1 Neil Carleton Grade 11 Pittsburgh Central Catholic High School PJAS 2012

Introduction: C2C12 2 Stem Cell -- unspecialized cell characterized by the capacity to give rise to various differentiated cell types To model human stem cells, C2C12 mus musculus (mouse) myoblast cell line used C2C12 cells used to model cell differentiation from stem cell state to skeletal muscle state through myotube structure formation Stem cells have uses in research and treatment Cancer, Type 1 Diabetes, Parkinson’s Disease, Huntington’s Disease, Celiac Disease, cardiac failure, muscle damage, neurological disorders

Introduction: MG63 3 Human cancer (osteosarcoma) cell line Cancerous bone tumor that usually develops during the period of rapid growth that occurs in adolescence, as a teenager matures into an adult.

Oxidative Stress 4 Free radicals of the reactive oxygen species (ROS) can damage cells in a process called oxidative stress Reactive species may account for the increase risk of cancer development and may promote malignancy Hydrogen Peroxide (H2O2) used to cause oxidative stress

Vitamin B 5 Supports and increases the rate of metabolism Maintain healthy skin, hair, and muscle tone Enhance immune and nervous system function Promote cell growth and division, including that of the red blood cells that help prevent anemia Reduce the risk of some cancers

Objective 6 Investigate the main effects and interaction effect of oxidative stress (Hydrogen Peroxide) and Vitamin B on the survivorship, proliferation, and differentiation of murine myoblastic stem cell line (C2C12) and human cancer cell line (MG63). Survivorship & Proliferation: Effect measured by counting number of surviving stem cells after exposure to different concentrations of treatment products Differentiation: quantitatively measured by counting the number of myosin positive nuclei out of total nuclei in cell photomicrograph

Hypotheses 7 As oxidative stress increases, the number of surviving C2C12 cells will decrease when no vitamin is present, but the number of surviving MG63 cells will increase. Unknown vitamin B effect on number of surviving cells when no oxidative stress is present. As oxidative stress levels increase, introduction of a vitamin will have a moderating effect with the C2C12 cells–survivorship will increase even with a stressor present. As oxidative stress levels increase, introduction of a vitamin will decrease the survivorship of the MG63 cells—even though oxidative stress promotes cancer growth, the vitamin will impede their growth.

Hypotheses 8 Null Hypothesis: Stress would not significantly affect mammal cell proliferation or differentiation. Alternative Hypothesis: Stress would significantly effect mammal cell proliferation and differentiation.

Materials and Apparatus 9 3% concentrated H 2 O 2 Liquid Vitamin B C2C12 murine myoblastic stem cells MG63 Human osteosarcoma cell line Deionized sterile water 100 mL graduated cylinder Test tube rack Incubator (37.0°C) Macropippette with tips µL pipette 0.1 – 1 mL pipette 1 – 10 mL pipette 70% Ethanol (for sterilization) Felt-tip marker 15 mL sterile conical tubes 1 24-well plates DMEM media (10% calf serum & 1% calf serum) contains salts, amino acids, vitamins, & glucose Sterile pipette tips 0.22 micron syringe filters + 10 mL syringe 200 g scales 75 mL culture flasks cm 2 culture flasks 50 mL Trypsin-EDTA 32 mL PBS saline 32 mL 100% ice-cold ethanol Penn Strep Solution 2 Hemocytometers Light microscope Inverted microscope (with imaging capabilities) Class II Biosafety hood Labcoats, Eye Protection, Disposible Gloves Anti Myo D stain DAPI nuclear stain Vortexor Delicate task wipes Counter Aluminum foil

10 0 H2O2Low H2O2High H2O2 0 Vit. B0 H2O210 μL H2O2100 μL H2O2 0 Vit. B 4 mL media3.99 mL media3.9 mL media 1 mL cells Low Vit. B0 H2o210 μL H2O2100 μL H2O2 50 μL Vit. B 3.95 mL media3.94 mL media3.85 mL media 1 mL cells Total=5mL H2O2 Concentration Vit. B Concentration Experimental Design

Procedure 11 Preparation of Treatment Materials µL 3% H2O2 diluted with 9.89 mL sterilized deionized water to yield 1 mM concentration of H2O mL of liquid Vitamin B diluted with 9 mL sterilized deionized water to yield 1 mM concentration of vitamin B. Stem Cell Line and Cancer Cell Line Culture 1.1 mL aliquot of C2C12 and MG-63 cells from a cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mL culture flask yielding a cell density of approximately 10 6 to 2*10 6 cells 2.Media changed with 15 mL fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4*10 6 to 5*10 6 cells/mL was reached 3.The culture was passed into 3 75 mL culture flasks in preparation for experiment (48 hours before)

Procedure (contd.) 12 Treatment Application (Proliferation and Differentiation: Day 0) cm 2 culture flasks were labeled - 24 for proliferation/toxicity 2. Treatment materials and other materials pipetted into each of 12 flasks in biosafety hood then left to incubate for 24 hours (see table)

Procedure (contd.) 13 Cell Counting (Proliferation: Day 1 and 3) 1. For proliferation assay, aspirated off current media 2. Added 2mL trypsin and aspirate immediately 3. Added 1mL trypsin and incubate for 4 minutes 4. Smacked side of flasks hard twice to detach cells from flask bottom 5. Transfered 1 mL of cells to 1 mL tubes in rack using pipette 6. Cleaned hemocytometer using 70% ethanol and delicate task wipes 7. Inserted 25 µL of cell solution into each end of hemocytometer making sure solution wicks across hemocytometer face in an even coating 8. Gently placed cover slip on hemocytometer and examined hemocytometer grid 9. Using the counter, counted and recorded the number of cells in the 3 mm by 3 mm grid

Procedure (contd.) 14 Media Replacement (Proliferation: Day 2) 1. For all cells, aspirated off current media in the process removing cell waste products 2. Added 3.9 mL of media and appropriate concentration of treatment materials to each group as specified in the previous table, 3. Gently shook to spread cells and left cells in incubator for 24 hours Serum Starvation (Differentiation: Day 2) 1. Aspirated off media 2. Added 3.9 mL of 1% serum media to cells in flask 3. Added appropriate concentration of degradation materials to each group, gently shook to spread cells, and left cells in incubator for 12 hours

Procedure (contd.) 15 Well Plate Transfer (Differentiation: Day 2) 1. For differentiation assay cells, repeated steps 1 through 4 of cell counting (using trypsin to detach cells from flask wall) 2. For each group, labeled three wells on the 24-well plates 3. Pippetted 0.4 mL of cell solution from each flask to each of the three corresponding wells 4. Added 1.6 mL of 1% serum media to each well and appropriate concentration of treatment materials to each group in a 2/5 ratio to the volumes specified in the previous table 5. Gently shook to spread cells and left cells in incubator for 36 hours

Procedure (contd.) 16 Cell Photomicrography (Differentiation) 1. Turned on inverted microscope optical imaging system and connected computer, opened imaging software 2. Wiped condensation off lid of well plates with delicate task wipes 3. Adjust focus, white balance, and exposure as necessary 4. For each differentiation well took and labeled six micrographs with attached camera, three with UV light filter to excite DAPI nuclear stain (blue) and three with blue light filter to excite myosin stain (green) 5. Obtained quantitative result by creating ratio of myosin positive nuclei (number of nuclei within green myosin stain) to total nuclei in cell photomicrograph

Procedure (contd.) 17 Treatment Product Preparation and Stem Cell Line cultured (both experiments) Treatment Application Cell Count Taken Media Replacement Cell Count Taken Serum Starvation and Well Transfer Cells Fixed and Stained Cell Photomicrography

Results: C2C12 Proliferation – Day 1 18 Vit. B Concentrations P-value:

Results: C2C12 Proliferation – Day 3 19 Vit. B Concentrations P-value:

Results: MG63 Proliferation – Day 1 20 Vit. B Concentrations P-value:

Results: MG63 Proliferation – Day 3 21 Vit. B Concentrations P-value: 6.35E-08

Conclusion: Proliferation 22 C2C12MG63 Day 1Insignificant Synergistic Effect Day 3Significant Synergistic Effect

Differentiation Assay 23 Control Day 1Day 3

Differentiation Assay 24 Low Concentration of H2O2 + Vit. B Day 1 Day 3

Differentiation Assay 25 High Concentration of H2O2 + Vit. B Day 1 Day 3

Conclusion 26 Proliferation: After 3 days of cell proliferation, there seemed to be a significant synergistic effect between H2O2 stress and Vitamin B remediation on both the C2C12 and MG63 cells. Differentiation: Based upon the images gathered from the inverted microscope, although some differentiation was observed, the analysis was qualitative and variance could not be statistically compared. Speculatively, it appeared that little difference was noticed between the control and the various concentrations.

Limitations Murine stem cells may not have provided accurate representation of human stem cells 2. Constant and direct exposure to only hydrogen peroxide may not accurately represent oxidative stress process in the human body 3. Constant and direct exposure to only Vitamin B may not accurately represent vitamin remediation process in the human body 4. Qualitative Differentiation Assay

Experiment Extensions Use human stem cells instead of murine stem cells 2. Test a wider range of oxidative stressors to mimic more closely oxidative stress in the human body 3. Test a wider range of antioxidants to mimic more closely antioxidant remediation in the human body

Acknowledgements 29 Mr. Mark Krotec Dr. Conrad Zapanta