The Effects of UVC Light
on C2C12 Stem Cells
Cory Soltys
Pittsburgh Central Catholic High School
Grade 12
PJAS 2015
Ultraviolet Rays
●  Light waves that have
shorter wavelengths, thus
greater energy, than visible
light
●  Range from 400nm to
100nm
●  Given off by the sun but
most are absorbed by the
ozone layer
Ultraviolet-C Light
●  Shorter wave than UVA or UVB light
●  Typically range around 280nm-100nm
●  Completely absorbed by the ozone layer and
atmosphere
●  Commonly used for Ultraviolet Germination
Irradiation (UVGI)
●  Disinfection method involving the use of UVC light to
kill microorganisms
Damage Caused by UV Light
●  Damage includes skin burn,
sun poisoning, skin
irritation, redness, photoaging, nausea, and possibly
skin cancer
●  FDA Protection methods
include sun screen, hats,
and radiation-blocking
clothing
●  Can cause DNA to form
dimers, leading to
replication errors,
mutations
C2C12 Stem Cell Line
●  Derived from the mus musculus
(mouse) myoblast cell line.
●  A model type of stem cell line that
was discovered in 1977 through
experimentation of murinae thigh
muscle growth after a crush injury.
●  Differentiates rapidly, forming
contractile myotubes and produces
characteristic muscle proteins.
Purpose
●  The purpose of this experiment was to test the
effects of Ultraviolet-C light radiation on C2C12
Stem Cells
Hypotheses
●  Null Hypothesis- Ultraviolet-C light radiation will
NOT have a significant effect on C2C12 cell
proliferation and differentiation
●  Alternative Hypothesis- Ultraviolet-C light radiation
WILL have a significant effect on C2C12 cell
proliferation and differentiation
Materials
●  Cryotank
●  Four 75mm2 tissue culture treated flasks
●  Twelve 25 mm2 tissue culture treated flasks
●  Fetal bovine serum (FBS)
●  C2C12 Myoblastic Stem Cell Line
●  Trypsin-EDTA
●  Pen/Strep
●  Power macropipette
●  Sterile macropipette tips (5mL, 10mL)
●  Incubator
Materials (continued)
●  Inverted microscope
●  Laminar flow hood
●  Laminar flow hood UV Sterilizing Lamp
●  Hemocytometer
●  Ethanol (70%)
●  Nitrile gloves
●  DMEM Media - 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])
●  Micropipettes and sterile tips
Procedure
●  The culture of cells was passed into four 75mm2 flasks in
preparation for experiment and incubated for 2 days at
37° C.
●  After trypsinization, cells from2 all of the flasks were
pooled into 1 common 75mm flask (cell density of
approximately 1 million cells/mL).
●  0.1mL of the cell suspension2 pool was added to each of
the eight proliferation 25mm tissue culture treated
flasks containing 5mL of DMEM media, creating a lower
concentration for cell counting
●  0.2mL of the cell suspension pool
was added to each of
2
the four differentiation 25mm tissue culture treated
flasks containing 5mL of DMEM media, creating a higher
cell density for more accurate and effective imaging
Procedure (continued)
●  Cell cultures (Flasks) were exposed to UVC radiation
(laminar flow tissue culture hood) for the following
time intervals: control (0 minutes), 2 minutes, 5
minutes, 10 minutes with three replicates for each
duration
●  Cells were trypsinized prior to counting to release
them from the bottom of the flask. 0.5mL of trypsin
was inserted into each flask and allowed to incubate
for five minutes and 2mL of media was added to stop
the reaction.
●  Twenty μL of trypsinized cells were loaded into
hemocytometers. Eight cell counts were performed
per group.
Hemocytometer Picture
P-Value=2.0759E-12
Day 1 Dunnett’s Test
T-Crit= 2.88
Comparison
Significant?
T-Value
Control vs. 2 Minutes
No
2.107
Control vs. 5 Minutes
Yes
8.181
Control vs. 10 Minutes
Yes
14.842
P-Value=4.86278E-14
Day 3 Dunnett’s Test
T-Crit=2.88
Comparison
Significant?
T-Value
Control vs. 2 Minutes
Yes
10.519
Control vs. 5 Minutes
Yes
17.887
Control vs. 10 Minutes
Yes
23.446
Day 1 Differentiation
Control
2 Minutes
Day 1 Differentiation
(continued)
5 Minutes
10 Minutes
Conclusion
●  Proliferation
●  Based on ANOVA and Dunnett's statistical analysis, the null
hypothesis has been rejected. The UVC light radiation had a
significant negative effect on the proliferation of the C2C12
stem cell line
●  Differentiation
●  It appears that the addition of extreme amounts (10
minutes) of Ultraviolet-C radiation has a significant negative
effect on the myotube differentiation of C2C12 stem cells.
Limitations
●  Use of plastic flasks (block radiation)
●  Hemocytometer counts
●  Differentiation test is qualitative
20
Future Extensions
●  Use well plates in place of plastic flasks
●  Multiple cell lines
●  More time durations
21
Day 1 Counting Results
Control
2 Minutes
5 Minutes
10 Minutes
594
572
381
335
564
557
397
362
580
562
451
408
564
548
403
339
584
521
290
283
651
572
319
311
607
560
346
323
596
512
344
327
Day 3 Counting Results
Control
2 Minutes
5 Minutes
10 Minutes
1392
634
250
174
1033
602
252
178
1566
656
235
165
1233
627
261
171
1440
698
266
162
1406
502
195
159
961
531
210
177
1021
576
237
154
Day 1 ANOVA
Day 3 ANOVA
References
●  Mark Krotec, PTEI
●  http://www2.centralcatholichs.com/biology/
26