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

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

Stressed Cells Stressing a cell Stresses include: Apoptosis 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.

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.

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 Varying magnetic fields apply energy to the body as opposed to the static fields of normal magnetic therapy.

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.

Trion:z Electromagnetic Therapy 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.

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.

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.

Purpose To examine the effects of Trion:Z magnets on the proliferation, differentiation, and survivorship of normal and UV stressed C2C12 cells.

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 normal or stressed C2C12 cells.

Materials 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

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.

Procedure (Proliferation Experiment contd.) Experimental Groups UV Stressed Unstressed Magnets 4 No Magnets 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.

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.

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

Proliferation Stressed + Magnets vs. Unstressed + Magnets

Proliferation Stressed vs. Stressed + Magnets

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 and stress had no significant synergistic interaction and suggests the magnets had no significant remediation effects on the cells.

Differentiation Stressed vs. Unstressed

Differentiation Stressed + Magnets vs. Unstressed + Magnets

Differentiation Stressed vs. Stressed + Magnets

Qualitative Summary of Differentiation Stressed vs. Unstressed Appearance- Significant Stressed-Low density; Unstressed-high density+ myotube formation 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 Apparent myotube formation was difficult to assess because the density was so low due to the stress

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.

Limitations and Extensions 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

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