steroid influence on myoblast stem cell line nathan lampenfeld cchs grade 11 pjas 2015
TRANSCRIPT
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)