EFFECTS OF METHYLHEXANAMINE (DMAA) ON

C2C12 AND 3T3 STEM CELLS

Cameron Franz

Pittsburgh Central Catholic High School

Grade 11

Tissue Engineering

• TE is 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

• 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 supplementing muscle tissue.

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Stem Cells• Unspecialized cells capable

of renewing themselves through cell division.

• Under certain physiologic or experimental conditions, they can be induced to become tissue or organ specific cells with special functions.

• Stem cells offer a number of new potentials for treating disease

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C2C12 Stem Cells• Subclone of the mus

musculus (mouse) myoblast cell line.

• A model type of stem cell line that was discovered in 1977 through experimentation of murine thigh muscle growth after a crush injury.

• Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

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3T3 Stem Cells

• Cell line established from Swiss mouse embryo tissue

• standard fibroblast cell line• Do not differentiate, produce ECM

components in connective tissue • Often used in the cultivation of keratinocytes,

with the 3T3 cells secreting growth factors favorable to these kinds of cells.

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Methylhexanamine (DMAA)• Commonly marketed as a nutritional supplement or workout aid

• It has recently been subject to multiple bans by both governments and Sports Authorities

• The US Food and Drug Administration declared that methylhexanamine was potentially dangerous and did not qualify as a legal dietary supplement

• It warned supplement makers that it was illegal to market the drug

• However, it is still found in many supplements

• According to the FDA Numerous adverse events and at least 5 deaths have been reported in association with methylhexanamine-containing dietary supplements

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DMAA (Continued)

• It is an indirect sympathomimetic drug• Acts as a stimulant • It constricts blood vessels and thus has effects

on the heart, lungs, and reproductive organs• It also causes bronchodilation, inhibits

peristalsis in the intestines, and has diuretic effects

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Purpose

• To determine the effect of DMAA exposure on C2C12 and 3T3 cell proliferation

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Hypothesis

Null: DMAA WILL NOT have an effect on the proliferation of C2C12 cells and 3T3 cells

Alternative: DMAA WILL significantly effect the proliferation of C2C12 cells and 3T3 cells

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Materials • Cryotank• Two 75mm2 tissue culture• treated flasks• 24 25mm2 tissue culture treated flasks• Fetal bovine serum (FBS)• C2C12 Myoblastic Stem Cell Line• 3T3 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])

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• Methylhexanamine (DMAA) Powder 99.89% • 75 mL culture flask• Incubator• Nikon Inverted Microscope• 24 well plate• Laminar Flow Hood• Laminar Flow Hood UV Sterilizing Lamp• Labeling Tape• Hemocytometer• Sterile PBS• Ethanol (70% and 100%)• Purple Nitrile gloves

Procedure

• A 1 mL aliquot of C2C12 cells and 3T3 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in two 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 two sets of 2 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

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Procedure (Continued)• After trypsinization, cells from all of the flasks were pooled

into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL).

• 0.2 mL of the cell suspension was added to eight 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask.

• Concentrations of 0.1x, 1x, and 10x DMAA were created based on a 20mg dose by diluting DMAA Powder in media. The resulting solutions where sterile filtered.

• 2 T25 flasks of each concentration where created for both cell lines.

Cell Counts where obtained through the following:• The cells were trypsinized and collected

into cell suspension. • 25 μl aliquots were transferred to a

Hemocytometer for quantification (eight counts per flask).

• Counts were then taken on days 1 and 3

Stat Analysis • ANOVA

• Short for Analysis of Variance

• Statistical test to find variance between and within groups

• If the P-Value is smaller than the Alpha Value (0.05), the analysis is significant

• Dunnett’s Test•Follow up to an ANOVA

•Statistical test to find the source of variance

•If the T-value is larger then the T-Crit, the effect is significant

Effects of DMAA on 3T3 Stem Cells

P-value=0.272P-value=0.0097 (SIG.)

0x 0.1x X 10x0

70

140

210

280

Day 1Day 3

Concentration of DMAA

Ave

rag

e C

ell

Co

un

t

Dunnett’s Test: Day 3 3T3

  Variable Concentrations

T-Value Interpretation

T-Crit=2.306      DMAA

 

0.1X 0.167 not significant

X 0.712 not significant

10X 3.12 SIGNIFICANT

Effects of DMAA on C2C12 Stem Cells

0X 0.1x X 10X0

75

150

225

300

375

Day 1 Day 3

Concentration of DMAA

Ave

rag

e C

ell

Co

un

ts

P-value=0.0179 (SIG.)P-value=0.0001 (SIG.)

Dunnett’s Test: Day 1 C2C12

  Variable Concentrations

T-Value Interpretation

T-Crit=2.306      DMAA

 

0.1X 0.502 not significant

X 3.16 SIGNIFICANT

10X 0.743 not significant

Dunnett’s Test: Day 3 C2C12

  Variable Concentrations

T-Value Interpretation

T-Crit=2.306      DMAA

 

0.1X 0.275 not significant

X 1.15 not significant

10X 2.654 SIGNIFICANT

Conclusions

• The null hypothesis may be rejected for both 3T3 and C2C12 stem cells based on the results of the ANOVAs

• The Dunnett’s Tests show significant variation in the 10X for 3T3 on Day 3, in the 1X for C2C12 Day 1, and the 10X for C2C12 Day 3

Limitations

• Variation in counts using Hemocytometers• Cell clumping• Limited Number of DMAA exposures• Limited number of replicates

Extensions

• More trials • Studying effects on cell differentiation • Different models• Adding more variable concentrations

and exposure times• Synergistic effects of different variables

(i.e. Caffeine and DMAA)

Resources & Acknowledgments • Mr. Mark Krotec, PTEI• Dr. Phil Campbell• http://www.fda.gov/Food/DietarySupplements/QA

DietarySupplements/ucm346576.htm• http://www.nytimes.com/2013/04/13/business/fda

-issues-warning-on-workout-booster.html?_r=0• http://www.nutrivitashop.com/dmaa.html• http://www.forbes.com/sites/robertglatter/2013/0

4/12/is-dmaa-dangerous-to-your-health/

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