Manipulating Cell

Migration Using
Amphiphiles
Final Presentation

Background
Amphiphiles are molecules that are both hydrophilic and
lipophilic
Examples include cholesterol, fatty acids, and some
alcohols

Previous research performed by the Mechanobiology

Laboratory at Penn State suggests that amphiphiles can
modify a cells ability to adhere to a surface

Hypothesis
If amphiphiles are applied to cellular media, then the
number of cells that migrate across a demarcation line
will be different compared to a control because
amphiphiles can manipulate the grouping of cellular
adhesion molecules.
Amphiphile to be tested:
Benzyl Alcohol

Experimental Setup
Cells are grown until confluent
Cells removed from region, samples are imaged
Amphiphiles applied to half of samples
Cells migrate for 8 hours and are then imaged again
Number of cells that migrate
are counted
Numbers from samples
containing amphiphiles
compared to controls

Trial 1: Razor Blade Assay

DAPI

Phase

Demarcation line created using razor blade. Cells then

scratched away with blade
Number of migrated cells counted
Benzyl alcohol and ethanol tested

Trial 1 Analysis
Ethanol correlated with control as expected
High numbers from Benzyl Alcohol experiment suggested
cells were not being effectively scraped away
New Experimental design was needed

Trial 2: Pipet Wound

Pipet scratched along surface of cell culture to remove
cells
Cells imaged and then location of image is marked on
lid
After migration, cells are imaged again
Region
of
interest

Pre-migration

Post-migration

DAPI

*Incorrect
excitation filter
used resulting in
no DAPI image*

Trial 2 Analysis
Numbers conformed to expectations but experimental
setup had issues
Cells removed much more effectively than in razor
wound
Borders of region of interest much more poorly defined
Location of imaging difficult to mark pre-migration
and post-migration images probably not the same

Trial 3: PDMS Square

Square of PDMS material
placed on bottom of
culture before cells are
seeded
Once cells become
confluent, PDMS is
removed revealing open
region
Images taken before and
after migration

Trial 3 Analysis
Experiment cut short by contamination due to
improperly sterilized PDMS
Results encouraging

Trial 4: PDMS Columns

Experiment designed to mirror typical cell migration kits
that can be purchased

Columns made of PDMS placed in contact with bottom

on one end and lid on other
Cells grow to confluence, then columns removed

Homemade Columns

Micropipet containers filled with PDMS. Micropipets

placed in containers and then filled with PDMS
Cut to size of wells in well plate

Trial 4 Results
Images stitched together using ImageJ software
Number of cells migrated in Benzyl Alcohol sample
barely different from Control
Ctrl Pre-migration

Ctrl Post-migration

BA Pre-migration

BA Post-migration

Trial 4 Issues

There are cells here that

were not captured in an
image
Entire region of interest not
captured by images area of
each image not held constant

In order to be a valid
experiment, entire area
created by PDMS columns would
need to be imaged

Trial 5: Smaller Columns

Originally tried just using small pipet tips not effective

Filled pipets with PDMS, let small bit drip out bottom
New columns taped to bottom of well plate

Trial 5 Analysis
Boundaries not very well-defined
Some wells unusable - not enough to complete
experiment

Experimental design
getting too complicated
introducing new variables

Trial 6 Overview
Repeat of trial 2 but modified
Experiment performed in well plate instead of cell
culture
Wells marked underneath with marker so it is easy to
match up pre- and post-migration images

Benzyl Alcohol Results

3 wells, 3 images from each well
Total # of migrated cells: 206
Average # of migrated cells per well: 69
Pre-migration

Post-migration

Control Results
Pre-migration

Post-migration

3 wells, 3 images from each well

Total # of migrated cells: 296
Average number of migrated cells per well: 99

Conclusions
90 more cells migrated in the control model than in the
Benzyl Alcohol model
On average, 30 more per well
Wells treated with BA saw 30% less migration

This experiment confirms the hypothesis that

amphiphiles affect cell migration, most likely by
manipulating adhesion
More trials can be completed to confirm results