Luca Consalvi 9 th 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 tissueor organ specific cells wit ID: 775048
Download Presentation The PPT/PDF document " The Effects of Aspartame on Mammalian S..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
The Effects of Aspartame on Mammalian Stem Cells
Luca Consalvi
9
th
Grade
Central Catholic High school
Slide2What 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.
Slide3C2C12 Stem Cells
C2C12 cells differentiate rapidly, forming contractile
myotubes
and produces characteristic muscle proteins.
They are Pluripotent cells.
Slide4An 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.
Slide5Purpose
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.
Slide6Hypotheses
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
Slide7Materials
Two 75mm culturing flasks Thirteen 25mm culturing flasksTrypsin Hemocytometers 10% diff media PBS P1000 pipette P100 pipette Sterile tipsMicro pipetteMacro pipettes IncubatorEvos computer imaging system
Laminar Sterilized hood
Sterile gloves
70% ethanol
Media
C2C12 Cells
Marker/pen and notebook
Slide8Procedure
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
Slide9Procedure 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.
Slide10Procedure continued
3. Stock variables
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
Slide13Images (High Concentration flasks one, two, and three: Day one)
Slide14Images (Low Concentration flask one, two, and three: day two)
Slide15Images (Low Concentration flask one, two, and three day two)
Slide16Images (High Concentration Flask one, two, and three: day two)
Slide17Anova: Single Factor
Slide18Conclusion
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.
Slide19Limitation and Further Studies
Limitation
Future Studies
Test
greater concentrations of
Aspartame
T
est
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
Slide20Sources
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