The Effects of Ibuprofen On Stem Cell Development Kevin Pfau Central Catholic High School Grade 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.

Hypotheses Ibuprofen 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  75mm 2 tissue culture treated flasks  25 mm 2 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 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells.  The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached.  The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.  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 K/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 0X10X 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 5X10 5 cells/mL) was added to 12 wells along with 900uL of 1% media .3mL of cell suspension (with concentration of 5X10 5 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 0X10X 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.  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)  Multiply by 10 3 to reach cells/mL

Statistical Analysis Proliferation P-Values C2C12 DAY 1 C2C12 Day 3 C2C12 Total 3T3 Day 13T3 Day 33T3 Total 1.65E E E E-05 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