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

Thesis. The University of Pittsburgh, 2014. - D-Scholarship@Pitt. The University of Pittsburgh, 4 Aug.

2014. Web. 07 Nov. 2015.

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SYSTEMS. Master’s Thesis, University not Cited.

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Proliferation and Insulin-producing-cell Differentiation of ARIP Cells." Scientific Research 2.4

(2009): 216-26. Effects of Extracellular Matrix Proteins on Expansion, Proliferation and

Insulinproducing-cell Differentiation of ARIP Cells. Scientific Researcher, Aug. 2009. Web. 07 Nov.

2015.

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