The Effects of Isotretinoin on C2C12 Stem Cells Colm Parrish Pittsburgh Central Catholic HS Grade 11

Tissue Engineering 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. Has the potential to replace or supplement the function of tissues TE has great potential for supplementing damaged or destroyed muscle tissue.

Adult Stem Cells Undifferentiated cells multiply to replenish dying cells. Somatic stem cells, can be found in juvenile and adult animals and humans. Self-renew indefinitely, and generate all cell types of the organ from which they originate.

C2C12 Stem Cells Subclone of the mus musculus (mouse) myoblast cell line. Common TE model Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

Isotretinoin A naturally occurring retinoic acid Supplemented for treatment of severe acne Can also be used in the treatment of skin cancer Prescribed under the names Roaccutane(formerly Accutane), Amnesteem, Claravis and others

Controversy over Isotretinoin Very powerful teratogen –In 2006, the FDA responded with iPledge A link between depression/anxiety has been suggested, but there is no causal evidence

Purpose To determine the effects of Isotretinoin supplementation on proliferation and differentiation of C2C12 stem cells

Hypotheses Null Hypothesis: Isotretinoin supplementation will have no significant effect on the proliferation and differentiation of C2C12 stem cells. Alternative Hypothesis: Isotretinoin supplementation will significantly increase proliferation and differentiation of C2C12 stem cells.

Materials Isotretinoin solution (Amnesteem) 75mm 2 tissue culture treated flasks Twenty 25 mm 2 tissue culture treated flasks C2C12 Myoblastic Stem Cell Line Trypsin-EDTA Pen/strep Power pipette+ sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM Serum - 1% and Complete 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]) 75 mL culture flask Incubator Nikon Inverted Microscope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water

Procedure 1.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 10 6 to 2x10 6 cells 2.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 4x10 6 to 5x10 6 cells/flask was reached 3.The suspended cells from the culture were passed into 20 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2

4.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 5.4 mL of 10% DMEM media was added to each T25 flask mL of cell suspension was transferred to 18 T25 flasks. Flasks were placed back into incubator and cells were allowed to attach for several hours 7.0.4g of Isotretinoin was added to 10mL of media. This created a stock from which a High Concentration (8x10 -4 ) and a Low Concentration (4x10 -4 ) were derived 8.T25 flasks were removed from incubator and variable added to reach desired concentrations

9.Cell densities were determined as follows: I.The cells were trypsinized and collected into cell suspension. II.25 μl aliquots were transferred to a Hemocytometer for quantification (eight counts per flask). III.Counts were taken on days 1 and 3 10.Images were taken on a Nikon Inverted Microscope I.Two flasks were used per concentration with two images taken per flask per day

Statistical Analyses ANOVA Short for Analysis of Variance Statistical test to find variance between and within groups If the P-Value is smaller than the Alpha Value (0.05), the analysis is significant Dunnett’s Test Follow up to an ANOVA Statistical test to find the source of variance If the T-value is larger then the T-Crit, the effect is significant

The Effects of Isotretinoin on C2C12 Proliferation Concentrations of Isotretinoin Cell Count (Cells/Flask) P-Value 2.26E-9 P-Value 0.E+0

Dunnett’s Test T-CritT-ValueVariation Day 1 Low Not Significant Day 1 High Significant Day 3 Low Significant Day 3 High Significant

Differentiation- Day 1 ControlLowHigh

Day 3 Control LowHigh

Day 8 LowHighControl

Differentiation Analysis Qualitative Analysis- Low concentration enhances myotube formation Control Low

Conclusions The null hypothesis was rejected, suggesting that Isotretinoin supplementation significantly increased the proliferation of C2C12 stem cells. The null hypothesis was rejected as qualitative analysis suggests that supplementation of Isotretinoin enhance the myotube formation of C2C12 stem cells

Limitations Hemocytometer counts can vary Cell Clumping Lag Time Low number of replicates and concentrations

Extensions Increase number of replicates and concentrations Test the effects of Isotretinoin on other cell lines (3T3, MG63) Use antibodies to quantify differentiation CyQUANTTM Cell Proliferation Assay –More quantitative than counting cells on a hemocytometer –Fluorescent dye binds to nucleic acid in cell Test the effects of Isotretinoin on oxidatively stressed cell lines

Resources Mr. Mark Krotec, PTEI Dr. Phil Campbell Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University Dr. Mark Seraly l/isotretinoin

Analysis of Variance (One-Way) Summary GroupsSample sizeSumMeanVariance Control 169, , Low 168, , High 1620,206.1, , ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups 5,587, ,793, E Within Groups3,927, , Total 9,515, Day 1

Analysis of Variance (One-Way) Summary GroupsSample sizeSumMeanVariance Control 169, ,221.4 Low 1613, , High 1624,833.1, , ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups8,256, ,128, E Within Groups1,125, , Total 9,382, Day 3