1. CELL ENGINEERING OF SKIN:
CELL EXTRACTION VIA ENZYMATIC TECHNIQUES AND INVESTIGATING CELL MORPHOLOGY AND WOUND HEALING OF
KERATINOCYTE MONOLAYERS WITH AND WITHOUT TRANSFORMING GROWTH FACTOR - BETA 2 (TGF -2)
Agbabiaka Oluwadamilola, UB no: 12017127, BEng Medical Engineering, Supervisor: Dr. M. Youseffi.
1. INTRODUCTION
Cell Engineering has made an impact on wound healing
over the years, tissue engineered skin drafts of various
types have been developed [Chen et al., 2009]. This is
achieved by cultivation of skin cells (keratinocytes) in vitro,
either in a 3D scaffold or culture flasks. The role of TGF -2
of the transforming growth factor beta superfamily is to
regulate proliferation, growth, differentiation and motility
of cells in vivo. [Gazaerly et al., 2013]
Primary keratinocyte (skin) cells were extracted from a 4
day old neonate Swiss white mouse. A wound was then
imitated by scratching the cell monolayer with a sterile
serological pipette. Next, the growth factor (TGF -2) was
added with supplemented media for nutrition and the
distance between the edges of the wound (wound width),
were monitored over the next 94 hours. This experiment
was also performed without the presence of the growth
factor TGF -2 (control experiment).
The cell morphology of keratinocytes monolayers was also
monitored during the experiment and the keratinocytes
had developed fibroblast-like morphologies in vitro. The
image analysis and observations were carried out using the
Visicapture software. The Image J software was used to
measure the distance between the wound edges between
the experiments including the control.
2. AIM
The main aim of this project is the cell engineering of skin
and the main objectives are development of own various
protocols with respect to cell extraction, cell culture, cell
passaging, imaging and counting and also to test the above
hypothesis that TGF -2 alters the rate at which wound
heals.
4. METHODOLOGY
This study was designed to evaluate the effects of Transforming
growth factor beta 2 on wound healing of keratinocytes. A
monolayer of Swiss white mouse keratinocytes was wounded by
a scratch for this purpose and the wound closure was observed
over time. An analysis was also done to examine whether TGF -2
may have influenced the wound closure process.
The lab procedures performed during this study are:
• Cell Extraction and Isolation
• Enzymatic digestion
• Cell Passage
• Scratch Assay
• Cell Counting
5. RESULTS AND ANALYSIS
• The Images below show attempt on scratch assay
• The images below show wound healing over time
• The Table and chart below show the wound healing analysis
6. DISCUSSION AND CONCLUSION
The chart below shows a comparison between Wound 1, 2 and 3
on wound closure over a period of time. From the information
derived, wounds 2 and 3 closed before wound 1. The R2 value,
mean, standard deviation were calculated in this comparison.
Also a T-Test was performed to justify the hypothesis. This
analysis was performed with the use of Microsoft Excel.
The original hypothesis states “TGF -2 alters the wound healing
process of mouse keratinocyte monolayers”, therefore the NULL
hypothesis would state TGF -2 does not make a difference. The
P value is the probability the NULL hypothesis is true and the P
value is always compared with the P critical value which is 0.05.
The conditions are if the P value is less than 0.05 REJECT NULL
hypotheses and if the P value is more than 0.05 ACCEPT NULL
hypotheses [mathworks, 2015]. Two paired sample T-Tests were
carried out, one between wound1 and wound2 while the other
was between wound1 and wound3. From the table above, both
comparisons were more than the critical value 0.05 (W1 vs W2 =
0.73) (W1 vs W3 = 0.76), then it is concluded that for both tests,
there is no statistical difference between the means and
standard deviations.
3. OBJECTIVES
• Development of cell culture, cell passage, cell imaging, cell counting
and cell isolation skills.
• Preparation and investigation of the wound closure response.
• Understanding the wound healing process.
• Good comparison between TGF - 2 and control with respect to wound
healing.
• Investigation of cell morphology in vitro.
REFERENCES
Chen, M. Przyborowski, M. Berthiaume, F., (2009).[Online] ‘Stem
Cells for Skin Tissue Engineering and Wound Healing’.The centre
for engineering medicine, USA.[NCBI]
Gazaerly, H.E. Elbardisey, D.M. Eltokhy, H.M. Teaama, D. (2013),
‘Effect of Transforming Growth Factor Beta 1 on Wound Healing in
Induced Diabetic Rats’. Int. J. Health Sci. (Qassim), Qassim
University. [NCBI]
Images were taken using the Image J software.