Steroid Influence on Myoblast Stem Cell Line Nathan Lampenfeld CCHS Grade 11 PJAS 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 CEL LS EC M BLOOD SUPPL Y SIGNA LS INJURY Regeneratio n 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: C 27 H 46 O 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: C 18 H 24 O 2 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 model o 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.

Hypotheses Null: 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 75mm 2 tissue culture treated flasks Twenty 25 mm 2 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 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/flask was reached. The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

Procedure (Variable creation) 0 Estradiol10 -8 Estradiol10 -6 Estradiol 0 CholesterolControl10 -8 Estradiol10 -6 Estradiol Cholesterol Estradiol Cholesterol Estradiol Cholesterol Cholesterol Estradiol Cholesterol Estradiol Cholesterol

Procedure (Variable addition on Day 0) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 0.1 mL of the cell suspension was added to mm 2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 10 5 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% CO 2 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 1 o 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 6 o Using the Nikon Inverted Microscope, images of 9 representative areas of each flask were taken

Procedures (Differentiation) Day 1 and Day 6 o 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.

Dunnett’s Test Concentration Group DAY 1 T-ValueVariation: T- Crit Estradiol SIGNIFICANT Cholesterol SIGNIFICANT Estradiol NOT Significant Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Concentration Group DAY 3 T-ValueVariation: T- Crit Estradiol SIGNIFICANT Cholesterol NOT Significant Estradiol SIGNIFICANT Cholesterol NOT Significant Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT Estradiol Cholesterol SIGNIFICANT

Data (Differentiation) Control Day 1Control Day 3Control Day 8 Control Day 8 Z- Out LE 0C Day 1LE 0C Day 3LE 0C Day 8 LE 0C Day 8 Z- Out

Data (Differentiation) LC 0E Day 1LC 0E Day 3LC 0E Day 8 LC 0E Day 8 Z- Out LC LE Day 1LC LE Day 3LC LE Day 8 LC LE Day 8 Z- Out

Data (Differentiation) 0C HE Day 10C HE Day 30C HE Day 8 0C HE Day 8 Z- Out HC 0E Day 1HC 0E Day 3HC 0E Day 8 HC 0E Day 8 Z- Out

Data (Differentiation) 0C HE Day 10C HE Day 30C HE Day 8 0C HE Day 8 Z- Out HC 0E Day 1HC 0E Day 3HC 0E Day 8 HC 0E Day 8 Z- Out

Data (Differentiation) LC HE Day 1LC HE Day 3LC HE Day 8 LC HE Day 8 Z- Out HC LE Day 1HC LE Day 3HC LE Day 8 HC LE Day 8 Z- Out

Data (Differentiation) HC HE Day 1HC HE Day 3HC 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: g was added to 10 mL of ethanol (100%) to create a Molar solution o The sub stock Molar was created by pipetting 0.1 mL of the Molar solution into 9.9 mL of ethanol Estradiol: g was added to 10 mL of ethanol (100%) to create a Molar solution o The sub stock Molar was created by pipetting 0.1 mL of the Molar solution into 9.9 mL of ethanol 5 µl of the M Estradiol solution to 3 flasks 5 µl of the M Estradiol solution to 3 flasks 5 µl of the M Cholesterol solution to 3 flasks 5 µl of the M Cholesterol solution to 3 flasks 5 µl of the M Estradiol M Cholesterol solution to 3 flasks 5 µl of the M Estradiol M Cholesterol solution to 3 flasks 5 µl of the M Estradiol M Cholesterol solution to 3 flasks 5 µl of the M Estradiol M Cholesterol solution to 3 flasks 5 µl of ethanol to 3 flasks (Control)