A Toxicity Analysis of Vascular Scaffold Degradation

1Vitamin B Effect on Stressed Mammalian CellsNeil CarletonGrade 11Pittsburgh Central Catholic High SchoolPJAS 20121Introduction: C2C122Stem Cell -- unspecialized cell characterized by the capacity to give rise to various differentiated cell typesTo model human stem cells, C2C12 mus musculus (mouse) myoblast cell line usedC2C12 cells used to model cell differentiation from stem cell state to skeletal muscle state through myotube structure formationStem cells have uses in research and treatmentCancer, Type 1 Diabetes, Parkinsons Disease, Huntingtons Disease, Celiac Disease, cardiac failure, muscle damage, neurological disorders

Found in embryos and adults of many multi-cellular organisms, stem cells have the remarkable ability to develop into many different specialized cell types in the body. In my experiment, as a model for human stem cells, C2C12 mouse myoblastic stem cells were used. C2C12 cells differentiate by individual myoblast cells fusing to form myotubes; this myogenisis is triggered when the serum concentration is decreased from 10% to 1%. The potency properties give stem cells the potential to treat many currently incurable complications and diseases such as cancer, Type 1 diabetes, Parkinsons, Huntingtons, and Celiac Disease, cardiac failure, muscle damage and neurological disorders, along with many others.2Introduction: MG63 3Human cancer (osteosarcoma) cell lineCancerous bone tumor that usually develops during the period of rapid growth that occurs in adolescence, as a teenager matures into an adult.

Oxidative Stress4Free radicals of the reactive oxygen species (ROS) can damage cells in a process called oxidative stressReactive species may account for the increase risk of cancer development and may promote malignancyHydrogen Peroxide (H2O2) used to cause oxidative stress

Oxidative stress is caused by the damaging effect of reactive oxygen species (free radical and peroxides) on cells. Reactive oxygen species or ROS are free radicals, molecules with unpaired electrons and without a full valence shell, that contain oxygen. The electron configuration of the molecules makes them highly reactive and potentially damaging in a biological environment. These free radicals form naturally as part of the bodys natural metabolization process. External factors such as pollution, sunlight and smoking also trigger the production of free radicals. ROS can cause damage to DNA oxidation of fats, proteins, and enzymes inactivating them. Though ROS also has beneficial effects in low concentrations such as cell signaling and slowing aging oxidative stress, it can cause atherosclerosis, Parkinson's disease, Heart Failure, Myocardial Infarction, Alzheimer's disease, Fragile X Syndrome and chronic fatigue syndrome. In my experiment hydrogen peroxide (H2O2), a volatile oxidant, was used to model the effect of oxidative stress on the stem cells.4Vitamin B5

Supports and increases the rate ofmetabolismMaintain healthy skin, hair, andmuscle toneEnhanceimmuneandnervous systemfunctionPromotecell growthanddivision, including that of thered blood cellsthat help preventanemiaReduce the risk of some cancers

Cells do have a defense to free radical damage, however, in the form of antioxidants. Antioxidants, such as vitamin E, vitamin C, and beta carotene, essentially neutralize the free radicals making them harmless to the cell. Antioxidants can stop the oxidative reactions by removing free radical intermediates. As with many, if not all, substances, antioxidants have damaging effects at high concentrations. In my experiment ascorbic acid (Vitamin C) is used to combat the effects of oxidative stress. As I had mentioned earlier, oxidative stress is potentially harmful to cells and can cause major complications and problems in a stem cell transplant. Also, antioxidants are known to be used to combat these effects.5Objective 6Investigate 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 productsDifferentiation: quantitatively measured by counting the number of myosin positive nuclei out of total nuclei in cell photomicrographSo my objective is to investigate the main effects and interaction effect of oxidative stress and antioxidants on mouse myoblastic stem cells, in terms of cell proliferation, differentiation, and toxicity. Toxicity and proliferation effects were measured by counting the number of surviving stem cells after exposure to different concentrations of degradation products. Differentiation was quantitatively measured by counting the number of myosin positive nuclei out of the total nuclei in a cell photomicrograph. Myosin are the motor proteins that provide contractile force of a muscle. In the C2C12 myoblastic stem cells their confluence indicates differentiation.6Hypotheses7As 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 cellssurvivorship will increase even with a stressor present.As oxidative stress levels increase, introduction of a vitamin will decrease the survivorship of the MG63 cellseven though oxidative stress promotes cancer growth, the vitamin will impede their growth. *say something7Hypotheses8Null Hypothesis: Stress would not significantly affect mammal cell proliferation or differentiation.

Alternative Hypothesis: Stress would significantly effect mammal cell proliferation and differentiation.

Materials and Apparatus93% concentrated H2O2Liquid Vitamin BC2C12 murine myoblastic stem cells MG63 Human osteosarcoma cell lineDeionized sterile water100 mL graduated cylinderTest tube rackIncubator (37.0C)Macropippette with tips100 - 1000 L pipette0.1 1 mL pipette1 10 mL pipette70% Ethanol (for sterilization)Felt-tip marker15 mL sterile conical tubes1 24-well platesDMEM media (10% calf serum & 1% calf serum) contains salts, amino acids, vitamins, & glucoseSterile pipette tips0.22 micron syringe filters + 10 mL syringe200 g scales75 mL culture flasks12 25 cm2 culture flasks50 mL Trypsin-EDTA32 mL PBS saline32 mL 100% ice-cold ethanolPenn Strep Solution2 HemocytometersLight microscopeInverted microscope (with imaging capabilities)Class II Biosafety hoodLabcoats, Eye Protection, Disposible GlovesAnti Myo D stainDAPI nuclear stainVortexorDelicate task wipesCounterAluminum foil This is a list of the materials and apparatus used in my experiment. The main materials are the hydrogen peroxide, the ascorbic acid and the C2C12 stem cells. 9100 H2O2Low H2O2High H2O20 Vit. B0 H2O210 L H2O2100 L H2O20 Vit. B0 Vit. B0 Vit. B4 mL media3.99 mL media3.9 mL media1 mL cells1 mL cells1 mL cellsLow Vit. B0 H2o210 L H2O2100 L H2O250 L Vit. B50 L Vit. B50 L Vit. B3.95 mL media3.94 mL media3.85 mL media1 mL cells1 mL cells1 mL cellsTotal=5mLH2O2 ConcentrationVit. B ConcentrationExperimental DesignProcedure11Preparation of Treatment Materials113 L 3% H2O2 diluted with 9.89 mL sterilized deionized water to yield 1 mM concentration of H2O21 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 Culture1 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 106 to 2*106 cellsMedia changed with 15 mL fresh media to remove cryo-freezing fluid and incubated (37 C, 5% CO2) for 2 days until a cell density of approximately 4*106 to 5*106 cells/mL was reachedThe culture was passed into 3 75 mL culture flasks in preparation for experiment (48 hours before)The first part of the procedure treatment materials. This involved diluting both hydrogen peroxide and ascorbic acid to a 10 micromolar concentration. The second part involved culturing the C2C12 cells and incubating for 2 days to reach a cell density of approximately 4 to 5 million cells per mL. 11Procedure (contd.)12Treatment Application (Proliferation and Differentiation: Day 0)12 25 cm2 culture flasks were labeled - 24 for proliferation/toxicityTreatment materials and other materials pipetted into each of 12 flasks in biosafety hood then left to incubate for 24 hours (see table)

The third part was to prepare the groups. This was done for proliferation and differentiation on Day 0

12Procedure (contd.)13Cell Counting (Proliferation: Day 1 and 3)For proliferation assay, aspirated off current mediaAdded 2mLtrypsin and aspirate immediatelyAdded 1mL trypsin andincubate for 4 minutesSmacked side of flasks hard twice to detach cells from flask bottomTransfered 1 mL of cells to 1 mL tubes in rack using pipetteCleaned hemocytometer using 70% ethanol and delicate task wipesInserted 25 L of cell solution into each end of hemocytometer making sure solution wicks across hemocytometer face in an even coatingGently placed cover slip on hemocytometer and examined hemocytometer gridUsing the counter, counted and recorded the number of cells in the 3 mm by 3 mm grid

The fourth part involved counting the cells. On Days 1 and Day 3 for the proliferation assay, the cells were trypsinized to detach them from the flask wall and 25 L aliquots were transferred to each end of a hemocytometer for quantification. 13Procedure (contd.)14Media Replacement (Proliferation: Day 2)For all cells, aspirated off current media in the process removing cell waste productsAdded 3.9 mLof media and appropriate concentration of treatment materials to each group as specified in the previous table,Gently shookto spreadcells and left cells in incubatorfor 24 hoursSerum Starvation (Differentiation: Day 2)Aspirated off mediaAdded 3.9 mL of 1% serum media to cells in flaskAddedappropriate concentration of degradation materials to each group,gently shookto spreadcells, and left cells in incubatorfor 12 hoursThe fifth part involved media replacement. This process exchanges the media to remove cell waste products. The sixth part dealt with the differentiation experiment. On day 3, the 10% serum media was replaced with 1% serum media to induce myotube differentiation.14Procedure (contd.)15Well Plate Transfer (Differentiation: Day 2)For differentiation assay cells, repeated steps 1 through 4 of cell counting (using trypsin to detach cells from flask wall)For each group, labeled three wells on the 24-well platesPippetted 0.4 mL of cell solution from each flask to each of the three corresponding wellsAdded 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 tableGently shook to spread cells and left cells in incubator for 36 hours

In the seventh part of the experiment, the differentiation cells were transferred to well plates in preparation for the staining process.15Procedure (contd.)16Cell Photomicrography (Differentiation)Turned on inverted microscope optical imaging system and connected computer, opened imaging softwareWiped condensation off lid of well plates with delicate task wipesAdjust focus, white balance, and exposure as necessaryFor 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)Obtained quantitative result by creating ratio of myosin positive nuclei (number of nuclei within green myosin stain) to total nuclei in cell photomicrographThe final part of the differentiation was to take the cell photomicrographs of the fluorescing stains by exciting it them with blue and UV light and using an inverted microscope imaging system. A quantitative result of differentiation was obtained by creating ratio of myosin positive nuclei, that is the number of nuclei within green myosin stain, to total nuclei in cell photomicrograph.16Procedure (contd.)17 Days30124Experiment: Survivorship/ProliferationTreatment Product Preparation and Stem Cell Line cultured (both experiments)Treatment ApplicationTreatment ApplicationCell Count TakenMedia ReplacementCell Count TakenSerum Starvationand Well TransferCells Fixedand StainedExperiment:DifferentiationCell PhotomicrographyThis timeline shows the process of the toxicity/proliferation and differentiation experiments.

17Results: C2C12 Proliferation Day 118Vit. B Concentrations P-value: 0.0929918Results: C2C12 Proliferation Day 319Vit. B Concentrations P-value: 0.000222As you can see there were more cells in the treatment groups A and B compared to the control groups at the end of the five-day period.19Results: MG63 Proliferation Day 120Vit. B Concentrations P-value: 0.17034320Results: MG63 Proliferation Day 321Vit. B Concentrations P-value: 6.35E-08 21Conclusion: Proliferation22C2C12MG63Day 1Insignificant Synergistic EffectInsignificant Synergistic Effect

Day 3Significant Synergistic EffectSignificant Synergistic EffectDifferentiation Assay23

Control

Day 1Day 3Differentiation Assay24Low Concentration of H2O2 + Vit. B

Day 1Day 3Differentiation Assay25High Concentration of H2O2 + Vit. BDay 1Day 3

Conclusion26Proliferation: 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.

Limitations27Murine stem cells may not have provided accurate representation of human stem cellsConstant and direct exposure to only hydrogen peroxide may not accurately represent oxidative stress process in the human bodyConstant and direct exposure to only Vitamin B may not accurately represent vitamin remediation process in the human bodyQualitative Differentiation AssayPossible sources of error and limitations include, first, the fact that murine stem cells may not have provided an accurate representation, second, in the human body oxidative stress may not only be caused by a constant exposure of cells to hydrogen peroxide and antioxidant remediation may not only be caused by a constant exposure to Vitamin C.

27Experiment Extensions28Use human stem cells instead of murine stem cellsTest a wider range of oxidative stressors to mimic more closely oxidative stress in the human bodyTest a wider range of antioxidants to mimic more closely antioxidant remediation in the human body

The experiment may be improved and extended by human stem cells in place murine stem cells, testing a wider range of oxidative stressors that mimic more closely oxidative stress in the body and testing wider range of antioxidants that mimic more closely antioxidant remediation in the body28Acknowledgements29Mr. Mark KrotecDr. Conrad ZapantaThank you. I would now like to answer any questions that you may have at this time.

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