Magnetic Stress Remediation on Mammalian Cell Lines

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

2. Electromagnetic Effects On Life • Scientific studies • Variations in life forms exposed to electromagnets • Positive results others have shown harmful effects • Most show no results at all • Electromagnetic effects on life • Controversial • Ongoing discussion

3. Stressed Cells • Stressing a cell • 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 • Apoptosis • Programmed cell death • Cell’s response • 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 • More favorable • Still not proven to modern scientific researchers • Electromagnetic therapy • Varying magnetic fields 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 • 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: • “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 • Medical grade magnets • Alternating North-South Polarity Orientation (ANSPO) • Maximizes 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. • 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.) • 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 normal and UV stressed C2C12 cells.

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

12. Cryotank Three 75mm2 tissue culture treated flasks Twenty-four 25 mm2 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 Materials

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 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 cyro-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached. • The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2. Procedure (Proliferation Experiment) • After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 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 25mm2 culture flasks containing 4 mL of fresh media. • 1 mL aliquots from the initial cell suspension (non UV-stressed) were transferred to the twelve 25mm2 culture flasks.

14. 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% CO2), 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 hemacytometer for quantification. Experimental Groups

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 mm2 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 Zeis Inverted Compound Optical imaging system.

16. ProliferationStressed vs. Non stressedNo Magnets Unstressed Stressed- 60 Sec. UV Light P= 0.0009

17. ProliferationStressed + 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 significanteffect 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 and stress had no significant synergistic interaction and suggests the magnets had no significantremediation effects on the cells.

21. Differentiation Stressed vs. Unstressed

22. DifferentiationStressed + Magnets vs. Unstressed + Magnets

23. Differentiation Stressed vs. Stressed + Magnets

24. Stressed vs. Unstressed Appearance- Significant Stressed-Low density; Unstressed-high density+ myotube formation Stressed + Magnets vs. Unstressed + Magnets Appearance of Remediation Effects- Insignificant Stressed-Low density; Unstressed-high density+ myotube formation Stressed vs. Stressed + Magnets Appearance -ability of Magnets to help cells survive the stressor- Insignificant Apparent myotube formation was difficult to assess because the density was so low due to the stress Qualitative Summary of Differentiation

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

26. Limitations and Extensions • Limitations • Amount (length) of UV light stress • No clear quantitative measure of differentiation • Extensions • Quantitative measure of differentiation (ex. MyoD expression) • Different magnet strengths • Different types of stressors • C2C12 Migration on TE Scaffolds • CyQUANT™ Cell Proliferation Assay • More quantitative than counting cells on a Hemocytometer • Fluorescent dye binds to nucleic acid in the cell

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 • Trion:Z • Ramey DW. Magnetic and electromagnetic therapy. Scientific Review of Alternative Medicine 2(1):13-19, 1998 • Mayrovitz HN and others. Assessment of the short-term effects of a permanent magnet on normal skin blood circulation via laser-Doppler flowmetry. Scientific Review of Alternative Medicine 6(1):9-12, 2002. • Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University