1. L-Theanine Effects on C2C12 Stem Cells
By: Sonali Dadoo
School: Pine-Richland High School
Grade: 10
Category: Biology
L-Theanine Effects on C2C12 Stem Cells

2. Tissue Engineering (TE)
TE is the science concerned with improving, restoring, or maintaining the quality of life via artificially created tissues, organs, and other genetically engineered biological substitutes.
TE revolves around three main aspects: cells, scaffolds, and signals
Experimental focus – muscle stem cell proliferation and differentiation
A porous scaffold is injected with cells and signals to allow cells to differentiate and become specialized

3. Phil Campbell, Carnegie Mellon
Principles of Tissue Engineering
Cells
ECM
Defect
Regeneration
TE scientists combine some of the following components in their research. TE scientists are working hard to determine what is the most effective type of cell for a given tissue. They also must determine what type of scaffold or environment would best support seeded stem cells. Furthermore, they must find a signaling molecule that is best suited for the particular stem cell line. This may be a growth factor or a hormone. Finally, TE scientists must ensure proper angiogenesis which is proper supply of blood to new engineered tissue
Blood
Supply
Hormones
Phil Campbell, Carnegie Mellon

4. Stem Cells
Un-specialized cells that can differentiate into various body tissues and organs
Have potential to cure or treat diseases such as heart disease, and diabetes

5. C2C12 Stem Cell Line
Pluripotent myoblastic line which derived from mice; Subclone of the mus musculous (mouse myoblast) stem cell line
Differentiate rapidly to form myotubes, the cells that make up the myofibers of muscle tissue.
A useful model to study the differentiation from stem cells to mature skeletal muscle cells and tissues
Used in tissue engineering experiments to test for proliferation, migration, attachment, and differentiation
Humans consume many different types of products. Scientists are concerned with the ways in which these products alter cells in the body or power cells such as stem cells

6. Variable: L-Theanine Why L-Theanine? commonly ingested in green tea
Ongoing research into its effects

7. Variable: L-Theanine What is L-Theanine?
Amino acid found in tea leaves
Theorized Uses
Relaxant – alpha brain waves, binds glutamate receptors
Stroke preventative
Cancer treatment, mainly in animals
Lowers blood pressure
L-Theanine is most commonly used as a relaxant. Contrary to popular belief, L-Theanine does not make the user drowsy, it just returns the person to a calm and relaxed state which can be attained by sleeping or meditating.
cancer: increase antitumor activity of chemotherapy

8. Purpose
To examine the effect(s) of L-Theanine on proliferation, survivorship, and differentiation of C2C12 murine adult stem cells .

9. Hypotheses Null Hypothesis
The addition of the L-Theanine will not affect the proliferation, differentiation, and survivorship of the C2C12 stem cells.
Alternate Hypothesis
The addition of the L-Theanine will significantly reduce the proliferation, differentiation, and survivorship of C2C12 stem cells.

10. Materials Cryotank Ethanol (70% and 100%)
One 75mm2 tissue culture treated flasks
Distilled water
Nikon Inverted Microscope
Six 25 mm2 tissue culture treated flasks
10% fetal bovine serum
Hemocytometer
C2C12 Myoblastic Stem Cell Line
Permanent marker
Trypsin-EDTA
24 well plate
Pen/strep
Test tube rack
Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)
Sterile Filter
Lint wipes
Micropipettes + sterile tips
L-Theanine Powder
DMEM media -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])
Incubator
Aspirating Vacuum Line
Nikon Inverted Compound Optical Scope
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Labeling Tape
Sterile PBS

11. Procedure: Preparing the Cells
A 1 mL sample of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask.
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 106 to 2x106 cells/mL was reached.
The culture was passed into 6 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

12. Procedure: Proliferation
Made up sterile stock solution of 1000x variable where x represents the average dose of L-Theanine in 5L of blood
Seeded 6 flasks with C2C12 cells from the original flasks in 4mL of 10% DMEM media each
Allowed cells to incubate in CO2 incubator overnight and adhere to bottom of flask
Added 50μL of variable and removed 50μL of media from 2 flasks
Added 5μL of variable and removed 5μL of media from 2 flasks
2 flasks for control
Allowed flasks to proliferate in incubator overnight

13. Procedure: Proliferation (Continued)
Next day, trypsinized cells and performed cell counts on the cell suspension
Rinsed with 1mL of trypsin, pipetted out
Added 1mL trypsin, incubate for 5 minutes
Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask
Quenched reaction with 5mL media. Re-added variable.
Re-suspended cells with 1mL trypsin wash before taking counts using pipette
Loaded hemocytometer with 25uL from flask
Took 8 counts per flask
Counted cells in field of view of hemocytometer under Nikon inverted microscope and multiplied count by 103 for total cells/mL
Repeated on Day 3

14. Procedure: Differentiation
Seeded 12 wells: 6 with 50μL of cells
and 6 with 150 μL of C2C12 cells in 1mL 10%
DMEM media
Allowed cells to proliferate until reached 80% confluence
Changed media to 1% DMEM to induce differentiation
Removed 10μL of media and added 10μL variable stock to 4 wells [10x concentration]
Removed 1μL media and added 1μL of variable stock to 4 wells [x concentration]
Kept 4 control wells
Took images of plates with Nikon Inverted Microscope Day 1, Day 3, Day 5, and Day 7
Compared number of myotubes formed on each plate
Same as proliferation assay
Don’t say I or my
Ex) “The variable was made up”

15. Proliferation Results
P-Value:
P-Value: 5.15E-5
Each bar represents an average of 8 replicates. The P-values are less than 0.5, so this shows that there was significant variation between the averages or means x x x

16. Dunnett’s Test Day 1 Concentration T-Value T-Critical Significance 1X
Difference of Average of Experimental Group and Average of Control Group
T-Critical Value
Square Root of two times the MS Value divide by the number of replicates
Day 1
Concentration
T-Value
T-Critical
Significance 1X
0.1742
2.67 No
10X
2.6131
Since the ANOVA revealed that at least two of these means varied significantly, a further pairwise test, the Dunnetts Test, was performed to see what concentration might have produced significant variation when compared to the control. The equation is []. I then took my T-Value from the Dunnett’s test and compared it to my T-critical value which was determined by my number of replicates and groups. If the T-value is greater than my T-critical value, then the data is significant, otherwise it is not. Significant data is data that supports or rejects my hypothesis. It means that the data collected actually showed significant results as opposed to results that occurred just by chance.
Day 3
Concentration
T-Value
T-Critical
Significance 1X
1.0178
2.67 No
10X
4.3706
Yes

17. Differentiation: 50 μL Cells, Control
Day 1
Day 7
Don’t show them all, just one of 50 and one of 150 and ask them if they would like to see more

18. Differentiation: 50 μL Cells, Low Concentration (1x)
Day 1
Day 7

19. Differentiation: 50 μL Cells, High Concentration (10x)
Day 1
Day 7
SHOW THIS ONE

20. Differentiation: 150 μL Cells, Control
Day 1
Day 3
Day 5
Day 7

21. Differentiation: 150 μL Cells, Low Concentration (1x)
Day 1
Day 3
Day 5
Day 7

22. Differentiation: 150 μL Cells, High Concentration (10x)
Day 1
Day 3
Day 5
Day 7
SHOW THIS ONE

23. Conclusion Proliferation:
L-Theanine significantly reduced C2C12 stem cell growth by Day 3 of the High Concentration. The Low Concentration, no significant effect, must accept the null hypothesis
Differentiation:
L-Theanine appeared to have little effect on differentiation based on qualitative analysis of images
Proliferation results from ANOVA tests
Differentiation results just based on images

24. Limitations and Extensions
Two concentrations of variable used
Two days of cell counting
Cell counts have small, inherent errors
Differentiation analysis was qualitative
Extensions
Wider range of variable concentrations
Earlier exposure to test effects on cell attachment
Utilize a quantitative differentiation assay (fluorescent antibody assay)
Explore synergistic effects of L-Theanine with other drugs

25. Acknowledgements Mr. Mark Krotec
Pittsburgh Tissue Engineering Initiative
Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University
"About Herbs, Botanicals & Other Products." Memorial Sloan-Kettering Cancer Center. Web. 29 Jan <
“Meeke, Fiona. "L-Theanine A Therapeutic." Bio Concepts. Web. 29 Jan <
"Product Description." ATCC: The Global Bioresource Center. Web. 29 Jan <
Thank you very much.
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