1. Adrenaline Effects on Adult Stem Cells
Christopher McNulty
Pittsburgh Central Catholic High School
Grade 11
PJAS 2011

2. Tissue Engineering What is TE? Why is TE important?
The development and manipulation of artificial implants, laboratory- grown tissues, and genetically engineered cells and/or molecules to replace or support the function of defective or injured body parts
Why is TE important?
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 supplementing compromised muscle tissue.

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

4. C2C12 Cells
Muscle derived adult stem cell line which originated in mice
Frequently used as a model in tissue engineering experiments.
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 the adrenergic receptor (AR).
AR- DNA binding transcription factor which regulates gene expression.

5. 3T3 Cells Mouse derived fibroblastic stem cell line
Standard line used to simulate human fibroblasts
Has a wide variety of receptors and messenger systems
Cells proliferate extremely rapidly, but growth stops when cell-to-cell contacts are formed
Widely used cell line in research

6. Adrenaline
Adrenaline (Epinepherine) is a hormone and neurotransmitter widely used throughout the body
Produced in the adrenal glands in the body and released during times of stress (fight or flight response).
Wide-ranging effects on the body:
Causes contraction or relaxation of smooth muscle

7. Purpose
To assess the effect(s) of the hormone and neurotransmitter Adrenaline on the proliferation, differentiation, and survivorship of C2C12 and 3T3 stem cells.

8. Hypotheses Null: Alternative:
Adrenaline will not have an effect on the proliferation and differentiation of C2C12 cells and the proliferation of 3T3 cells
Alternative:
Adrenaline will have an effect on the proliferation and differentiation of C2C12 cells and the proliferation of 3T3 cells

9. Materials Cryotank Three 75mm2 tissue culture treated flasks
Six 25 mm2 tissue culture treated flasks
C2C12 Myoblastic Stem Cell Line (CMU)
3T3 Myoblastic Stem Cell Line (CMU)
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, mg/L glucose, 1 mM sodium pyruvate, and mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete])
Materials
10% fetal bovine serum
Trypsin-EDTA
Pen/strep
75 mL culture flask
Incubator
Nikon Inverted Compound Optical Scope
Aspirating Vacuum Line
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Labeling Tape
Adrenaline
Hemacytometer
Sterile PBS
Ethanol (70%)
Distilled water
10% Goat serum
Antibody staining system
Fluorescent sensitive microscope

10. Creating Variable Substock
The Adrenaline contained in the Autoinjector was at a concentration of 10-4
By adding 50µL of adrenaline to 5mL of media, the concentration would decrease to 10-6
A substock was created with 10µL adrenaline and 990µL PBS to create a dilution of 10-8.
Normal body concentration of Adrenaline is ~500ng/L during times of stress, or 10-11)

11. 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.

12. 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 six 25 mm2 tissue culture treated flasks, creating a cell density of approximately 2x10 5 cells per flask.
50µL of variable was added to the 4 variable flasks to form 10-6 and 10-8
The cells were incubated (37°C, 5% CO2) for one day.
Cell counts (hemacytometer) were performed on Days 1 and 3

13. Procedure (Antibody Assay)
Three glass bottom plates were seeded with 3mL of cell solution, serum starved with 1% media, and allowed to grow until myotubes had formed (7 days)
The three plates were fixed by washing with PBS and fixing with ice-cold methyl alcohol (MeOH)
The plates were blocked with 10% goat serum for 1 hour and washed with PBS
Stain plates with Primary and Secondary Desmin, Primary and Secondary GFP, and Dapi.
Images were recorded utilizing an fluorescent inverted view microscope .

14. Adrenaline Effects on C2C12 Proliferation
P = 2.0E-08

15. Adrenaline Effects of 3T3 Proliferation
P = 1.97E-05

16. C2C12 Proliferation Control Day 1 10-6 Day 1 10-8 Day 1 Control Day 3

17. 3T3 Proliferation Control Day 1 10-6 Day 1 10-8 Day 1 Control Day 3

18. Results of Statistical Analyses
Statistical Analyses Results
Results of Statistical Analyses
C2C12 Proliferation
Day 1
Day 3
10-6
3.53
SIGNIFICANT
14.7
10-8
2.22
INSIGNIFICANT
8.55
t-crit = 3.03
3T3 Proliferation
Day 1
Day 3
10-6
3.17
SIGNIFICANT
1.4
INSIGNIFICANT
10-8
2.17
9.86
t-crit = 3.03

19. Antibody Assay (Control Plate)
Normal
Dapi Stain
Desmin Stain
GFP Stain

20. Antibody Assay Results
Variable Concentration
% Differentiation
Nuclei/Myotube
Control
20.755%
3.666
10-6
26.887%
4.385
10-8
26.866%
2.571
An ANOVA was performed on the data, and the f-value (65535) was larger than the f-crit (5.143), so the results are SIGNIFICANT.

21. Conclusions
The Null was rejected for the C2C12 Proliferation (10-6 and 10-8), 3T3 Proliferation
(10-8) and the Differentiation assay.
The Null was accepted for the 3T3 Proliferation (10-6)
Supports Previous Findings
Supports Previous Findings

22. Limitations and Extensions
Possibility of errant cell counts
Quantitative assay such as CyQUANTTM Proliferation assay would reduce the chance of error
Over trypsinization
Damages cells and has a negative effect on colony health
Extensions:
More concentrations of adrenaline
Different cell lines

23. References Nina Diprimio, Ph.D Carnegie Mellon University
Conrad M. Zapanta, Ph.D BiomedicalEngineering Laboratory, Carnegie Mellon University
Mark Krotec, PTEI
Phil Campbell, Ph.D Carnegie Mellon University

24. Cross-Sectional View of Muscle

26. ANOVA Statistical Analysis
Analysis of variance
Compares variation within groups to variation between groups
If variation between groups is larger than the variance within groups, the results were most likely obtained by action of the variable (outside of chance)
Cutoff value of p=.05 (95% chance that results were obtained outside of chance)

27. Dunnett’s Test
To determine which variable groups had significant variance from the control group, a Dunnett’s Test is performed
The Dunnett’s test individually compares each variable groups against the control, to see if that particular variable groups varied significantly from the control
The resulting value is checked against a chart of critical values; if the t-value is larger than the t- crit, the results are significant.