Transcript Krishnasamy PJAS 14

ALOE EFFECTS ON 3T3 CELL
PROLIFERATION AND SURVIVORSHIP
Raashmi Krishnasamy
Peters Township High School
Grade 12
6th 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-tocell 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
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Scupula
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Balance
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Aloe Leaf
Distilled Water
Cryotank
One 75mm2 tissue culture
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treated flasks
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Eight 25 mm2 tissue culture
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treated flasks
10% fetal bovine serum
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3T3 Cell Line
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Trypsin-EDTA
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Pen/strep
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Macropipette + sterile
macropipette Tips (1 mL, 5 mL, •
10, mL, 20 mL)
Micropipettes + sterile tips
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DMEM media (4 mM L•
glutamine, 4500 mg/L glucose, •
1 mM sodium pyruvate, and
1500 mg/L sodium
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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:
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑎𝑙𝑜𝑒 𝑔𝑒𝑙
× 100 = 1.00%
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑑𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝑤𝑎𝑡𝑒𝑟 + 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑎𝑙𝑜𝑒 𝑔𝑒𝑙
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
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 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
Low
Medium
High
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
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 3T3 cells.
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
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
T-Critical
Significance
Yes
3.29
Yes
Yes
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.
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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.
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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): 47784. 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 Krishnasamy
Peters Township High School
Grade 12
6th Year in PJAS