Effect of Microwave Radiation on C2C12 Stem Cells

Anthony TironeGrade 11

Central Catholic HSSecond PJAS

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

C2C12 Stem Cells

• Subclone of the mus musculus (mouse) myoblast cell line

• Immortalized • Differentiates rapidly,

forming contractile myotubes and produces characteristic muscle proteins

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

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.

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

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

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• Hemocytometer

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

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 20 25 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.

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.

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

Results of C2C12 Proliferation Analysis

Control 5 second 10 second 15 second0

100000

200000

300000

400000

500000

600000

Day 1Day 3

Microwave Exposure Length

Cell

Coun

t (Ce

lls P

er F

lask

)

Day # Day 1 Day 3P - Value 6.28 E-23 2.42 E-28

Dunnett’s Test Results

MicrowaveExposure

T - Value T - Crit Variation

Day 1 - - -

5 seconds 10.834 2.88 Significant10 seconds 23.805 2.88 Significant

15 seconds 31.563 2.88 Significant

Day 3 - - -5 seconds 12.087 2.88 Significant

10 seconds 30.314 2.88 Significant

15 seconds 50.257 2.88 Significant

Differentiation Day 1

0 seconds

5 seconds

10 seconds

15 seconds

Differentiation Day 11

0 seconds

5 seconds

10 seconds

15 seconds

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

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.

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.

• http://www.fda.gov/radiation-emittingproducts/• https://www.osha.gov/SLTC/radiofrequencyradiation/