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

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

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

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

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

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

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

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

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.

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

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.

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

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

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”

Proliferation Results P-Value: 0.018227 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

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

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

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

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

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

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

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

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

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

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. 2012. <http://www.mskcc.org/>. “Meeke, Fiona. "L-Theanine A Therapeutic." Bio Concepts. Web. 29 Jan. 2012. <http://www.neuroconcepts.memberlodge.org/>. "Product Description." ATCC: The Global Bioresource Center. Web. 29 Jan. 2012. <http://www.atcc.org/>. Thank you very much. DO NOT READ THIS SLIDE