Light Wavelength Influence on Stem Cell Behavior

Clarke Bacharach12th GradeCentral Catholic High SchoolLight Wavelength Influence on Stem Cell Behavior

Stressed CellsStressing a cell is defined as interfering with the cells 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 WavelengthsDifferent colors of light are known to affect photosynthetic activity

Unknown effects on numerous cellsResearch needed Stem cells (?)

C2C12 Cell LineSubclone 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.

4C2C12 (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 StudiesAdult stem cell research - relatively new fieldNeed to grow faster and healthier stem cells

AdiStem Laser Activation3 activate stem cells, 2 inhibit themTrials are ongoing

Proliferation v. DifferentiationProliferation- cell divisionWhen 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.

PurposeTo 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

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

Key QuestionsWhich wavelength had the most dramatic effects?

What kinds of effects were exhibited?

Were the effects of both the wavelengths and serum starvation synergistic?

MaterialsCryotankDifferent colored transparent films (clear, pink, blue, green)10 petri-dishes2-75mm2 tissue culture treated flasksC2C12 Myoblastic Stem Cell Line (approximately 100,000 cell/mL)10% fetal bovine serum20 -25 mm2 tissue culture treated flasksMicro and macro pipettesTrypsin-EDTA2 Floodlights

75 mL culture flaskIncubatorZeis Inverted Compound Optical ScopeAspirating Vacuum LineLaminar Flow HoodSterile 60x100 mm Petri dish12 Microtubes3 HemocytometersEthanol (70%)

11Procedure (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 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells.The media was replaced with 15 mL of fresh media to remove cyro-freezing fluid and incubated (37 C, 5% CO2) 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% CO2.

Common Procedure For Both Proliferation and DifferentiationAfter trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 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 25mm2 flasks. A total of 20 flasks.

ProcedureProliferationDifferentiationThe cells were incubated (37C, 5% CO2), 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 l 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 GroupsProliferationDifferentiation Control22Clear22Pink22Blue22Green22After 20 Minutes of Exposure to Wavelength P=0.00882P=0.069P=1.00P=0.0372Average Cell CountsP=0.0372P=1.00P=0.069P=0.00882Day 3 CountsP=0.012P=0.104P=0.000986Day 1 v. Day 3 CountsCell CountsPhotographic Data

Blue Proliferation Day 1Clear Proliferation Day 1

Control Proliferation Day 1

Green Proliferation Day 1

Pink Proliferation Day 1ANOVA Statistical Analyses Day 1 P-ValueDay 3 P-Value Clear0.00882 Significant0.012 SignificantPink0.069 Insignificant0.104 InsignificantBlue1.00 Insignificant0.000986 SignificantGreen0.0372 Significant0.186 Insignificant Data AnalysisSignificant 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 groups cell counts went up

Differentiation

Pink Day 10

Blue Day 10

Clear Day 10

Green Day 10Control Day 10Qualitative AnalysisControl vs. Experimental Groups

Change in appearance- Insignificant

Density- Very Similar

Myotube formation- Very Similar

Key Questions AnsweredWhich 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.

ConclusionsNull 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 & ExtensionsLimitationsLength of the exposureNot all wavelengths were testedOne test subject

ExtensionsGrow entirely under experimental wavelengthsTest more wavelengthsExplore the long-term effectsInclude more test groups

Sources