1. Magnetic Stress Remediation on Mammalian Cell Lines J.R. Stragar Grade 11 Pittsburgh Central Catholic High School

2. Electromagnetic Effects On Life Numerous scientific studies have shown variations in life forms (i.e. plants) that have been exposed to electromagnets. Some of these studies have favored positive results while others have clearly shown harmful effects. Electromagnetic effects on life is a very controversial and ongoing discussion in modern science.

3. Stressed Cells Stressing a cell is defined as interfering with the cell’s ability to carry out basic functions. Stresses include: prolonged exposure to heat, deprivation of food, pollutants, various chemicals, infection,UV radiation. Some cells respond to stress through apoptosis. UV light exposure to cells was used to stress cells in this experiment.

4. Magnet Therapy Practitioners claim that subjecting certain parts of the body to magnetic fields have beneficial health effects. The magnetic therapy industry > $300 million U.S. Magnet therapy items include: magnetic bracelets, jewelry, magnetic straps for wrists and ankles, back magnets, shoe insoles, mattresses, blankets, and even magnetized water.

5. Magnetic Therapy in Modern Science Magnet therapy is generally considered pseudoscience by modern scientific standards. Researchers at the VA Medical Center in Prescott, Arizona conducted a randomized, double-blind, placebo-controlled, crossover study involving 20 patients with chronic back pain. Patients were exposed to real and sham bipolar permanent magnets during alternate weeks, for 6 hours per day, 3 days per week for a week, with a 1-week period between the treatment weeks. No difference in pain or mobility was found between the treatment and sham-treatment periods. Electromagnetic therapy is considerably more favorable, but still has not been proven to modern scientific researchers. Electromagnetic therapy uses varying magnetic fields to apply energy to the body as opposed to the static fields of normal magnetic therapy.

6. Electromagnetic Therapy in Modern Medicine The University of California Medical Center, Moore Cancer Center, offers electromagnetic treatments to cancer patients. However, the UC Moore Cancer Center clearly states that "there is no scientific evidence available that any electromagnetic therapies work." The American Cancer Society says that "relying on electromagnetic treatment alone and avoiding conventional medical care may have serious health consequences." Electromagnetic devices have never been scientifically proven in the treatment of disease.

7. Trion:z Electromagnetic Therapy Trion:Z uses medical grade magnets in a unique Alternating North-South Polarity Orientation (ANSPO) to maximize magnetic field flow. Trion:Z says “this ANSPO orientation increases the effective area of the magnetic field, and penetrates deeper into tissue fibers and muscle.” According to this quote, the Trion:Z magnets are more able to effect the tissue and muscle cells in the body.

8. C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line. The C2C12 cell line differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. Mouse stem cell line is used as a model in many tissue engineering experiments.

9. C2C12 Cell Line (contd.) C2C12 cells are a useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells. Expresses muscle proteins and the androgen receptor (AR).  AR- DNA binding transcription factor which regulates gene expression.

10. Purpose To examine the effects of Trion:Z magnets on the proliferation, differentiation, and survivorship of the C2C12 mammalian cell line.

11. Hypotheses The Trion:Z magnets will have a remediation effect on the survivorship, proliferation, and differentiation of the C2C12 cell line exposed to UV radiation. The null hypothesis is that the Trion:Z magnets will have no effect on the survivorship, proliferation, or differentiation of the C2C12 cell line.

12. Materials Cryotank Three 75mm 2 tissue culture treated flasks Twenty-four 25 mm 2 tissue culture treated flasks 10% fetal bovine serum C2C12 Myoblastic Stem Cell Line Trypsin-EDTA Pen/strep 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]) 75 mL culture flask Incubator Zeis Inverted Compound Optical Scope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Sterile 60x100 mm Petri dish 48 Trion:Z Magnets Labeling Tape 12 Microtubes Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water Toluidine Blue stain

13. Procedure (Stem Cell Line Culture) A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells. The media was replaced with 15 mL of fresh media to remove cyro- freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached. The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2. Procedure (Proliferation Experiment) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 7 mL of cell suspension was transferred into a sterile 60x100 mm Petri dish within a laminar flow hood. The lid was removed and the cells were subjected to 60 seconds of UV light. 1 mL aliquots of cell suspension were transferred to 12 25mm 2 culture flasks containing 4 mL of fresh media. 1 mL aliquots from the initial cell suspension (non UV-stressed) were transferred to the twelve 25mm 2 culture flasks.

14. Experimental Groups Procedure (Proliferation Experiment contd.) 1 Trion:z magnet was attached to each side of the respective flask using tape. The cells were incubated (37°C, 5% CO 2 ), and cell densities were determined at day 3 and day 6 as follows:  The cells were trypsinized and collected into cell suspension.  50 µl aliquots were transferred to a hemocytometer for quantification. UV StressedUnstressed Magnets44 Non Magnets44

15. Procedure Serum Starvation (Differentiation) The differentiation experiment was identical to the proliferation experiment with the following exceptions:  After the first day of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation. Procedure (Cell Fixing and Staining) The cells were fixed and stained in preparation for photo microscopy on day 8 as follows:  The media from each 25 mm 2 cell culture flask was removed and 2 mL of Sterile PBS was added to each flask, swirled around, and removed.  2 mL of ice cold 100% ethanol was added to each flask, swirled and removed.  The excess ethanol was allowed to evaporate for 5 minutes.  1 mL of Toluidine Blue stain was added to each flask followed by a rinse with distilled water. Photomicrographs were taken using a _____ optical imaging system.

16. Proliferation Stressed vs. Unstressed No Magnets Stressed- 60 Sec. UV Light Unstressed P= 0.0009

17. Proliferation Stressed + Magnets vs. Unstressed + Magnets P=0.001

18. Proliferation Stressed vs. Stressed + Magnets P=0.82

20. Summary of Proliferation Data Pair wise ANOVA Single Factors- Stress vs. Unstressed P= 0.07, 0.007, 0.0009, and 0.001. All P values <.01- Stress had a significant effect on the survivorship of the cells. Double Factor ANOVA with replication Sample P value -Stressed vs. Stressed + Magnets AND Unstressed vs. Unstressed + Magnets- P= 0.66 and 0.08. Magnets had no significant effect on the survivorship of the cells. Double Factor ANOVA with replication comparing the interaction of both variables on the survivorship and remediation of the cells. P= 0.30 and 0.07. Magnets had no significant remediation effects on the cells.

21. Differentiation Stressed vs. Unstressed

22. Differentiation Stressed + Magnets vs. Unstressed + Magnets

23. Differentiation Stressed vs. Stressed + Magnets

24. Qualitative Summary of Differentiation Stressed vs. Unstressed  Appearance- Significant Stressed + Magnets vs. Unstressed + Magnets  Appearance of Remediation Effects- Insignificant Stressed vs. Stressed + Magnets  Appearance -ability of Magnets to help cells survive the stressor- Insignificant

25. Conclusion The evidence (P= >.05) strongly supports the null hypothesis. The alternative hypothesis is rejected. The Trion:Z magnets had no significant effects on the survivorship, proliferation, or differentiation of the C2C12 cell line.

26. Limitations and Extensions

27. Sources Collacott EA and others. Bipolar permanent magnets for the treatment of chronic low back pain. JAMA 283:1322- 1325, 2000. - VA Medical Center in Prescott, Arizona