Upload
sam-boland
View
19
Download
0
Embed Size (px)
DESCRIPTION
yee
Citation preview
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