Manipulating Cell Migration Using

AmphiphilesFinal Presentation

BackgroundAmphiphiles are molecules that are both

hydrophilic and lipophilicExamples include cholesterol, fatty acids, and some

alcohols

Previous research performed by the Mechanobiology Laboratory at Penn State suggests that amphiphiles can modify a cell’s ability to adhere to a surface

HypothesisIf 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 SetupCells are grown until confluentCells removed from region, samples are imagedAmphiphiles applied to half of samplesCells 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

Demarcation line created using razor blade. Cells then scratched away with blade

Number of migrated cells countedBenzyl alcohol and ethanol tested

DAPI Phase

Trial 1 AnalysisEthanol correlated with control as expectedHigh 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 cellsCells imaged and then location of image is marked

on lidAfter migration, cells are imaged again

Pre-migration Post-migration DAPIRegion of interest

*Incorrect excitation filter used resulting in no DAPI image*

Trial 2 AnalysisNumbers conformed to expectations but

experimental setup had issuesCells removed much more effectively than in razor

woundBorders of region of interest much more poorly

definedLocation of imaging difficult to mark – pre-

migration and post-migration images probably not the same

Trial 3: PDMS SquareSquare 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 AnalysisExperiment cut short by contamination due to

improperly sterilized PDMSResults encouraging

Trial 4: PDMS ColumnsExperiment 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 ResultsImages stitched together using ImageJ softwareNumber 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

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

There are cells here that were not captured in an image

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 AnalysisBoundaries not very well-definedSome wells unusable - not enough to complete

experiment

• Experimental design getting too complicated – introducing new variables

Trial 6 OverviewRepeat 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

Pre-migration Post-migration

• 3 wells, 3 images from each well

• Total # of migrated cells: 206

• Average # of migrated cells per well: 69

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

Conclusions90 more cells migrated in the control model than

in the Benzyl Alcohol modelOn average, 30 more per wellWells 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