Clarke Bacharach 12 th Grade Central Catholic High School Light Wavelength Influence on Stem Cell Behavior

Stressed Cells Stressing a cell is defined as interfering with the cell’s cycle to carry out basic functions. Some different types of cell stress include prolonged exposure to heat, UV Light, and deprivation of food. However, to a certain extent of stress, not all cells will undergo apoptosis. Exposure to variable wavelengths was the stressor in this project

Effects of Different Wavelengths Different colors of light are known to affect photosynthetic activity Unknown effects on numerous cells Research needed Stem cells (?)

C2C12 Cell Line Subclone of the mus musculus (mouse) myoblast cell line. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. Juvenile mouse stem cell line is used as a model in many tissue engineering experiments.

C2C12 (Continued) 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.

Recent Studies Adult stem cell research - relatively new field Need to grow faster and healthier stem cells AdiStem Laser Activation 3 activate stem cells, 2 inhibit them “Trials are ongoing”

Proliferation v. Differentiation Proliferation- cell division  When a cell population proliferates, the population should increase twofold. Differentiation- a cell transforms to a more specialized state. This occurs several times as multicellular organisms change from single zygotes to complex series of tissues and cell types.

Purpose To examine the effects of variable wavelengths (different colored light) on proliferation, differentiation, and survivorship of normal C2C12 cells. To find a more efficient way to grow stem cells

Hypotheses Null - Different wavelengths will not significantly affect proliferation, survivorship, or differentiation of C2C12 stem cells.

Key Questions Which wavelength had the most dramatic effects? What kinds of effects were exhibited? Were the effects of both the wavelengths and serum starvation synergistic?

Materials Cryotank Different colored transparent films (clear, pink, blue, green) 10 petri-dishes 2-75mm 2 tissue culture treated flasks C2C12 Myoblastic Stem Cell Line (approximately 100,000 cell/mL) 10% fetal bovine serum mm 2 tissue culture treated flasks Micro and macro pipettes Trypsin-EDTA 2 Floodlights 75 mL culture flask Incubator Zeis Inverted Compound Optical Scope Aspirating Vacuum Line Laminar Flow Hood Sterile 60x100 mm Petri dish 12 Microtubes 3 Hemocytometers Ethanol (70%)

Procedure (Stem cell 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 cyro-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 100,000 cells/mL was reached. The culture was passed into 2 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

Common Procedure For Both Proliferation and Differentiation After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 2mL of cell suspension was transferred into 10 sterile Petri along with 4mL of 10% fetal bovine serum (media). 2 plates were set aside Clear film was placed over 2 plates. Pink film was placed over 2 plates. Blue film was placed over 2 plates. Green film was placed over 2 plates. The plates which were covered by film were then placed under floodlights for 20 minutes. The petri-dishes were then divided in two and pipetted into 25mm 2 flasks. A total of 20 flasks.

Procedure ProliferationDifferentiation The cells were incubated (37°C, 5% CO 2 ), and cell densities were determined at day 1 and day 3 as follows: Pictures were taken using the inverted microscope. The cells were trypsinized and collected into cell suspension. 25 µl aliquots were transferred to a hemacytometer for quantification. After the first day of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

Experimental Groups ProliferationDifferentiation Control22 Clear22 Pink22 Blue22 Green22

After 20 Minutes of Exposure to Wavelength P= P=0.069 P=1.00 P=0.0372

Average Cell Counts P= P=1.00 P=0.069 P=

Day 3 Counts P=0.012 P=0.104 P=

Day 1 v. Day 3 Counts Cell Counts

Photographic Data Blue Proliferation Day 1 Clear Proliferation Day 1 Control Proliferation Day 1 Green Proliferation Day 1Pink Proliferation Day 1

ANOVA Statistical Analyses Day 1 P-ValueDay 3 P-Value Clear Significant0.012 Significant Pink0.069 Insignificant0.104 Insignificant Blue1.00 Insignificant Significant Green Significant0.186 Insignificant

Data Analysis Significant effects shown for clear light on day 1 and 3 Pink insignificant effects insignificant day 1 and day 3 Blue insignificant day 1, significant day 3 Green significant day 1, insignificant day 3 After day 1 the experimental groups’ cell counts went down Post day 1 the control group’s cell counts went up

Differentiation Pink Day 10 Blue Day 10 Clear Day 10 Green Day 10 Control Day 10

Qualitative Analysis Control vs. Experimental Groups Change in appearance- Insignificant Density- Very Similar Myotube formation- Very Similar

Key Questions Answered Which wavelength had the most dramatic effects? Clear light showed the most significant effects What kinds of effects were exhibited? Increased and decreased survivorship, smaller cells Were the effects of both the wavelengths and serum starvation synergistic? No synergy was exhibited.

Conclusions Null I- Different wavelengths will not affect the stem cells. Rejected Null II- Synergy will not exist between serum starvation and wavelength exposure will not affect the stem cells. Accepted

Limitations & Extensions Limitations Length of the exposure Not all wavelengths were tested One test subject Extensions Grow entirely under experimental wavelengths Test more wavelengths Explore the long-term effects Include more test groups

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