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