1. Creatine Effects on Oxidative-Stressed Stem Cells
Anthony DeRenzo
Pittsburgh Central Catholic HS
Grade 11
February 6, 2010

2. What is Tissue Engineering?
Broadly Defined: Tissue Engineering is the development and manipulation of artificial implants, laboratory-grown tissues, genetically engineered cells and/or molecules to replace or support the function of defective or injured parts of the body.

3. Principles of Tissue Engineering
Cells
ECM
Defect
Regeneration
Blood
Supply
Hormones
Phil Campbell, Carnegie Mellon

4. Overview of Skeletal Muscle Structure
*Muscle Fiber Bundles (fascicles) and the ECM
*Muscle Fibers (multinucleated structures)
*Basal Lamina Membrane
*Satellite Cells (mixed population)
*Sarcolemma Membrane
*Sarcoglycan Complex
*Dystrophin
*Actin-Myosin Complex

5. C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line.
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.
Mouse stem cell line is used as a model in many tissue engineering experiments.

6. C2C12 Cell Line (contd.)
Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.
Expresses muscle proteins and the androgen receptor (AR).
AR- DNA binding transcription factor which regulates gene expression.

7. Creatine
An organic acid naturally synthesized from amino acids (methionine, glycine, arginine) primarily in the kidney and liver, then transported in the blood for use by muscles.
Approximately 95% located in the skeletal muscle.
Direct relation to ATP production and storage.

8. Supplemental Creatine
Most popular bodybuilding supplement on the market.
Has such an impact because it super-hydrates muscle cells with water.
Enhances muscle growth, and strengthens fibers.
Increased energy levels, strength, and recovery rates. Accelerates weight loss and builds lean body mass.

9. Oxidative Stress and Hydrogen Peroxide
Caused by an imbalance between the reproduction of reactive oxygen and the ability to repair toxic damage.
All life forms maintain reducing environment within their cells.
If disturbed, peroxides and free radicals are produced.
These cause harm to cell components (protein, lipids, DNA).
Apoptosis
Hydrogen peroxide stress
Two electron reduction state
Formed by dismutation of O2- or direct reduction of O2
Lipid soluble
Can be caused through overproduction of oxygen during physical exercise

10. Purpose
To examine the effects of Creatine Monohydrate on the proliferation, differentiation, and survivorship of normal and H2O2 stressed C2C12 cells.

11. Hypotheses
Nulls: 1. Creatine Monohydrate will not have an effect on the proliferation, differentiation, and survivorship of normal and H2O2 stressed C2C12 cells. 2. There will be no evidence of synergy between Creatine Monohydrate and peroxide stress. Alternates: 1. Creatine Monohydrate will have an effect on the proliferation, differentiation, and survivorship of normal and H2O2 stressed C2C12 cells. 2. There will be significant evidence of synergy between Creatine Monohydrate and peroxide stress.

12. Materials Cryotank Three 75mm2 tissue culture treated flasks
Twenty-four 25 mm2 tissue culture treated flasks
10% fetal bovine serum
C2C12 Myoblastic Stem Cell Line
Trypsin-EDTA
Pen/strep
Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)
Micropipettes + sterile tips
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])
75 mL culture flask
Incubator
Zeis Inverted Compound Optical Scope
Aspirating Vacuum Line
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Labeling Tape
Creatine Monohydrate
Hydrogen Peroxide
Hemocytometer
Sterile PBS
Ethanol (70% and 100%)
Distilled water

13. Procedure (Stem Cell Line Preparation)
A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells.
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 4x106 to 5x106 cells/mL was reached.
The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

14. Procedure (Proliferation)
After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL).
5 ml of the cell suspension was added to mm2 tissue culture treated flasks, creating a cell density of approximately 10 5 cells per flask.
1 mM stock concentration of hydrogen peroxide created by adding 0.1 ml of peroxide to 8.7 ml of sterile water.
The following concentrations of variable (next page) were added to the flasks. 4 flasks for each group (2 for proliferation, 2 for differentiation)
The cells were incubated (37°C, 5% CO2) for the remainder of the study.
Procedure (Proliferation)

15. Experimental Groups 0% Peroxide 10 uM Peroxide 0% Creatine
0.6 ml sterile water
4.4 ml cell culture and medium
0.05 ml peroxide
0.55 ml sterile water
.01% Creatine
0.05 ml Creatine
0.5 ml sterile water
1% Creatine
0.5 ml Creatine
0.1 ml sterile water
0.05 ml sterile water

16. Procedure (Differentiation)
The differentiation experiment was identical to the proliferation experiment with the following exceptions:
On Day 3 of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.
Imaging
On Days 1, 3, and 6 pictures of two areas of each flask were taken with a Nikon Inverted Microscope.
These pictures are a visual representation of the proliferation and differentiation of the cells.

17. Proliferation
Cell Count (104 cells/flask)

18. P Values (Single /Double Factor)
Stressed vs. Non Stressed
0.01% Creatine
0.1% Creatine
2.23 E-07 (SF)
Significant
(SF)
(DF)
Insignificant
(S)
(DF)
Conclusions:
The first null hypothesis can be rejected in every case.
The second null hypothesis can be accepted in both cases.

19. Proliferation Unstressed vs. Stressed 0% Creatine
Unstressed Stressed
Day 1
Day 3

20. Proliferation Unstressed vs. Stressed 0.01% Creatine
Unstressed Stressed
Day 1
Day 3

21. Proliferation Unstressed vs. Stressed 0.1% Creatine
Unstressed Stressed
Day 1
Day 3

22. Differentiation Unstressed vs. Stressed 0% Creatine Day 6
Unstressed Stressed

23. Differentiation Unstressed vs. Stressed 0.01% Creatine Day 6
Unstressed Stressed

24. Differentiation Unstressed vs. Stressed 0.1% Creatine Day 6
Unstressed Stressed

25. Qualitative Analysis (Differentiation)
Unstressed vs. Stressed 0% Creatine
Appearance - Significant
Evidence of myotube formation in unstressed test group.
Unstressed vs. Stressed 0.01% Creatine
Unstressed vs. Stressed 0.1% Creatine
Appearance - Significant
In all examples, creatine was not able to remediate the oxidative stress. Therefore, there were no synergistic effects.

26. Limitations and Extensions
Only used qualitative assay of differentiation / Utilize quantitative assay
Test more variations of concentrations
Use other types of stress (UV, heat, infection, various chemicals)

27. References
John Wilson, Biostatistician for the University of Pittsburgh
Conrad M. Zapanta, Ph.D BiomedicalEngineering Laboratory, Carnegie Mellon University
Mark Krotec, PTEI