The Effects of Isotretinoin on C2C12 Stem Cells Colm Parrish Pittsburgh Central Catholic HS Grade...
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Transcript of The Effects of Isotretinoin on C2C12 Stem Cells Colm Parrish Pittsburgh Central Catholic HS Grade...
The Effects of Isotretinoin on C2C12 Stem Cells
Colm ParrishPittsburgh 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)• 75mm2 tissue culture
treated flasks• Twenty 25 mm2 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 106 to 2x106 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 4x106 to 5x106 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 300-500K/mL. T75 flasks were incubated for 4 minutes at 37° C
5. 4 mL of 10% DMEM media was added to each T25 flask
6. 0.5 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 MicroscopeI. 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
Control Low (4x10^-4) High (8x10^-4)0
20,000
40,000
60,000
80,000
100,000
120,000
140,000
160,000
180,000
Day 1
Day 3
Concentrations of Isotretinoin
Cell
Cou
nt
(Cells
/Fla
sk)
P-Value 2.26E-9
P-Value 0.E+0
Dunnett’s TestT-Crit T-Value Variation
Day 1 Low 2.61 0.801791049 Not Significant
Day 1 High 2.61 6.493310793 Significant
Day 3 Low 2.61 4.57040739 Significant
Day 3 High 2.61 17.51374747 Significant
Differentiation- Day 1
Control Low High
Day 3
Control Low High
Day 8
Low HighControl
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
www.PTEI.orghttp://www.ncbi.nlm.nih.gov/pubmed/19126049https://www.aad.org/dermatology-a-to-z/diseases-and-treatments/i---l/isotretinoinhttp://www.nlm.nih.gov/medlineplus/druginfo/meds/a681043.htmlwww.ipledgeprogram.com
Analysis of Variance (One-Way)
Summary
Groups Sample size Sum Mean Variance
Control 16 9,354. 58400.625 3,773.85Low 16 8,014. 50000.875 5,083.85
High 16 20,206. 1,26200.875 252,995.85
ANOVA Source of Variation SS df MS F p-level F crit
Between Groups
5,587,632.66667 2 2,793,816.33333 32.00815 2.69E-9 4.27271
Within Groups 3,927,803.25 45 87,284.51667
Total9,515,435.9166
7 47
Day 1
Analysis of Variance (One-Way)
Summary
Groups Sample size Sum Mean Variance
Control 16 9,164. 57200.75 2,221.4
Low 16 13,253. 828.003125 8,360.62917
High 16 24,833. 1,55200.0625 64,458.4625
ANOVA
Source of Variation SS df MS F p-level F crit
Between Groups 8,256,955.875 2 4,128,477.9375 165.05001 0.E+0 4.27271
Within Groups 1,125,607.375 45 25,013.49722
Total 9,382,563.25 47
Day 3