The Effect of Resveratrol on 3T3 Cell Survivorship. By Anthony Nese Central Catholic High School Grade 11

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1 The Effect of Resveratrol on 3T3 Cell Survivorship By Anthony Nese Central Catholic High School Grade 11

2 Resveratrol Natural phenol that can be extracted from Japanese Knotweed roots and the skin of red grapes. Reported to lengthen life of organisms like yeast, fruit flies. Has failed to extend lifespan of lean, genetically normal mice. Long term effects of supplementation in humans largely unknown.

3 3T3 cells Come from a cell line established in 1962 by two NYU scientists. Originally obtained from Swiss albino mouse tissue. They have become the standard fibroblast (connective tissue) cell line. Plays a large role in wound healing. They do not differentiate, produce ECM and structural proteins.

4 Purpose To determine the effects of different concentrations of Resveratrol on cells of the 3T3 line.

5 Hypotheses Null hypothesis: The Resveratrol will not have a significant effect on 3T3 cell survivorship. Alternate hypothesis: Resveratrol will have a significant effect on 3T3 cells.

6 Materials Cryotank Incubator 25mm^2 tissue culture treated flasks 75mm^2 tissue culture treated flasks Distilled water Ethanol (70% and 100%) Macropipette and sterile tips (1mL, 2mL, 5mL, 10mL, 20mL) Micropipettes and sterile tips Trypsin-EDTA 150 mg Resveratrol Capsules 3T3 Cells 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]) Sterile PBS Fetal Bovine Serum Nikon Inverted Microscope Hemocytometer Labeling Tape Purple Nitrile Gloves Laminar Flow Hood and Sterilizing UV Lamp

7 Cell Culture Procedure A 1 ml aliquot of 3T3 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/ml was reached The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37 C, 5% CO2

8 Addition of Variable on Day 0 Procedure Cells from a T75 flask were resuspended after trypsinization to a density of approximately K/mL. T75 flasks were incubated for 4 minutes at 37 C 4 ml of 10% DMEM media was added to each T25 flask 0.5 ml of cell suspension was transferred to 9 T25 flasks. Flasks were placed back into incubator and cells were allowed to attach for several hours A 1% Resveratrol solution was added to the flasks in different amounts to create 3 flasks in 3 different concentrations.

9 Concentrations 0% 0.001% 0.01% Media 6 ml ml 5.94 Resveratrol 0 ml 6 µl 60 µl Total 6mL 6mL 6mL

10 Cell Counting Procedure For both Day 1 and Day 3 Cell densities were determined by trypsinizing cells in all 9 flasks and collecting them into cell suspension and then 25 µl aliquots were transferred to a Hemocytometer for quantification. 8 counts were taken per flask.

11 Cell Counts Blue=0% Green=.001% Turq.=.01% Day 1 Day 3

12 ANOVA Results Alpha:.05 Day 1 p-value: 7.57 x 10^-34 Day 3 p-value: 2.06 x 10^-34

13 Dunnett's Test Concen. T-Value T-Critical Variation Day % Sig 0.01% Sig Day % Sig 0.01% Sig

14 Conclusions The ANOVA and Dunnett's test show that the Resveratrol had a significant, negative effect on 3T3 cell growth. The null hypothesis can be rejected and the alternative hypothesis accepted.

15 Extensions The cells can be stressed with H2O2, or another variable could be added to weigh variables against each other. Other cell lines could be used. (C2C12, etc.) A wider range of concentrations could be used.

16 Limitations Hemocytometer counts can be inaccurate. Low flask number Cell clumping Lag time CELL HEALTH?

17 Acknowledgments/ Works Cited Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University Mark Krotec, PTEI supplements/lifestyle-guide-11/supplement-guide- resveratrol

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