The Effects of Ibuprofen On Stem
Cell Development
Kevin Pfau
Central Catholic High School
Grade 11
Tissue Engineering
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What is TE?
 The development and manipulation of artificial implants, laboratory-grown
tissues, and genetically engineered cells and/or molecules to replace or
support the function of defective or injured body parts
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Why is TE important?
 It has the potential to replace or supplement the function of tissues
destroyed or compromised in any variety of ways, including:
 Inherent design flaws
 Hereditary/congenital defects or conditions
 Disease
 Trauma
 Damage from an individual’s environment
 Aging
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TE has great potential for supplementing muscle tissue.
C2C12 Stem Cells
Subclone of the mus musculus (mouse) myoblast cell line.
 Differentiates rapidly, forming contractile myotubes and
produces characteristic muscle proteins.
 Mouse stem cell line is used as a model in many tissue
engineering experiments.
 Useful model to study the differentiation of non-muscle
cells (stem cells) to skeletal muscle cells.
 Expresses muscle proteins and the androgen receptor
(AR).
AR- DNA binding transcription factor which regulates
gene expression.
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3T3 Cells
Subclone of embryonic mus musculus fibroblast cells
 Do not differentiate, produce ECM parts and structural
proteins
 Often used to cultivate and study keratinocytes
 Useful for studying of stem cell proliferation in nonmuscle cells
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Ibuprofen
NSAID used to treat arthritis, primary dysmenorrheal, and
fever; also serves as an analgesic.
 Inhibits cyclooxygenase- produces prostaglandins that
promote inflammation, pain, and fever.
 Non selective of the isoforms of cyclooxygenase it inhibits.
Inhibition of COX-2 enzyme leads to the antiinflammatory properties
COX-1 inhibition affects platelet aggregation and the
gastrointestinal tract
Side effects of this drug include: upset stomach,
mild heartburn, diarrhea, constipation; bloating, gas;
dizziness, headache, nervousness; chest pain, weakness of
heart, slurred speech; rapid weight gain; nausea; fever;
bruising, muscle weakness; and sensitivity to light.

Purpose
Determine the effects of Ibuprofen on
proliferation and differentiation in C2C12 and 3T3
cell lines.
Hypotheses
Ibuprofen will significantly increase the proliferation of both the
C2C12 and 3T3 cell lines.
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Ibuprofen will significantly increase the differentiation of the
C2C12 stem cell line.
Null Hypotheses
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It is hypothesized for this experiment that the variable will
not significantly increase the proliferation of the C2C12 and
3T3 stem cell lines.
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It is hypothesized for this experiment that the variable will
not significantly increase the differentiation of the C2C12
stem cell line.
Materials
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75mm2 tissue culture treated flasks
25 mm2 tissue culture treated flasks
10% fetal bovine serum
C2C12 Myoblastic Stem Cell Line
3T3 Fibroblastic Cell Line
Trypsin-EDTA
Pen/strep
Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)
Micropipettes + sterile tips
DMEM media (1% + 10% serum concentrations)
Sterile PBS
70% Ethanol
Laminar Flow Culture Hood
Hemocytometers
Ibuprofen suspension
Microscopes (Zeiss Inverted Scope/imaging system)
Incubator
24 Well Culture Plates
Procedure (Cell Line Culture)
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A 1 mL aliquot of C2C12 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.
The above procedure was repeated for 3T3 cells
Procedure (Addition of Variable on Day 0)
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Cells from a T75 flask were resuspended after trypsinization to a density of
approximately 300-500K/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 theT25 flasks. Flasks were placed back
into incubator and cells were allowed to attach for several hours
5mL of ibuprofen suspension with a concentration of 20mg/mL were added to 45mL of
sterile PBS
T25 flasks were removed from incubator and variable was added to reach desired
concentrations
Above steps were repeated for 3T3 cell line
0
X
10X
0 uL ibuprofen
solution
5 uL ibuprofen
solution
50 uL ibuprofen
solution
1mL 10% DMEM
media
995 uL 10% DMEM
media
950 uL 10% DMEM
media
Procedure (Day 0 Differentiation)
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Cell Suspension was aspirated out of 1 T75 flasks
2mL of trypsin was add as a wash to T75 flask
After wash trypsin was removed and 1 mL of fresh trypsin was added to
each flask
T75 flasks were incubated for 4 minutes at 37° C
.1mL of cell suspension (with concentration of 5X105 cells/mL) was added
to 12 wells along with 900uL of 1% media
.3mL of cell suspension (with concentration of 5X105 cells/mL) was added
to 12 wells different wells 700uL of media
5mL of ibuprofen suspension with a concentration of 20mg/1mL were added
to 45mL of sterile PBS
Well plate was removed from incubator and variable was added to reach
desired concentrations
0
X
10X
0 uL ibuprofen
solution
1 uL ibuprofen
solution
10 uL ibuprofen
solution
50uL 10% DMEM
media
49 uL 10% DMEM
media
40 uL 10% DMEM
media
Procedure (Proliferation Experiment)
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Day 1 and Day 3
 Cell densities were determined as follows:
 The cells were trypsinized and collected into cell suspension.
 25 µl aliquots were transferred to a Hemocytometer for quantification (eight
total counts).
 Using the Nikon Inverted Microscope, images of eight representative areas of each
flask were taken.
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Day 1, Day 3
Using the Nikon Inverted Microscope, images of eight representative
areas of each of the well plates were taken.
Day 2
 The original media was removed and replaced with 1% DMEM media
(serum starvation) to induce myotube differentiation.
 Images were captured on days 0, 1, 3, and 6 to evaluate cell
differentiation.
Results (proliferation)
C2C12 01 C2C12 L1 C2C12 H1
10
64
64
15
69
62
10
55
65
12
68
59
13
60
49
16
65
36
18
58
65
17
48
61
3T3 01
3T3 L1
27
20
18
19
17
21
22
15
3T3 H1
20
21
16
19
16
17
17
19
C2C12 03 C2C12 L3 C2C12 H3
112
125
22
119
101
11
109
96
16
99
87
31
80
91
25
106
97
19
85
98
16
101
96
18
3T3 03
10
11
14
15
12
15
15
12
 Multiply by 103 to reach cells/mL
3T3 L3
16
24
18
20
21
13
18
13
3T3 H3
10
15
13
13
11
15
12
16
7
13
14
11
17
16
16
18
C2C12 Proliferation
Number of Cells (Average)
120
100
80
60
40
20
0
C2C12 01
C2C12 L1
C2C12 H1
C2C12 03
C2C12 L3
C2C12 H3
T3 L3
T3 H3
3T3 Proliferation
Number of Cells (Average)
25
20
15
10
5
0
T3 01
T3 L1
T3 H1
T3 03
Statistical Analysis
Proliferation P-Values
C2C12
DAY 1
C2C12
Day 3
C2C12
Total
3T3 Day 1
3T3 Day 3
3T3 Total
1.65E-11
3.65E-13
1.57E-24
0.000101
0.019789
2.24E-05
Significant
Significant
Significant
Significant
Not
Significant
Significant
Conclusions
First null hypothesis can be rejected
in most cases.
Differentiation (control)
Day 1
Day 3
Differentiation continued (X
concentration) Day 1
Day 3
Differentiation continued (10X
concentration) Day 1
Day 3
Differentiation Conclusion
 Cells
show significant myotube formation
under low concentrations of variable
 Control group and high variable
concentrations show insignificant
differentiation
 Cell death and poor cells growth was a
problem in some groups
 Not enough significant data to reject null
hypothesis
Project Limitations and Extensions
 Only used qualitative assay of differentiation /
Utilize quantitative assay (MyoD expression)
 Test more variable concentrations
 Test other NSAID’s or polypeptide hormones
 CyQUANT™ Cell Proliferation Assay
 More quantitative than counting cells on a
hemocytometer
 Fluorescent dye binds to nucleic acid in cell