effect of microwave radiation on c2c12 stem cells anthony tirone grade 11 central catholic hs second...
TRANSCRIPT
![Page 1: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/1.jpg)
Effect of Microwave Radiation on C2C12 Stem Cells
Anthony TironeGrade 11
Central Catholic HSSecond PJAS
![Page 2: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/2.jpg)
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
![Page 3: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/3.jpg)
C2C12 Stem Cells
• Subclone of the mus musculus (mouse) myoblast cell line
• Immortalized • Differentiates rapidly,
forming contractile myotubes and produces characteristic muscle proteins
![Page 4: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/4.jpg)
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
![Page 5: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/5.jpg)
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.
![Page 6: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/6.jpg)
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
![Page 7: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/7.jpg)
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
![Page 8: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/8.jpg)
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
![Page 9: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/9.jpg)
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
![Page 10: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/10.jpg)
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.
![Page 11: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/11.jpg)
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.
![Page 12: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/12.jpg)
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
![Page 13: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/13.jpg)
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
![Page 14: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/14.jpg)
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
![Page 15: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/15.jpg)
Differentiation Day 1
0 seconds
5 seconds
10 seconds
15 seconds
![Page 16: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/16.jpg)
Differentiation Day 11
0 seconds
5 seconds
10 seconds
15 seconds
![Page 17: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/17.jpg)
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
![Page 18: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/18.jpg)
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.
![Page 19: Effect of Microwave Radiation on C2C12 Stem Cells Anthony Tirone Grade 11 Central Catholic HS Second PJAS](https://reader033.vdocuments.mx/reader033/viewer/2022051619/56649e2f5503460f94b1f915/html5/thumbnails/19.jpg)
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/