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

kcohara@vt.edukcohara@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

Making AdjustmentsMaking Adjustments

Beads FITC-D

outlet(withdraw)

Beads FITC-D

outlet(withdraw)

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

CONTAMINATION!!!CONTAMINATION!!!

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

Tracking CellsTracking Cells

Project GoalProject Goal

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)

Questions?Questions?