ALOE EFFECTS ON 3T3 CELL PROLIFERATION AND SURVIVORSHIP

Raashmi KrishnasamyPeters Township High School

Grade 126th Year in PJAS

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, PMN

Macrophages, Fibroblasts,

Capillaries

Purpose

To examine the effects of Aloe on 3T3 cell proliferation and survivorship

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 3T3 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 3T3 cells, when compared with the other two concentrations tested.

BACKGROUND INFORMATION

3T3 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 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• One 75mm2 tissue culture

treated flasks• Eight 25 mm2 tissue culture

treated flasks• 10% fetal bovine serum• 3T3 Cell Line• Trypsin-EDTA• Pen/strep• Macropipette + sterile

macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)

• Micropipettes + sterile tips• DMEM 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])

• 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• Hemocytometer• Permanent marker• Test tube rack

Procedure: Preparing Variable Stock• Extracted gel from aloe plant – cut leaf open and scrape gel out using scupula

• Record mass of an empty weighing tray

• Place gel into plastic weighing tray

• Add gel and enough distilled water into beaker to create a 1.00% of Aloe Solution

• Calculate mass percent using:

Procedure: Preparing the Cells• A 1 mL sample of 3T3 cells from a Cryotank was used to inoculate 30 mL of 10%

serum DMEM media in a 75mm2 culture flask.

• 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 106 to 2x106 cells/mL was reached.

• The culture was passed into 5 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

Procedure: Proliferation• Seeded 20 flasks with 3T3 cells from the original flasks in 5mL of 10% DMEM media

each

• Allowed cells to incubate in CO2 incubator overnight and adhere to bottom of flask

• Allowed flasks to proliferate in an incubator overnight

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

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 suspension• Rinsed with 1mL of trypsin, pipetted out• Added 1mL trypsin, incubate for 5 minutes• Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask• Quenched reaction with 5mL media. Re-added variable.• Re-suspended cells with 1mL trypsin wash before taking counts using pipette

• Loaded hemocytometer with 20uL 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/mL

• Repeated on Day 3

RESULTS

Proliferation Pictures: Day 1 Control Low Medium High

Proliferation Pictures: Day 3

Control LowMedium High

Proliferation Results

Control Low Medium High 0

100

200

300

400

500

600

Cell Counts

Day 1Day 3

Variable Concentration

Cell C

ounts

(in

thousands)

STATISTICAL ANALYSIS

ANOVA Test

• Day 1 p-value: 3.03 E-09 • Day 3 p-value: 3.05 E -10

Test Conclusion:

There is very strong evidence that the addition of Aloe will significantly increase the proliferation and survivorship of 3T3 cells.

Control Low Medium High 0

100

200

300

400

500

600

Cell Counts

Day 1Day 3

Variable Concentration

Cell C

ounts

(in

thousands)

Dunnett’s TestT-Value

Difference of Average of Experimental Group and Average of Control Group

Square Root of two times the MS Value divide by the number of replicates

Concentration T-Value T-Critical Significance

Low – 10-8 4.986

3.29

Yes

Medium - 10-6 11.596 Yes

High - 10-4 18.167 Yes

Concentration T-Value T-Critical Significance

Low – 10-8 9.159

3.29

Yes

Medium - 10-6 16.288 Yes

High - 10-4 16.981 Yes

Day 1

Day 3

Conclusion• In conclusion, the data supports my hypothesis that if Aloe is applied to 3T3 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 3T3 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 3T3 cell growth and enhancethe wound healing process.

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

phytochemicals on 3T3 Cells, such as those from the Neem plant. Also, I would explore the synergistic effects of Aloe with other phytochemicals on 3T3 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. 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): 477- 84. Web.

"Wound Care Is an Important Part of Recovery." Alarys Home Health Care RSS. N.p., 16 Jan. 2013. Web.

ALOE EFFECTS ON 3T3 CELL PROLIFERATION AND SURVIVORSHIP

Raashmi KrishnasamyPeters Township High School

Grade 126th Year in PJAS