1. The Impact of Red Food Coloring on C2C12 Stem Cells Christian Ford Grade 12 Central Catholic High School PJAS 2015

2. C2C12 Stem Cells Subclone of the mus musculus (mouse) myoblast cell line. Mouse stem cell line is frequently used as a model in tissue engineering experiments. 1.Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. 2.Useful model to study the differentiation of non- muscle cells (stem cells) to skeletal muscle cells.

3. Stem Cell Lineage Every cell in the human body arises from stem cells. Example: Bone marrow-derived mesenchymal stem cells.

4. Blood Vessels Nerves…

5. Ingredients in McCormick Red Food Dye Water Propylene Glycol – Colorless and odorless. Miscible in water, acetone, and chloroform. 0.1% Propylparaben – Preservative used in cosmetics, pharmaceuticals and foods. Found in insects and plants, or synthetically made. Fd&C Red 40 Fd&C Red 3

6. Red Food Coloring Red 40 Allura Red AC Commonly used in processed food. Known to cause allergic reactions in some people Red 3 Erythrosine Mildly carcinogenic Not widely used since discovery of carcinogenic

7. Purpose The purpose of this experiment is to observe how Red Food Coloring effects the proliferation and differentiation of C2C12 Stem Cells

8. Null Hypothesis Red Food Coloring (Mixture of Red 40 and Red 3) will not have a significant effect on the proliferation and differentiation of C2C12 Stem Cells Alternative Hypothesis Red Food Coloring (Mixture of Red 40 and Red 3) will have a significant effect on the proliferation and differentiation of C2C12 Stem Cells

9. Materials Cryotank 75mm 2 tissue culture treated flasks Twenty 25 mm 2 tissue culture treated flasks Fetal bovine serum (FBS) C2C12 Myoblastic Stem Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (5 mL, 10, 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]) McCormick Red Food Coloring (Mixture of Red 40 and Red 3) 75 mL culture flask Incubator EVO Imaging System Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemacytometer Ethanol 70%

10. 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 cryo-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 5 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

11. Procedure (Addition of Variable on Day 0)  Proliferation  After trypsinization, cells from four normally populated flasks were pooled into 1 common 75mm 2 flask (cell density of between 2-4x10 5 cells/mL).  1 mL of the cell suspension was added to ten 25 mm 2 tissue culture treated flasks containing 4 mL of 10% DMEM media, creating a cell density of between 2-4x10 5 cells per flask.  Differentiation  After trypsinization, the reaction in a highly populated flask was stopped the the remaining 10 mL of the normally populated suspension to create a cell density of 0.5-1.0x10 6 cells/mL.  1 mL of the new cell suspension was added to ten 25 mm 2 tissue culture treated flasks containing 4 mL of 10% DMEM media, creating a cell density of between 0.5-1.0x10 6 cells per flask.  An unopened bottle of McCormick Red Food Dye was designated to be the 100% stock solution of Red Food Coloring.

12. Concentrations Control10^-6%10^-4%10^-2%1% Stock 0µL50µL @ 10^-4%50µL @ 10^-2%50µL @ 1%50µL Media 5mL4.950mL Total 5mL

13. Procedure (Addition of Variable on Day 0) (cont.)  The 1%, 10 -2 %, 10 -4 %, and 10 -6 % concentrations were created from the stock, through serial dilution.  The 4 experimental groups and the control group were created by adding:  20 µL of the 1% solution to 4 flasks  20 µL of the 10 -2 % solution to 4 flasks  20 µL of the 10 -4 % solution to 4 flasks  20 µL of the 10 -6 % solution to 4 flasks  20 µL of DMEM to 4 flasks (Control)  The cells were incubated at 37°C, 5% CO 2 for the remainder of the study.  Three flasks from each group were used in the Proliferation Experiment and two flasks from each group were used in the Differentiation Experiment.

14. Procedure (Proliferation Experiment) Day 1 and Day 3 Using one flask from each group, cell densities were determined as follows: The cells were trypsinized and collected into cell suspension. 20 µl aliquots were transferred to a Hemacytometer for quantification (six counts per flask). Day 3 Using the EVO Imaging System, images of representative areas of each flask were taken. Procedure (Differentiation Experiment)  Day 6 and Day 8  Using the EVO Imaging System, images of 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.

15. Statistical Analysis Analysis of Variance (ANOVA) Compares variation within groups to variation between groups If a P-Value less than.05 is generated (significant variation), the null hypothesis is rejected Dunnett’s Test Used to find which of the experimental groups significantly varies from the control. If the t-value found through the Dunnett’s test is higher than the t-critical the experimental group significantly varies from the control.

17. Day 1 and Day 3 Dunnett’s Tests GroupT-ValueT-CriticalInterpretation Day 1 10^-6%2.7912413.02Not Significant Day 1 10^-4%6.1233163.02Significant Day 1 10^-2%8.5416993.02Significant Day 1 1%6.577583.02Significant Day 3 10^-6%6.0892883.02Significant Day 3 10^-4%6.9059663.02Significant Day 3 10^-2%6.831033.02Significant Day 3 1%6.961973.02Significant

18. Differentiation Results Control10^-6%10^-4% 10^-2%1%

19. Conclusion Proliferation Based upon the ANOVA, graphic trends, and Dunnett’s Tests, it appears that Red Food Coloring has a significant negative effect on the proliferation of C2C12 stem cells at all concentrations expect 10^-6% on Day 1 Differentiation It appears that Red Food Coloring does have an effect (negative) on the myotube formation of C2C12 stem cells.

20. Future Changes Limitations Hemacytpometer counts in proliferation are subject to technique/clumping errors Differentiation test was qualitative Extentions Obtain LD-50 of Red Food Coloring Wider range of concentrations More cell types More replicates Quantitative proliferation (MTT assay, etc.)

21. References Mark Krotec, PTEI http://www.allergykids.com/blog/seeing-red/ http://fox2now.com/2014/01/08/mom-wants-artificial- food-coloring-removed-from-mms/ http://fox2now.com/2014/01/08/mom-wants-artificial- food-coloring-removed-from-mms/ http://www.easylunchboxes.com/blog/bye-bye-dye- healthier-alternatives-to-artificial-food-coloring/ http://www.easylunchboxes.com/blog/bye-bye-dye- healthier-alternatives-to-artificial-food-coloring/