1. Optimizing Usage of Microfluidic Chemotaxis Chambers for Cancer Research
Kathleen O’Hara
Virginia Polytechnic Institute and State University
Biological Sciences and Psychology, Spring 2006
Mentor: Dr. Noo Li Jeon
Department of Biomedical Engineering

2. The Big Picture
Our microfluidic devices can be used to study chemotaxis, directed migration, of cell systems such as cancer
For example, it has been shown that breast cancer metastasizes based on epidermal growth factor (EGF) released from secondary target organs - we are able to study the details of this 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

3. The Big Picture, cont.
The device I have been working with is easier to use than previous devices and is ideal for isolating individual cells
Isolating cells assists in studies that might target metastatic versus non-metastatic cells for gene research

4. Microfluidic Chemotaxis Chambers
Soft 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.

5. Hoffman Modulation Contrast
Microscopes
Inverted
Differential Interference Contrast (DIC)
Hoffman Modulation Contrast

6. Using the device Making gradients
-Use fluorescent beads and fluorescein isothiocyanate-dextran (FITC-Dextran) to image flow
-Altered device assembly, timing of solution input, and flow rates to create gradients

7. Making Adjustments outlet outlet Beads FITC-D Beads FITC-D (withdraw)

8. Adding cells Cell culture Cell Passaging Experimentation
Growth of cells in vitro on an artificial medium for experimental research
Cell Passaging
The process of passing or maintaining a group of microorganisms or cells through a series of hosts or cultures
Dilutions, confluence
Experimentation
Trypsinization of adherent cells
Cell counting
Cell starvation
Addition of epidermal growth factor (EGF)

9. Adding cells, cont.
Modeled protocol of paper our lab group published in 2004 using MDA-MB-231 metastatic breast cancer cells (~15-20 µm)
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
Slow movement of cells requires long experiment times – constant flow
Found that the high speeds needed to create gradient used up reservoir too quickly

10. Expanding reservoirs Punch larger inlets Thicker devices
Extend upwards - bonding reservoir walls
Tubing to attach pipette tips

11. CONTAMINATION!!!

12. Collaboration *Bonus points
Neutrophils
-A type of white blood cell, specifically a form of granulocyte, filled with neutrally-staining granules, tiny sacs of enzymes that help the cell to kill and digest microorganisms it has engulfed by phagocytosis.
*Movie

13. Neutrophils  Use devices with smaller channels
Smaller (~10-15 µm)
Fast moving
- Shorter experiment run times
Short lived
- Lots of tests in one day
 Use devices with smaller channels
 How to add more solution during test
 Make LOTS of devices

14. New Cell Lines
MTLn3 CFP (rat mammary adenocarcinoma cells with cyan fluorescent protein markers)
-Background research
-Changes in culturing
-Impact of confluence
-Alter experiment protocol

15. Changes in Protocol Coating devices (cell attachment)
- Fibronectin, Collagen I, Collagen IV
- concentrations
- Bovine Serum Albumin (BSA)
Cell starvation (induce movement)
Inside or outside device
How long
Channel size
Inducing chemotaxis
- Growth factors, chemokines, extracellular matrix components
- Concentrations
Cell speed
Experiment running time

16. Optimization Experiments
Cell adhesion
Cell movement
Cell shape
Cell velocity

17. Optimization Experiments
Diffusion versus suction for coating and cells
Getting cells into the channels
Number of cells

18. Altering the Device outlet outlet Buffer EGF + FITC-D Buffer
(withdraw)
outlet
(withdraw)

19. Where to now
Now that set up appears to be consistent and cells are moving
-Find optimal ranges of: EGF concentration; collagen concentration; cell number

20. Tracking Cells

21. Project Goal

22. Importance of this research
Design devices with which to study these systems
Make devices user friendly and design consistent protocols so data can be replicated
Technology later applied to other fields
Documentation of the optimal gradients, chemotactic characteristics and limiting factors in these systems will aid the development of treatment options to limit the directing effects of identified soluble factors

23. Acknowledgements Mentor: Dr. Noo Li Jeon
Lab: Delaram Sahebzamani, Bobak Mosadegh, Madelyn Luttgen, Wajeeh Saadi, Bonggeun Chung, Jeong Won Park, Cyrus Roushan
IM SURE: Said Shokair, Jerry McMillan, Goran Matijasevic
National Science Foundation (NSF)

24. Questions?