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

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.

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.

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.

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

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

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

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

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 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 Grape seed extract Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water

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.

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 13 25 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.

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

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.

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

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

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

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

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

Differentiation Low Concentration – 0.0001% Control Flask 1 Flask 2

Differentiation High Concentration – 0.01% Control Flask 1 Flask 2

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

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

Sources: http://www.altmedrev.com/publications/8/4/442.pdf http://www.ncbi.nlm.nih.gov/pubmed/10693912 http://www.ncbi.nlm.nih.gov/pubmed/15161187 https://jgryall.wordpress.com/muscle-methods/working-with-the-c2c12-cell-line/ https://genome.ucsc.edu/ENCODE/protocols/cell/mouse/C2C12_Wold_protocol.pdf http://ashwanimittal.com/Upload/upload/2%20cell%20proliferation%20differentitation.pdf

ANOVA Example