ALOE EFFECTS ON 3 T 3 CELL PROLIFERATION

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ALOE EFFECTS ON 3 T 3 CELL PROLIFERATION AND SURVIVORSHIP Raashmi Krishnasamy Peters Township

ALOE EFFECTS ON 3 T 3 CELL PROLIFERATION AND SURVIVORSHIP Raashmi Krishnasamy Peters Township High School Grade 12 6 th Year in PJAS

Introduction to Wound healing What is a scar? • Body’s natural healing mechanism in

Introduction to Wound healing What is a scar? • Body’s natural healing mechanism in response to injury, trauma, etc. • Tissue formed during healing process Scar Formation Complications • Collagen fibers arranged randomly as opposed to linear, parallel formation

Wound healing: “Pathophysiology of Tissue Repair and Transfer” By Edoardo Austoni, Vincenzo Giannoccaro Platelets,

Wound healing: “Pathophysiology of Tissue Repair and Transfer” By Edoardo Austoni, Vincenzo Giannoccaro Platelets, PMN Macrophages, Fibroblasts, Capillaries

Purpose To examine the effects of Aloe on 3 T 3 cell proliferation and

Purpose To examine the effects of Aloe on 3 T 3 cell proliferation and survivorship

Hypothesis • Aloe has been known to have positive effects on wound repair, however,

Hypothesis • Aloe has been known to have positive effects on wound repair, however, the extent at which it can be effective remains unclear. • If Aloe is applied to 3 T 3 cells in three different concentrations, cell proliferation will be significantly increased for all three of those concentrations. • Furthermore, the highest concentration of Aloe tested will have the most significantly varied results on the 3 T 3 cells, when compared with the other two concentrations tested.

BACKGROUND INFORMATION

BACKGROUND INFORMATION

3 T 3 Cells • Mouse derived fibroblastic cell line • Standard line used

3 T 3 Cells • Mouse derived fibroblastic cell line • Standard line used to simulate human fibroblasts • Cells proliferate extremely rapidly, but growth stops when cell-to-cell contacts are formed • Widely used cell line in research • Biologically immortalized cell line

Variable: Aloe Solution • What is it? • Succulent plant widely used in alternative

Variable: Aloe Solution • What is it? • Succulent plant widely used in alternative medicine • Use can be traced back to 6, 000 years to early Egypt, where plant was known as “plant of immortality” • Modern Use: • clear gel from Aloe plant is rubbed on skin as ointment to treat wounds and burns • green part of leaf surrounding gel can be used to produce a juice or a dried substance (called latex) that can be taken by mouth • Topical use is more common • Used as a folk or traditional remedy for diabetes, asthma, epilepsy, osteoarthritis, burns, sunburns and psoriasis • Topical use of aloe gel may help heal burns and abrasions • Aids in healing of deep surgical wounds

Materials • • • • Scupula • Balance Aloe Leaf Distilled Water Cryotank •

Materials • • • • Scupula • Balance Aloe Leaf Distilled Water Cryotank • One 75 mm 2 tissue culture treated • flasks • Eight 25 mm 2 tissue culture treated flasks • 10% fetal bovine serum • 3 T 3 Cell Line • Trypsin-EDTA • Pen/strep • Macropipette + sterile macropipette Tips (1 m. L, 5 m. L, • 10, m. L, 20 m. L) Micropipettes + sterile tips • Hemocytometer DMEM media (4 m. M L • Permanent marker glutamine, 4500 mg/L glucose, 1 m. M sodium pyruvate, and 1500 • Test tube rack mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) Incubator Aspirating Vacuum Line Nikon Inverted Compound Optical Scope Laminar Flow Hood Labeling Tape Sterile PBS Ethanol (70% and 100%) Distilled water Nikon Inverted Microscope

Procedure: Preparing Variable Stock •

Procedure: Preparing Variable Stock •

Procedure: Preparing the Cells • A 1 m. L sample of 3 T 3

Procedure: Preparing the Cells • A 1 m. L sample of 3 T 3 cells from a Cryotank was used to inoculate 30 m. L of 10% serum DMEM media in a 75 mm 2 culture flask. • The media was replaced with 15 m. L of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2) for 2 days until a cell density of approximately 106 to 2 x 106 cells/m. L was reached. • The culture was passed into 5 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

Procedure: Proliferation • Seeded 20 flasks with 3 T 3 cells from the original

Procedure: Proliferation • Seeded 20 flasks with 3 T 3 cells from the original flasks in 5 m. L of 10% DMEM media each • Allowed cells to incubate in CO 2 incubator overnight and adhere to bottom of flask Control group: • 5 flasks Low concentration of variable group: • 5 flasks • Removed 5μL media • Added 5μL variable Medium concentration of variable group: • 5 flasks • Removed 5μL media • Added 5μL variable • Allowed flasks to proliferate in an incubator overnight High concentration of variable group: • 5 flasks • Removed 5μL media • Added 5μL variable

Procedure: Proliferation(continued) • Next day, trypsinized cells and performed cell counts on the cell

Procedure: Proliferation(continued) • Next day, trypsinized cells and performed cell counts on the cell suspension • • • Rinsed with 1 m. L of trypsin, pipetted out Added 1 m. L trypsin, incubate for 5 minutes Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask Quenched reaction with 5 m. L media. Re-added variable. Re-suspended cells with 1 m. L trypsin wash before taking counts using pipette • Loaded hemocytometer with 20 u. L from flask • Took 8 counts per flask • Counted cells in field of view of hemocytometer under Nikon inverted microscope and multiplied count by 103 for total cells/m. L • Repeated on Day 3

RESULTS

RESULTS

Proliferation Pictures: Day 1 Control Low Medium High

Proliferation Pictures: Day 1 Control Low Medium High

Proliferation Pictures: Day 3 Control Low Medium High

Proliferation Pictures: Day 3 Control Low Medium High

Proliferation Results Cell Counts 600 Cell Counts (in thousands) 500 400 300 Day 1

Proliferation Results Cell Counts 600 Cell Counts (in thousands) 500 400 300 Day 1 Day 3 200 100 0 Control Low Medium Variable Concentration High

STATISTICAL ANALYSIS

STATISTICAL ANALYSIS

ANOVA Test Cell Counts 600 • Day 1 p-value: 3. 03 E-09 • Day

ANOVA Test Cell Counts 600 • Day 1 p-value: 3. 03 E-09 • Day 3 p-value: 3. 05 E -10 Cell Counts (in thousands) 500 400 300 Day 1 Day 3 200 100 0 Control Low Medium Variable Concentration High Test Conclusion: There is very strong evidence that the addition of Aloe will significantly increase the proliferation and survivorship of 3 T 3 cells.

Dunnett’s Test Difference of Average of Experimental Group and Average of Control Group T-Value

Dunnett’s Test Difference of Average of Experimental Group and Average of Control Group T-Value Square Root of two times the MS Value divide by the number of replicates Concentration Day 1 Low – 10 -8 4. 986 Medium - 10 -6 11. 596 High - 10 -4 18. 167 Concentration Day 3 T-Value Low – 10 -8 9. 159 Medium - 10 -6 16. 288 High - 10 -4 16. 981 T-Critical Significance Yes 3. 29 Yes Yes

Conclusion • In conclusion, the data supports my hypothesis that if Aloe is applied

Conclusion • In conclusion, the data supports my hypothesis that if Aloe is applied to 3 T 3 cells in three different concentrations, cell proliferation will be significantly increased for all three of those concentrations. My hypothesis was supported because the results indicated that Aloe significantly increased 3 T 3 cell proliferation. • However, the results of the Dunnett’s Test indicated that, contrary to my hypothesis that only the highest concentration will have the most significantly varied results, all three concentrations of Aloe tested varied significantly when compared to the control. • This shows that any concentration of Aloe can be used to promote 3 T 3 cell growth and enhancethe wound healing process.

Extensions • If I were to do this experiment again, I would test the

Extensions • If I were to do this experiment again, I would test the effects of other phytochemicals on 3 T 3 Cells, such as those from the Neem plant. Also, I would explore the synergistic effects of Aloe with other phytochemicals on 3 T 3 cells to see if they enhance cell counts. • This experiment is applicable to the field of medicine because caring for wounds of any kind, whether it is following a surgery, or it is following a traumatic accident, is a crucial part of the recovery process. Not only will proper wound care prevent infection and other complications, it will also help the wound heal faster with minimal scarring. From the results of this experiment, Aloe vera can be used as a solution to care for and dress wounds in an effective manner. • Additionally, as I mentioned earlier, this experiment directly relates to regenerative medicine, which is a keystone to modern medical treatment.

Acknowledgements and References • Mr. Mark Krotec • Mr. Keith Compeggie • Conrad M.

Acknowledgements and References • Mr. Mark Krotec • Mr. Keith Compeggie • Conrad M. Zapanta, Ph. D. Biomedical Engineering Laboratory, Carnegie Mellon University "Aloe Vera. " National Center for Complementary and Integrative Health. U. S. Department of Health & Human Services, n. d. Web. Austoni, Edoardo, and Vincenzo Giannoccoro. "Pathophysiology of Tissue Repair and Transfer. " Pathophysiology of Tissue Repair and Transfer(n. d. ): n. pag. Web. Knapp, Daniels, and Kaplan. "Pathologic Scar Formation. " National Center for Biotechnology Information. U. S. National Library of Medicine, Jan. 1977. Web. Velcheva, Margarita. "Aloe Vera Transformation: The Role of Amberlite XAD-4 Resin and Antioxidants during Selection and Regeneration. " In Vitro Cellular & Developmental Biology. Plant 46. 6 (2010): 47784. Web. "Wound Care Is an Important Part of Recovery. " Alarys Home Health Care RSS. N. p. , 16 Jan. 2013. Web.

ALOE EFFECTS ON 3 T 3 CELL PROLIFERATION AND SURVIVORSHIP Raashmi Krishnasamy Peters Township

ALOE EFFECTS ON 3 T 3 CELL PROLIFERATION AND SURVIVORSHIP Raashmi Krishnasamy Peters Township High School Grade 12 6 th Year in PJAS