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The Influence of
Scaffoldings on C2C12
Cell Behaviors 8th Grade Emily Mullen St. Mary of the Assumption School
Regenerative Medicine
Replacing injured tissues with tissue constructs fabricated for the needs of each patient.
Tissue Engineering
Principles
Cells
ECM
Hormones
Blood
Supply
Regeneration Defect
Tissue Scaffolds
Provide temporary framework for cell proliferation,
migration, and differentiation
Type/composition varies with purpose, seeded cells, and host
tissue compartment
Many different materials (natural and synthetic) have been
investigated for use in tissue regeneration
Scaffold Types
PLA Scaffolds– popularly used in tissue engineering because of its
biodegradability
PCL Scaffolds– A biodegradable scaffold material (neural engineering)
UBM Scaffolds—Urinary Bladder Matrix is an extracellular matrix (ECM) scaffold. It
is now used in wound care management of partial and full-thickness wounds
where conventional methods for wound care usually fail to give satisfactory
results.
Stem Cells Cell that can produce lineages more specialized than themselves and can renew itself
Spectrum of Stem Cell Behavior:
totipotent embryonic cells
pluripotent cells
multipotent cells
unipotent cells.
Un-specialized cells that can differentiate into various body
tissues and organs
Have potential to cure or treat diseases such as heart disease,
and diabetes
C2C12 Cell Line
Subclone of the mus musculus (mouse) myoblast cell
line.
A common TE experimental model
Differentiates rapidly, forming contractile
myotomes and produces characteristic muscle
proteins.
Useful model to study the differentiation of
nonmuscle cells (stem cells) to skeletal muscle
cells.
Objective
To compare stem cell behaviors on a synthetic and natural
scaffold
HYPOTHESIS
The decellularized matrix will promote the cell behaviors of attachment,
proliferation, and differentiation. A secondary hypothesis is that cells will have a
greater response to a growth factor cue on the decellularized matrix.
Materials PLA Scaffold Material DMEM Media (10% Serum)
C2C12 Cell Line DMEM Media (2% Serum)
Microscope Ethanol
Flasks Trypsin
Pipet Nikon inverted scope/computer interface
Water Growth Factor, FGF2
Rubber Gloves Test Tube Rack
Sterile Pipet Tips 100 mL Graduated Cylinder
Well Plates Decellularized Extracellular Matrix
Laboratory Tongs
Aspirator
Procedure
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. *Between 200,000 and
300,000 cells are in 1mL.
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/mL was reached.
The culture was passed into 2 flasks in preparation for experiment and incubated for 2 days at 37° C, 5%
CO2.
After trypsinization, cells from both of the flasks were pooled into 1 common 75mm2 flask (cell density of
approximately 1 million cells/mL).
1 ml of the cell suspension and 3 ml of media were added to 9 well plates, creating a cell density of
approximately 5.0 x 10^5 cells per flask.
1 ml of the cell suspension and 3 ml of media containing FGF-2 growth factor were added to 9 well plates,
creating a cell density of approximately 5.0 x 10^5 cells per flask.
The cells were incubated (37°C, 5% CO2) for the remainder of the study.
Images were taken on day 4
Before the experiment the UBM and PLA scaffolds were created and sterilized.
The UBM was spin coated in pepsin a technique that had never been done before.
After the experiment, the well plates were imaged using a Nikon computer interface on three areas on the
well plate.
Results
UBM Scaffold with FGF2
UBM Scaffold without FGF2
Control with FGF2
Control without FGF2
With FGF2 Without FGF2
Myotube
Formation Cells have not formed
nearly as many myotubes
Start After Trypsinization
Amount of Cells 200,000 1,000,000
200,000
1,000,000
0
200,000
400,000
600,000
800,000
1,000,000
1,200,000
AM
OU
NT
OF
CEL
LS
POINT IN EXPERIMENT
AMOUNT OF CELLSAmount of Cells Linear (Amount of Cells)
At Carnegie Mellon University during imaging.
Conclusion
The results indicate the following…
The use of bladder decellularized extracellular matrix digested with pepsin to make
into a hydrogel then spin coated onto coverslips had not been done before.
In differentiation media the C2C12 readily differentiated. In the presence of FGF2
there was significant cell growth, but not myoblast differentiation.
The growth factor affected the cells.
There was not a significant difference in cell growth or differention between UBM or
PLA matrix types.
As predicted in the hypothesis, well plates that were not treated with the FGF2 growth factor formed greater myotubes than the samples treated with the growth factor in the media
The hypothesis that stated that each scaffold was able to promote cell attachment,
proliferation and a response to a outside growth factor will be supported.
Possible Limitations
Due to the irregular 3D nature of the PLA construct, it could not be properly
imaged.
The incubator was shared so there is some risk of contamination.
No cell counts were performed.
Only imaged on one day.
Scaffold areas not indentical
No precise differention marker
Cell images may not reflect the cells overall.
Extensions
More consistent compensation and area of scaffold
Employ other types of stem cells
Other growth factors(sYthergistic effects)
Use of a differention marker
References
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Interviews
Campbell, P, Ph.D. Interviewed by E. Mullen. Central Catholic High School.
2:00 P.M., November 3, 2015
Hawranko, B. Interviewed by E. Mullen. Shadyside Place,
6:00 PM October 7, 2015
Krotec, M. Interviewed by E. Mullen. Central Catholic High School, Phone, and Email.
This source was contacted numerous times and acted as a mentor in my experiment.
University Thesis
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TEMPOROMANDIBULAR JOINT: UNIAXIAL COMPRESSION AND BIOCHEMICAL ANALYSIS." Master`s
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