the effects of ibuprofen on stem cell development kevin pfau central catholic high school grade 11
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The Effects of Ibuprofen On Stem Cell Development
Kevin PfauCentral Catholic High SchoolGrade 11
Tissue Engineering 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
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
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.
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 non-muscle cells
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 anti-inflammatory 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.
HypothesesIbuprofen will significantly increase the proliferation
of both the C2C12 and 3T3 cell lines.
Ibuprofen will significantly increase the differentiation of the C2C12 stem cell line.
Null Hypotheses It is hypothesized for this experiment that the
variable will not significantly increase the proliferation of the C2C12 and 3T3 stem cell lines.
It is hypothesized for this experiment that the variable will not significantly increase the
differentiation of the C2C12 stem cell line.
Materials 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)
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)
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) 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) 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.
Procedure (Differentiation Experiment) 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 C2C12 03 C2C12 L3 C2C12 H3
10 64 64 112 125 2215 69 62 119 101 1110 55 65 109 96 1612 68 59 99 87 3113 60 49 80 91 2516 65 36 106 97 1918 58 65 85 98 1617 48 61 101 96 18
Multiply by 103 to reach cells/mL
3T3 01 3T3 L1 3T3 H1 3T3 03 3T3 L3 3T3 H327 20 10 16 10 720 21 11 24 15 1318 16 14 18 13 1419 19 15 20 13 1117 16 12 21 11 1721 17 15 13 15 1622 17 15 18 12 1615 19 12 13 16 18
C2C12 01 C2C12 L1 C2C12 H1 C2C12 03 C2C12 L3 C2C12 H30
20
40
60
80
100
120
C2C12 Proliferation
Num
ber o
f Cel
ls (A
vera
ge)
T3 01 T3 L1 T3 H1 T3 03 T3 L3 T3 H30
5
10
15
20
25
3T3 Proliferation
Num
ber o
f Cel
ls (A
vera
ge)
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 Significa
nt
Significant
ConclusionsFirst 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 ConclusionCells 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