The Effects of Aspartame on Mammalian Stem Cells Luca Consalvi 9th Grade Central Catholic High school

What are Stem Cells ? Stem Cells are unspecialized cells capable of renewing themselves through cell division. Under certain physiological or experimental conditions, they can be induced to become tissue-or organ- specific cells with specific functions.

C2C12 Stem Cells C2C12 cells differentiate rapidly, forming contractile myotubes and produces characteristic muscle proteins. They are Pluripotent cells.

An Overview of Aspartame Aspartame has been acclaimed to cause different forms of cancer varying from brain cancer, pancreatic cancer, leukemia,and lyphmonia. Aspartame is composed of a sucralose base, and has phylalanine as an ingredient, which is the main contributor to a genetic disorder called PKU.

Purpose The purpose of this experiment is to determine the effects that aspartame used in equal sweetener, will have on the survivorship and differentiation on C2C12 Stem Cells.

Hypotheses Null Hypothesis- Aspartame will not have an effect on the survivorship/ differentiation of the C2C12 Stem Cells Hypothesis- Aspartame will have an effect on the cell survivorship and differentiation on the myotubes of the cell

Materials Two 75mm culturing flasks Thirteen 25mm culturing flasks Trypsin Hemocytometers 10% diff media PBS P1000 pipette P100 pipette Sterile tips Micro pipette Macro pipettes Incubator Evos computer imaging system Laminar Sterilized hood Sterile gloves 70% ethanol Media C2C12 Cells Marker/pen and notebook

Procedure Cell Culturing 1. 1ml of C2C12 Stem Cells was aliquoted into a 75ml culturing flask 2. 15ml of media was added to the first flask 3. the media was removed from the first flask, in which 1.5ml of trypsin was added, to trypsonize the reaction. 4. The trypsonized cells were then moved from the first flask to the other in which 15ml of new media was added to eliminate any contamination. 5. 1ml of the trypsonized reaction in the 75ml flask was transferred into each of the 25 ml culturing flasks. 4ml of fresh media was added to each 25ml flask making the total concentration 5ml per flask. 6. The flasks were incubated for 24 hours. 7. After bring incubated for 24 hours the variable was added

Procedure continued 2. Cell Passing 1.The media was removed from each of the 75ml flasks 2. 2ml of trypsin was added to each flask to wash the surface 3. After trypsin was removed, 1ml of fresh trypsin was added each, this process was called trypsinization. 4. The flasks were incubated for 5 minutes 5. 1ml of the cell suspension was added to 12 25hat contained mm flasks 4ml of media, yielding a media of 5ml per flask.

Procedure continued 3. Stock variables 4. Counting 1. Two stock solutions were created. The High concentration stock (Stock A) was 0.02% of aspartame, in which 0.1 ml was taken out of (10% of the total stock) and put into the low concentration (Stock B) to produce a low concentration of 0.0002%. 4. Counting 1. Each of the 25ml culturing flasks were counted. 2. All of the media was removed 3. 1.5ml of trypsin was added and swirled around for 30 seconds, then removed. 4. another 1ml of trypsin was added, then the flasks was incubated for five minutes. 5. 0.20 microliters were removed, then put onto a hemocytometer for counting. Counts went as eight counts per flask.

Variable solutions High Concentration *Note all variable concentration before being diluted came in 1g packets 1gm/5ml(100x)=1gm/50ml 50ml/5ml=x x=0.02% for the high concentration Low Concentration 0.1ml of the High concentration was diluted 0.1ml/9.9m=x 50ml/5ml (1/100)= 0.0002% for the Low concentration

Data and Results Cell Quantity

Images (High Concentration flasks one, two, and three: Day one)

Images (Low Concentration flask one, two, and three: day two)

Images (Low Concentration flask one, two, and three day two)

Images (High Concentration Flask one, two, and three: day two)

Anova: Single Factor

Conclusion The data from the Anova suggests that the aspartame does have a significant effect on the survivorship and proliferation of the stem cells, since it’s p-value is 1.76E-10 whish is above the 0.5 cut off, leaving it outside of error. Therefore the Null hypothesis is to be rejected.

Limitation and Further Studies Future Studies Test greater concentrations of Aspartame Test the effects of aspartame on different cell lines, or types of stem cells Conduct the experiment over a longer period of time Test the ingredients of aspartame individually

Sources Harvard Medical School Dash Scholars program “The History of Aspartame” Donley, 2000 Johns Hopkins University Johns Hopkins Cancer Center American Cancer Society National Institute of Cancer