Frank Wolf Pittsburgh Central Catholic High School Grade 11 PJAS 2010

 What is TE? ◦ 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  Why is TE important? ◦ It has the potential to replace or supplement the function of tissues destroyed or compromised in any variety of ways, including:  Inherent design flaws  Hereditary/congenital defects or conditions  Disease  Trauma  Damage from an individual’s environment  Aging  TE has great potential for muscle therapy.

Cells ECM Hormones Blood Supply Defect Regeneration Phil Campbell, Carnegie Mellon

 Direct Physical Trauma ◦ Laceration – cutting of tissue ◦ Contusion - bruise ◦ Strain – “pulling a muscle”  Biological (Inherited Abnormalities) ◦ Muscular Dystrophies ◦ Neurological ◦ Storage Diseases

 Main male sex hormone  Produced in the sex organs and adrenal cortex of the adrenal gland  Steroid hormone of the androgen group ◦ Steroid hormone – passes through cellular and nuclear and affects transcription ◦ Androgen – any hormone that stimulates/maintains male characteristics in vertebrates  Acts by binding to androgen receptor ◦ Androgen receptor – transcription factor  Stimulates myoblast formation (cell differentiation)  Particular testosterone molecule used: testosterone C-III ◦ Anabolic steroid  Drug meant to mimic the effects of bodily-produced testosterone  Increases skeletal muscle mass

 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.  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.

 The purpose of this study is observe the effects of the anabolic steroid testosterone C-III on the proliferation, differentiation, and survivorship of C2C12 stem cells.

 Alternative Hypothesis: The addition of testosterone C-III WILL affect the proliferation, differentiation, and survivorship of C2C12 stem cells.  Null Hypothesis: The addition of testosterone C-III WILL NOT affect the proliferation, differentiation, and survivorship of C2C12 stem cells.

 Cryotank  75mm 2 tissue culture treated flasks  Twenty 25 mm 2 tissue culture treated flasks  Fetal bovine serum (FBS)  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 Serum - 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])  Testosterone C-III 1 M stock solution  75 mL culture flask  Incubator  Nikon Inverted Microscope  Aspirating Vacuum Line  Laminar Flow Hood  Laminar Flow Hood UV Sterilizing Lamp  Labeling Tape  Hemacytometer  Sterile PBS  Ethanol (70% and 100%)  Distilled water

 A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells.  The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached.  The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

 After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL).  0.1 mL of the cell suspension was added to mm 2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 10 5 cells per flask.  The 1 M stock solution of testosterone C-III was created using 1 mL of ethanol and grams of testosterone C-III.  The M, M, and M concentrations were created from the stock, where M is the suggested working concentration of the steroid.  The 3 experimental groups and the control group were created by adding:  20 µl of the M solution to 5 flasks (Group A)  20 µl of the M solution to 5 flasks (Group B)  20 µl of the M solution to 5 flasks (Group C)  20 µl of ethanol to 5 flasks (Control)  The cells were incubated at 37°C, 5% CO 2 for the remainder of the study.  Three flasks from each group were used in the Proliferation Experiment and two flasks from each group were used in the Differentiation Experiment.

 Day 1 and Day 6 ◦ Using the Nikon Inverted Microscope, images of two representative areas of each flask were taken. ◦ Using one flask from each group, cell densities were determined as follows:  The cells were trypsinized and collected into cell suspension.  25 µl aliquots were transferred to a hemacytometer for quantification (six counts per flask).  Day 1  Using the Nikon Inverted Microscope, images of two representative areas of each of the flasks were taken.  Day 2  The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.  Day 6  Using the Nikon Inverted Microscope, images of eight representative areas of each of the flasks were taken.

 ANOVA ◦ p-value: 1.78E-08 ◦ REJECT NULL ◦ Significant variation  Dunnett’s Test ◦ 0.05 t-critical value: 2.71 Groupt-valueVariation M9.82Significant M3.11Significant M1.19Insignificant

Day 1Day 6

Day 1Day 6

Day 1Day 6

Day 1Day 6

Control Group A Group CGroup B

Results Consistent with Prior Study  Proliferation Experiment ◦ From the ANOVA and the subsequent Dunnett’s tests, the addition of testosterone C-III induced a statistically significant increase in proliferation in the C2C12 cells when it is added at its suggested working concentration, M, and one tenth this concentration, M.  Differentiation Experiment ◦ From the qualitative analysis of the images gathered from the flasks, it appears that the addition of testosterone C-III induced myotubule formation. This was especially apparent in the M, while the M and M concentrations showed less dramatic differentiation in comparison.

 Proliferation Experiment – some cell clumping occurred during passage, and counts were lower than expected  Differentiation Experiment ◦ Evaluation of images – qualitative and imprecise ◦ Solution – quantitative differentiation assay, e.g. MyoD tagging, myosin/nuclear staining ratio, etc.  Evaluate concentrations of testosterone C-III greater than suggested dosage to determine if the steroid is hazardous in excess

 Dr. Phil Campbell  Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University  Mark Krotec, PTEI  C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression – German Sport University, Cologne, Germany