Optimizing Usage of Microfluidic Chemotaxis Chambers for Cancer Research Kathleen O’Hara Virginia...
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Transcript of Optimizing Usage of Microfluidic Chemotaxis Chambers for Cancer Research Kathleen O’Hara Virginia...
Optimizing Usage of Optimizing Usage of Microfluidic Chemotaxis Microfluidic Chemotaxis
Chambers for Cancer Chambers for Cancer ResearchResearch
Kathleen O’HaraKathleen O’HaraVirginia Polytechnic Institute and State Virginia Polytechnic Institute and State
UniversityUniversityBiological Sciences and Psychology, Spring 2006Biological Sciences and Psychology, Spring 2006
[email protected]@vt.eduMentor: Dr. Noo Li JeonMentor: Dr. Noo Li Jeon
Department of Biomedical EngineeringDepartment of Biomedical Engineering
The Big PictureThe Big Picture
Our microfluidic devices can be used to Our microfluidic devices can be used to study chemotaxis, directed migration, study chemotaxis, directed migration, of cell systems such as cancerof cell systems such as cancer
For example, it has been shown that For example, it has been shown that breast cancer metastasizes based on breast cancer metastasizes based on epidermal growth factor (EGF) released epidermal growth factor (EGF) released from secondary target organs - we are from secondary target organs - we are able to study the details of this able to study the details of this chemotaxis chemotaxis
Chemotaxis: The characteristic movement or orientation of an organism or cell along a chemical concentration gradient either toward or away from the chemical stimulus
Metastasis: A secondary cancerous growth formed by transmission of cancerous cells from a primary growth located elsewhere in the body
The Big Picture, cont.The Big Picture, cont.
The device I have been working The device I have been working with is easier to use than previous with is easier to use than previous devices and is ideal for isolating devices and is ideal for isolating individual cellsindividual cells
Isolating cells assists in studies Isolating cells assists in studies that might target metastatic that might target metastatic versus non-metastatic cells for versus non-metastatic cells for gene researchgene research
Microfluidic Chemotaxis Microfluidic Chemotaxis ChambersChambers
Soft LithographySoft Lithography(a) A small patterning
polydimethylsiloxane (PDMS) piece with embossed surface pattern is placed on a substrate that is coated with a thin film.
(b) Exposure to reactive oxygen plasma selectively removes material in regions where the patterning piece does not contact the substrate.
(c) After the patterning PDMS piece is removed, well-defined surface micropatterns of cell-adhesive or non-adhesive materials that can be used for selective cell attachment and growth.
(d) A microfluidic PDMS piece with microchannel is aligned and bonded to the patterned substrate. The finished device can be used to culture patterned cells inside a microfluidic device.
Seog Woo Rhee, Anne M. Taylor, Christina H. Tu, David H. Cribbs, Carl W. Cotman and Noo Li Jeon. Patterned cell culture inside microfluidic devices. Lab on a Chip, 2004, 4.
MicroscopesMicroscopes
Inverted
Differential Interference Contrast (DIC)
Hoffman Modulation Contrast
Using the deviceUsing the device
Making gradientsMaking gradients
-Use fluorescent beads and -Use fluorescent beads and fluorescein isothiocyanate-fluorescein isothiocyanate-dextran (FITC-Dextran) to image dextran (FITC-Dextran) to image flowflow
-Altered device assembly, timing -Altered device assembly, timing of solution input, and flow rates to of solution input, and flow rates to create gradientscreate gradients
Adding cellsAdding cells
Cell cultureCell culture- Growth of cells in vitro on an Growth of cells in vitro on an
artificial medium for artificial medium for experimental researchexperimental research
Cell Passaging Cell Passaging - The process of passing or The process of passing or
maintaining a group of maintaining a group of microorganisms or cells microorganisms or cells through a series of hosts or through a series of hosts or culturescultures
- Dilutions, confluenceDilutions, confluence
ExperimentationExperimentation- Trypsinization of Trypsinization of
adherent cellsadherent cells- Cell countingCell counting- Cell starvationCell starvation- Addition of Addition of
epidermal growth epidermal growth factor (EGF)factor (EGF)
Adding cells, cont.Adding cells, cont.
Modeled protocol of paper our lab Modeled protocol of paper our lab group published in 2004 using MDA-group published in 2004 using MDA-MB-231 metastatic breast cancer cells MB-231 metastatic breast cancer cells (~15-20 µm)(~15-20 µm)Shurgen Wang, Wajeeh Saadi, Francis Lin, Connie Minh-Canh Nguyen, Noo Li Jeon. Differential effects of EGF gradient Shurgen Wang, Wajeeh Saadi, Francis Lin, Connie Minh-Canh Nguyen, Noo Li Jeon. Differential effects of EGF gradient
profiles on MDA-MB-231 breast cancer cell chemotaxis. Experimental Cell Research. 2004.profiles on MDA-MB-231 breast cancer cell chemotaxis. Experimental Cell Research. 2004.
Slow movement of cells requires long Slow movement of cells requires long experiment times – constant flowexperiment times – constant flow
Found that the high speeds needed to Found that the high speeds needed to create gradient used up reservoir too create gradient used up reservoir too quicklyquickly
Expanding reservoirsExpanding reservoirs
Punch larger inletsPunch larger inlets Thicker devicesThicker devices Extend upwards - bonding Extend upwards - bonding
reservoir wallsreservoir walls Tubing to attach pipette tipsTubing to attach pipette tips
Collaboration Collaboration *Bonus points*Bonus points
NeutrophilsNeutrophils--A type of white blood cell, specifically a form of A type of white blood cell, specifically a form of granulocyte, filled with neutrally-staining granules, tiny sacs granulocyte, filled with neutrally-staining granules, tiny sacs of enzymes that help the cell to kill and digest of enzymes that help the cell to kill and digest microorganisms it has engulfed by phagocytosis.microorganisms it has engulfed by phagocytosis.
*Movie*Movie
NeutrophilsNeutrophils
Smaller (~10-15 µm)Smaller (~10-15 µm)
Fast movingFast moving- Shorter experiment - Shorter experiment run timesrun times
Short livedShort lived- Lots of tests in one - Lots of tests in one dayday
Use devices with Use devices with smaller channelssmaller channels
How to add How to add more solution more solution during testduring test
Make LOTS of Make LOTS of devicesdevices
New Cell LinesNew Cell Lines
MTLn3 CFP (rat mammary MTLn3 CFP (rat mammary adenocarcinoma cells with cyan adenocarcinoma cells with cyan fluorescent protein markers)fluorescent protein markers)
-Background research-Background research
-Changes in culturing-Changes in culturing
-Impact of confluence-Impact of confluence
-Alter experiment protocol-Alter experiment protocol
Changes in ProtocolChanges in Protocol
Coating devices (cell attachment)Coating devices (cell attachment)- Fibronectin, Collagen I, Collagen IV- Fibronectin, Collagen I, Collagen IV
- concentrations- concentrations- Bovine Serum Albumin (BSA)- Bovine Serum Albumin (BSA)
Cell starvation (induce movement)Cell starvation (induce movement)– Inside or outside deviceInside or outside device– How longHow long
Channel sizeChannel size Inducing chemotaxisInducing chemotaxis
- Growth factors, chemokines, extracellular matrix - Growth factors, chemokines, extracellular matrix componentscomponents
- Concentrations- Concentrations Cell speedCell speed
– Experiment running timeExperiment running time
Optimization Optimization ExperimentsExperiments
Cell adhesionCell adhesion Cell movementCell movement Cell shapeCell shape Cell velocityCell velocity
Optimization Optimization ExperimentsExperiments Diffusion versus Diffusion versus
suction for suction for coating and cellscoating and cells
Getting cells Getting cells intointo the channelsthe channels
Number of cellsNumber of cells
Altering the DeviceAltering the Device
Buffer EGF + FITC-D
outlet(withdraw)
Buffer EGF + FITC-D
outlet(withdraw)
Where to nowWhere to now
Now that set up Now that set up appears to be appears to be consistent and consistent and cells are movingcells are moving
-Find optimal -Find optimal ranges of: EGF ranges of: EGF concentration; concentration; collagen collagen concentration; cell concentration; cell numbernumber
Importance of this Importance of this researchresearch
Design devices with Design devices with which to study which to study these systems these systems
Make devices user Make devices user friendly and design friendly and design consistent protocols consistent protocols so data can be so data can be replicatedreplicated
Technology later Technology later applied to other applied to other fields fields
Documentation of the Documentation of the optimal gradients, optimal gradients, chemotactic chemotactic characteristics and characteristics and limiting factors in limiting factors in these systems will aid these systems will aid the development of the development of treatment options to treatment options to limit the directing limit the directing effects of identified effects of identified soluble factorssoluble factors
AcknowledgementsAcknowledgements
Mentor: Dr. Noo Li JeonMentor: Dr. Noo Li Jeon Lab: Delaram Sahebzamani, Bobak Lab: Delaram Sahebzamani, Bobak
Mosadegh, Madelyn Luttgen, Mosadegh, Madelyn Luttgen, Wajeeh Saadi, Bonggeun Chung, Wajeeh Saadi, Bonggeun Chung, Jeong Won Park, Cyrus Roushan Jeong Won Park, Cyrus Roushan
IM SURE: Said Shokair, Jerry IM SURE: Said Shokair, Jerry McMillan, Goran Matijasevic McMillan, Goran Matijasevic
National Science Foundation (NSF)National Science Foundation (NSF)