells were incubated at 37°C, 5% CO2 for the remainder of the study
Two flasks from each exposure were used in the Proliferation Experiment and two flasks from each exposure were used in the Differentiation Experiment.

11. Procedure (Proliferation Experiment)
Day 1
Using one flask from each group, cell densities were determined as follows:
The cells were trypsinized and collected into cell suspension.
25 µl aliquots were transferred to a Hemocytometer for quantification (four counts per flask).
Day 1 and Day 3
Using the Nikon Inverted Microscope, images of eight representative areas of each flask were taken.
Procedure (Differentiation Experiment)
Day 1 and Day 11
Using the Nikon Inverted Microscope, images of eight representative areas of each of the flasks were taken.
Day 2
The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

12. Statistical Analyses of Proliferation Results
ANOVA
Compares variation within groups to variation between groups
Using the ANOVA, if a p-value less than the alpha of 0.05 is generated (significant variation), it suggests that the null hypothesis can be rejected
Dunnett’s Test
Compares each experimental group to control individually
A 0.05 alpha was used, and each generated T-value was compared to the T-critical value of 2.88

13. Results of C2C12 Proliferation Analysis
Day #
Day 1
Day 3
P - Value
6.28 E-23
2.42 E-28

14. Dunnett’s Test Results
Microwave
Exposure
T - Value
T - Crit
Variation
Day 1 -
5 seconds
10.834
2.88
Significant
10 seconds
23.805
15 seconds
31.563
Day 3
12.087
30.314
50.257

15. Differentiation Day 1
0 seconds
5 seconds
10 seconds
15 seconds

16. Differentiation Day 11
0 seconds
5 seconds
10 seconds
15 seconds

17. Conclusion
The null hypothesis is rejected for all exposure times as each one did significantly affect the proliferationof the stem cells
From the ANOVA and Dunnett’s tests, the exposure to microwave radiation induced a statistically significant decrease in proliferation in the C2C12 at all tested exposure times
From the qualitative analysis of the images gathered from the flasks, it appears that the exposure to microwave radiation inhibited myotubule formation. This was especially apparent as exposure times increased

18. Limitations & Extensions
Evaluation of differentiation images is qualitative and imprecise. A quantitative differentiation assay can be used, e.g. MyoD tagging.
CyQUANT™ Cell Proliferation Assay can be used. More quantitative than counting cells on a Hemocytometer. Fluorescent dye binds to nucleic acid in the cell.
Test additional microwave exposure lengths and intensities.

19. Sources & Acknowledgements
Mark Krotec, PTEI
C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression – German Sport University, Cologne, Germany
Liou, Kuo-Nan (2002). An introduction to atmospheric radiation. Academic Press. p. 2.