Effect of Microwave Radiation on C2C12 Stem Cells

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Presentation transcript:

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

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

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

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/