Electromagnetic Field Effects on MG-63 Cell Lines Napat Kulruchakorn Pittsburgh Central Catholic Grade 11

Electromagnetic Field Magnetic Field produced by electrically charged objects Common in today’s environment from wireless waves produced by technological devices

Electromagnetic Field in a Solenoid Uniform throughout the structure Flows in one direction Highly concentrated within the coil

An Overview of Cancer Cells Cancer cells are cells that grow and divide at an irregular, unregulated pace. Apoptosis does not occur in cancerous cells; their mutations are passed on to the second generation, eventually clustering and forming tumors. Tumors can be malignant (aggressive) or benign.

MG-63 Cancer Cell Line Human cancer cell line Osteosarcoma cells, an aggressive form of bone cancer Useful model to test the effects of variables on cancer cell proliferation

Past Studies In cancer cells, the membrane potential level is significantly lower than that of healthy cells Cancer cells are more detached and do not exhibit contact inhibition of their growth Therefore, could charged particles within the membrane of the cancer cells be influenced by external electromagnetic forces?

Purpose To examine the effect of electromagnetic fields on MG-63 cell proliferation and survivorship

Hypotheses Null: The different strengths of electromagnetic fields will not have a significant effect on the proliferation and survivorship of the MG-63 cells. Alternative: The different strengths of electromagnetic fields will have a significant effect on the proliferation and survivorship of the MG-63 cells.

Materials The Solenoid: Insulated Copper Wire 6V Lithium Battery Water Bottle (for structure) Aluminium Foil Cable Zip Ties Multimeter

Materials (cont’d) Cryotank Fetal Bovine Serum (FBS) MG-63 Cell Line Trypsin-EDTA Pen / Strep 75 mm2 tissue culture treated flasks 25 mm2 tissue culture treated flasks Macropipette + Sterile Macropipette Tips (1 mL, 5 mL, 10 mL, 20 mL) Micropipette + Sterile Micropipette Tips DMEM Serum - 1% and Complete Media (4mM 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 Nikon Inverted Microscope with Imaging Technology Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Hemacytometer Sterile PBS Ethanol (70%) Ethanol (70%) Sharpie Pen Sterile Water Purple Nitrile Gloves

Procedure (Constructing the Solenoid) For each solenoid: A plastic water bottle was used as a structure. The bottle was wrapped with one layer of Aluminum Foil A 61-meter insulated copper wire was coiled 296 times around the circumference, creating a cylindrical structure The wire coil was held together with cable zip ties The batteries were prepared to meet their exact charges. Each would run out after 10 hours of exposure time.

Procedure (Cell Line Culture) A 1 mL aliquot of MG63 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/mL was reached The culture was passed into 8 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2

Procedure (Addition of Variable on Day 0) Cells from a T75 flask were resuspended after trypsinization to a density of approximately 300- 500K/mL. 4 mL of 10% DMEM media was added to each T25 flask 0.5 mL of cell suspension was transferred to 8 T25 flasks. Flasks were placed back into incubator and cells were allowed to attach for several hours

Procedure (Addition of Variable on Day 0) Each end of the insulated copper wire was connected to the cathodes and anodes of the 6V and 12V batteries under a sterile environment. T25 flasks and solenoids were incubated at 37C and 5% CO2.

Procedure (Cell Counts) Day 1 and Day 3 Cell densities were determined as follows: The cells were trypsinized and collected into cell suspension. 20 μl aliquots were transferred to a Hemocytometer for quantification (eight total counts).

Strength of Magnetic Field Field Strength Levels Turn Density Strength of Magnetic Field Current Permeability (Air) = 4π × 10-7

Field Strength Levels High (12V) Low (6V) Control Tesla Gauss 2.2282 Gauss 2.2282 1.1141 2.2282 × 10−4 1.1141 × 10−4

Electromagnetic Field Effects on MG-63 Proliferation P-Value Day 1: 3.8x10-12 P-Value Day 3: 6.1x10-10

Dunnett’s Test Day 1 Day 3 Field Strength T Value T Crit Results High 19.3896 2.88 Significant Low 10.4769 Day 1 Field Strength T Value T Crit Results High 11.6699 2.88 Significant Low 6.7817 Day 3

Conclusion The null hypothesis was rejected. The data from Day 1 and Day 3 strongly suggested that both low and high strength levels of electromagnetic field have significant effects on the survivorship and proliferation of MG- 63 cells.

Limitations The batteries needed to be small enough to fit in the incubator, resulting in short exposure time Only two different strength levels tested Only one cell line used Low number of flasks Cell health not observed Cell counts can vary

Project Improvements An alternative power source that would allow longer exposure time More flasks Use multiple cell lines More strength levels of EM fields

Citations / Acknowledgements Mark Krotec, PTEI Bob Leonard Presman, A. S. Electromagnetic Fields and Life. New York: Plenum, 1970. Print. Steve Haltiwanger M.D., C.C.N. The Electrical Properties of Cancer Cells (2002): 216-24. Web. Cone CD. The role of surface electrical transmembrane potential in normal and malignant mitogenesis. Ann NY Acad Sci 1975;238:420-35.

ANOVA - Day 1 Analysis of Variance (One-Way) Summary Groups Sample size Sum Mean Variance Low 8 21,120,000 2,640,000 3.06257E+11 High 984,000 123,000 1,000,285,714.28571 C 44,790,000 5,598,750 6.49784E+11 ANOVA Source of Variation SS df MS F p-level F crit Between Groups 1.20196E+14 2 6.00978E+13 188.38613 3.86357612569555E-12 3.4668 Within Groups 6.69929E+12 21 3.19014E+11 Total 1.26895E+14 23

ANOVA - Day 3 Analysis of Variance (One-Way) Summary Groups Sample size Sum Mean Variance Low 8 24,030,000 3,003,750 2.02998E+11 High 633,000 79,125 951,267,857.14286 C 56,490,000 7,061,250 4.09164E+12 ANOVA Source of Variation SS df MS F p-level F crit Between Groups 1.96711E+14 2 9.83557E+13 68.69072 6.11515E-10 3.4668 Within Groups 3.00691E+13 21 1.43186E+12 Total 2.26781E+14 23

Concentrations (cells / mL) Day 1 Low High Control 1950000 144000 4320000 2160000 69000 6600000 3600000 129000 5490000 2760000 135000 4680000 3060000 6210000 2820000 99000 5850000 2670000 102000 6330000 2100000 171000 5310000 Day 3 Low High Control 3300000 105000 4530000 3270000 129000 4920000 2700000 30000 8190000 3750000 90000 9300000 2610000 51000 5340000 3120000 81000 6450000 2940000 66000 9720000 2340000 8040000