Steroid Influence on Myoblast Stem

Cell Line

Nathan LampenfeldCCHS

Grade 11PJAS 2015

Tissue Engineering• Tissue engineering 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

• Including: Congenital defects, disease, trauma, and aging.

• TE has the potential to revolutionize medicine through the technique of tissue culturing.

Tissue Engineering Principles

CELLS

ECM

BLOOD SUPPLY

SIGNALS

INJURY Regeneration

Repair

Muscle & Repair

Hormones• Chemicals used as

signals in an organism. • Deposited into the tissue

through the bloodstream and other fluids

• Used to stimulate a reaction in targeted tissue.

• Types of hormones include : Amines, Steroids, and peptides

Steroids

• Class of organic molecules naturally found in plants, animals, and fungi

• Lipid based, with receptors in cell cytoplasm

• Play very important roles in biochemistry and plasma membranes of eukaryotic cells

Cholesterol: C27H46O• Precursor for all

sterols• Primarily produced in

the liver• Passes through cell

and nuclear membranes to influence the cells

• Potentially influences myoblast proliferation and differentiation

Estradiol:C18H24O2

• A form of the female sex hormone estrogen

• Primarily produced in the sex organs and adrenal cortex

• Potentially influences myoblast proliferation and differentiation

C2C12 Stem Cell Line• Subclone of the mus

musculus (mouse) myoblast cell line

• This type of mouse stem cell is a common tissue engineering modelo Creates multinucleate

tissue and produces characteristic proteins

o Useful example of stem cell to skeletal muscle

Purpose

The purpose of this study is to observe the synergistic effects of

multiple concentrations the steroids estradiol and cholesterol

on the proliferation, differentiation, and survivorship

of C2C12 stem cells.

HypothesesNull:

Estradiol and Cholesterol will not individually effect proliferation or differentiation of

c2c12 cells, and they will not have a significant synergistic effect

Alternate:Estradiol and Cholesterol will individually effect proliferation

or differentiation of c2c12 cells, and they will have a significant synergistic effect

Materials• Cryotank• 75mm2 tissue culture treated

flasks• Twenty 25 mm2 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])• 75 mL culture flask• Incubator• Nikon Inverted Microscope• Aspirating Vacuum Line• Laminar Flow Hood• Laminar Flow Hood UV

Sterilizing Lamp• Labeling Tape• Hemocytometer• Sterile PBS• Ethanol (70% and 100%)• Distilled water• Estradiol (Powder)• Cholesterol (Powder)

Procedures (Cell Culture)• 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/flask was reached.

• The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

Procedure (Variable creation)

  0 Estradiol 10-8 Estradiol 10-6 Estradiol

0 Cholesterol Control 10-8 Estradiol 10-6 Estradiol

10-8

Cholesterol10-8 Cholesterol 10-8 Estradiol

10-8 Cholesterol10-6 Estradiol10-8 Cholesterol

10-6

Cholesterol10-6 Cholesterol 10-8 Estradiol

10-6 Cholesterol10-6 Estradiol10-6 Cholesterol

Procedure (Variable addition on

Day 0)• After trypsinization, cells from all of the flasks

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

• 0.1 mL of the cell suspension was added to 24 25 mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask.

• The 21 experimental groups and 3 control groups were created with varying concentrations of cholesterol and estradiol

• The cells were incubated at 37°C, 5% CO2 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.

Procedures (Proliferation)

• Day 1o 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 Hemocytometer

for quantification (4 counts per flask).• Day 1 and Day 6o Using the Nikon Inverted Microscope, images of 9

representative areas of each flask were taken

Procedures (Differentiation)

• Day 1 and Day 6o Using the Nikon Inverted Microscope, images of eight

representative areas of each of the flasks were taken.• Day 2

o The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

P-value: 3.2007E-27 P-value: 4.25309E-08

0C 0E 0C LE 0C HE LC 0E LC LE LC HE HC 0E HC LE HC HE0

100

200

300

400

500

600

700

Cell Proliferation

Day 1

Day 3

Variable

Cell

s (x

10

00

)/ m

L

Dunnett’s TestConcentration GroupDAY 1

T-Value Variation: T-Crit 2.12

10-8 Estradiol 8.627450607

SIGNIFICANT

10-8 Cholesterol 14.79596194

SIGNIFICANT

10-6 Estradiol1.00920276

8

NOT Significant

10-6 Cholesterol 20.37425981

SIGNIFICANT

10-8 Estradiol10-8 Cholesterol 15.67225891

SIGNIFICANT

10-8 Estradiol10-6 Cholesterol 12.41223057

SIGNIFICANT

10-6 Estradiol10-8 Cholesterol 15.67225891

SIGNIFICANT

10-6 Estradiol10-6 Cholesterol 15.86514985

SIGNIFICANT

Concentration GroupDAY 3

T-Value Variation: T-Crit 2.65

10-8 Estradiol26.358547

64

SIGNIFICANT

10-8 Cholesterol2.0560876

18

NOT Significant

10-6 Estradiol22.3790542

5

SIGNIFICANT

10-6 Cholesterol0.9482352

78

NOT Significant

10-8 Estradiol10-8 Cholesterol

7.347209527

SIGNIFICANT

10-8 Estradiol10-6 Cholesterol

3.301981784

SIGNIFICANT

10-6 Estradiol10-8 Cholesterol

3.602913942

SIGNIFICANT

10-6 Estradiol10-6 Cholesterol

14.40192761

SIGNIFICANT

Data (Differentiation)

Control Day 1 Control Day 3 Control Day 8 Control Day 8 Z-Out

LE 0C Day 1 LE 0C Day 3 LE 0C Day 8 LE 0C Day 8 Z-Out

Data (Differentiation)

LC 0E Day 1 LC 0E Day 3 LC 0E Day 8 LC 0E Day 8 Z-Out

LC LE Day 1 LC LE Day 3 LC LE Day 8 LC LE Day 8 Z-Out

Data (Differentiation)

0C HE Day 1 0C HE Day 3 0C HE Day 8 0C HE Day 8 Z-Out

HC 0E Day 1 HC 0E Day 3 HC 0E Day 8 HC 0E Day 8 Z-Out

Data (Differentiation)

0C HE Day 1 0C HE Day 3 0C HE Day 8 0C HE Day 8 Z-Out

HC 0E Day 1 HC 0E Day 3 HC 0E Day 8 HC 0E Day 8 Z-Out

Data (Differentiation)

LC HE Day 1 LC HE Day 3 LC HE Day 8 LC HE Day 8 Z-Out

HC LE Day 1 HC LE Day 3 HC LE Day 8 HC LE Day 8 Z-Out

Data (Differentiation)

HC HE Day 1 HC HE Day 3 HC HE Day 8 HC HE Day 8 Z-Out

Conclusion• Cholesterol and Estradiol had a significant

synergistic effect on the cell proliferation and differentiation

• The Variable has a significant effect on all concentrations except High Estradiol (Day 1), Low Cholesterol (Day 3), & High Cholesterol (Day 3)

• High Concentrations of Estrogen appear to have a negative effect on cell proliferation and differentiation

• Low Cholesterol, Low Estradiol, and Low Cholesterol and Estradiol had the most significantly positive effects

Limitations / Extensions

• CyQUANT™ Cell Proliferation Assay/Antibody Tagging• Gives more accurate data compared to a

hemocytometer• More quantitative differentiation study

• This experiment will be extended with other steroids (Aldosterone, Testosterone, Relaxin, & Progesterone), and other cell types (3t3 cells)

Acknowledgements & Sources

• Dr. Phil Campbell• Brother Don Alger FSC• Conrad M. Zapanta, Ph.D.

Biomedical Engineering Laboratory, Carnegie Mellon University• Mark Krotec, PTEI

• Cholesterol: 0.38665 g was added to 10 mL of ethanol (100%) to create a 10-3 Molar solutiono The sub stock 10-5 Molar was created by pipetting

0.1 mL of the 10-3 Molar solution into 9.9 mL of ethanol

• Estradiol: 0.272382g was added to 10 mL of ethanol (100%) to create a 10-3 Molar solutiono The sub stock 10-5 Molar was created by pipetting

0.1 mL of the 10-3 Molar solution into 9.9 mL of ethanol

• 5 µl of the 10-3 M Estradiol solution to 3 flasks• 5 µl of the 10-5 M Estradiol solution to 3 flasks• 5 µl of the 10-3 M Cholesterol solution to 3 flasks• 5 µl of the 10-5 M Cholesterol solution to 3 flasks• 5 µl of the 10-3 M Estradiol + 10-3 M Cholesterol solution

to 3 flasks• 5 µl of the 10-3 M Estradiol + 10-5 M Cholesterol solution

to 3 flasks• 5 µl of the 10-5 M Estradiol + 10-3 M Cholesterol solution

to 3 flasks• 5 µl of the 10-5 M Estradiol + 10-5 M Cholesterol solution

to 3 flasks• 5 µl of ethanol to 3 flasks (Control)