1. Grape Seed Extract Effects on C2C12 Cell Behavior
Luke Giannetta
Pittsburgh Central Catholic High School
Grade 11
3rd Year in PJAS

2. What is Tissue Engineering?
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. Stem Cells Undifferentiated cells multiply to replenish dying cells.
Somatic stem cells can be found in juvenile/adult animals and humans.
Self-renew indefinitely, and generate all cell types of the organ from which they originate.

4. C2C12 Cells Subclone of the Mus musculus (mouse) myoblast cell line.
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.
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.

5. Grape Seed Extract Antibacterial properties Contains Proanthocyanidins
Class of polyphenols
Chemically classified as oligomeric flavanoids
Found in a variety of fruits
Used in nature as chemical defense mechanisms against plant pathogens

6. Question
Will Grape Seed Extract Significantly affect the proliferation and differentiation of C2C12 cells?

7. Purpose
To determine the effects of grape seed extract on the proliferation of the C2C12 stem cell line
To determine the effects of grape seed extract on the differentiation of C2C12 cells into myotubes

8. Hypotheses Null Hypotheses: Alternate Hypothesis:
Grape seed extract will not have a significant effect on the proliferation of the C2C12 cells
The grape seed extract will not have a significant effect on the differentiation of C2C12 cells
Alternate Hypothesis:
Grape seed extract will have a significant effect on the proliferation of the C2C12 cells
The grape seed extract will have a significant effect on the differentiation of C2C12 cells

9. Materials Cryotank Two 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 10% Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 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
Grape seed extract
Hemocytometer
Sterile PBS
Ethanol (70% and 100%)
Distilled water

10. Proliferation Procedure (C2C12 Cell 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 2 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

11. Proliferation Procedure
After trypsinization, cells from both of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL).
1 ml of the cell suspension and 4 ml of media were added to mm2 tissue culture treated flasks, creating a cell density of approximately 10 5 cells per flask.
The following concentrations of variable (next page) were added to the flasks. 4 flasks for each group (2 for proliferation, 2 for differentiation) per day and one control
The cells were incubated (37°C, 5% CO2) for the remainder of the study.

12. Experimental Groups Control 0.001% 0.01% Cell Suspension 1 mL
10% Media
4 mL
Grape Seed Extract
0 uL
5 uL
50 uL

13. Differentiation Procedure
The differentiation experiment was identical to the proliferation experiment with the following exceptions:
On Day 2 of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.
On days 2 and 3 of the investigation pictures of four areas of each flask were taken with a Nikon Inverted Microscope.
These pictures served as a visual representation of the proliferation and differentiation of the cells.

14. Proliferation Results
Grape Seed Extract Effects on C2C12 Cell Proliferation
7.0 x 10^6
5.25 x 10^6
3.5 x 10 ^6
1.75 x 10^6
Day 1 ANOVA:
p-value = 0.311
Day 2 ANOVA:
P-value = 1.17E^-10
Cells Per Flask
Concentration of Grape Seed Extract

15. Statistical Analysis – Dunnett’s Test
Day 1 Low vs. Control
Day 1 High vs. Control
Day 2 Low vs. Control
Day 2 High vs. Control
T-value
1.6417
2.3958
3.434
5.7081
T-Critical
3.48
Interpretation
Not Significant
Significant

16. Proliferation Conclusions
The null hypothesis can be rejected for day 2 high concentration exposure
Low concentration did not appear to significantly influence proliferation on days 1 and 2
The null hypothesis can be accepted for all other concentrations
High concentration significantly increased proliferation on day 2
Cells potentially reproduced and overcame initial toxic effect

17. Proliferation Low Concentration – 0.001%
Day 2 Flask 1
Day 3 Flask 1
Day 2 Flask 2
Day 3 Flask 2

18. Proliferation High Concentration – 0.01%
Day 2 Flask 1
Day 3 Flask 1
Day 2 Flask 2
Day 3 Flask 2

19. Differentiation Low Concentration – 0.0001%
Control
Flask 1
Flask 2

20. Differentiation High Concentration – 0.01%
Control
Flask 1
Flask 2

21. Differentiation Conclusions:
Low Concentration
Appeared to inhibit myotube formation
Reject null hypothesis
High Concentration

22. Limitations and Extensions
Only used qualitative assay of differentiation / Utilize quantitative assay (MyoD expression)
Non-synchronous pipetting (application of variable, changing media)
Extensions
Test more variations of concentrations
Quantify ability of grape seed extract to remediate stressed cells
CyQUANT™ Cell Proliferation Assay
More quantitative than counting cells on a Hemocytometer
Fluorescent dye binds to nucleic acid in the cell

23. Sources: http://www.altmedrev.com/publications/8/4/442.pdf

24. ANOVA Example