steroid influence on myoblast stem cell line nathan lampenfeld cchs grade 11 pjas 2015
TRANSCRIPT
![Page 1: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/1.jpg)
Steroid Influence on Myoblast Stem
Cell Line
Nathan LampenfeldCCHS
Grade 11PJAS 2015
![Page 2: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/2.jpg)
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.
![Page 3: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/3.jpg)
Tissue Engineering Principles
CELLS
ECM
BLOOD SUPPLY
SIGNALS
INJURY Regeneration
Repair
![Page 4: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/4.jpg)
Muscle & Repair
![Page 5: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/5.jpg)
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
![Page 6: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/6.jpg)
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
![Page 7: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/7.jpg)
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
![Page 8: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/8.jpg)
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
![Page 9: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/9.jpg)
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
![Page 10: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/10.jpg)
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.
![Page 11: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/11.jpg)
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
![Page 12: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/12.jpg)
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)
![Page 13: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/13.jpg)
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.
![Page 14: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/14.jpg)
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
![Page 15: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/15.jpg)
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.
![Page 16: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/16.jpg)
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
![Page 17: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/17.jpg)
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.
![Page 18: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/18.jpg)
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
![Page 19: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/19.jpg)
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
![Page 20: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/20.jpg)
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
![Page 21: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/21.jpg)
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
![Page 22: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/22.jpg)
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
![Page 23: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/23.jpg)
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
![Page 24: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/24.jpg)
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
![Page 25: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/25.jpg)
Data (Differentiation)
HC HE Day 1 HC HE Day 3 HC HE Day 8 HC HE Day 8 Z-Out
![Page 26: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/26.jpg)
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
![Page 27: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/27.jpg)
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)
![Page 28: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/28.jpg)
Acknowledgements & Sources
• Dr. Phil Campbell• Brother Don Alger FSC• Conrad M. Zapanta, Ph.D.
Biomedical Engineering Laboratory, Carnegie Mellon University• Mark Krotec, PTEI
![Page 29: Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 2015](https://reader035.vdocuments.site/reader035/viewer/2022062719/56649ec05503460f94bcb419/html5/thumbnails/29.jpg)
• 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)