Transcript Leukocyte Adhesion - University of Oklahoma Physics

Investigation of Fluid Behavior
in Bifurcated Microfluidic
Chambers
Joel Ramey
Advisor: Dr. David Schmidtke
Outline
• Motivation
• Making the apparatus
• Experiments
• Results
• Implications
• Future work
8um diameter
http://www.ladinfo.org/wbc-rbc-large-B.jpg
Motivation
Monocytes
• What is a Leukocyte?
Endothelial cells
–White blood cell
• Why is adhesion important?
–Inflammation
–Thrombosis
–Atherosclerosis
•~1 million deaths/year
Smooth muscle cells
David W. Schmidtke
Pool of fat
http://en.wikipedia.org/wiki/Ima
ge:Carotid_Plaque.jpg
How do monocytes penetrate
Ley et al…Immunology 2007
Why bifurcations
Fox & Hugh, Heart 1966
Ku et al. Atherosclerosis. 1985
Research Aim
• Take advantage of established microfabrication
techniques to study fluid dynamics with different
geometries.
–Easy to implement
–Plethora of available geometries
David W. Schmidtke
Photolithography Procedure
Transfer of a geometric shape from a photomask to a substrate
• Substrate Cleaning (glass slide)
• Layer of Photoresist
(negative photoresist)
• Aligning the Mask
• UV Exposure
• Develop
David W. Schmidtke
PDMS stamps
• Clean pattern
• Pour PDMS over
over pattern
–Cure PDMS
• Peal from pattern
http://faculty.washington.edu/afolch/i
mages/Project_3DPDMS0.jpg
1st semester
• Created new masks
–Initially transparencies
–Inherent limitations
• Flow experiments
–Bead adhesion shows
promise
• More new masks
–Chrome
Apparatus
Bead suspension (100 x 103 beads/mL)
Polystyrene beads. 6, 10, 20 um diameters
Syringe
Pump
Flow
chamber
Video
Camera
Microscope
Controls shear stress
Monitor
Schmidtke
VCR
Computer
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Investigation of flow
• Ran initial flow experiments with 6um beads
• Ran with 30º and 120º bifurcations
• Analyze flow
Sarvepalli
QuickTime™ and a
Animation decompressor
are needed to see this picture.
2uL/min
500.00
450.00
400.00
Velocity um/s
350.00
300.00
6um 2uL/min 120deg
6um 2uL/min 30deg
10um 2uL/min 120deg
250.00
200.00
150.00
100.00
50.00
0.00
0.00
50.00
100.00
150.00
200.00
Distance from center um
250.00
300.00
5uL/min
1400.00
1200.00
Velocity in um/s
1000.00
800.00
5um 5uL/min 120deg
5um 5uL/min 30deg
10um 5uL/min 120deg
600.00
400.00
200.00
0.00
0.00
50.00
100.00
150.00
200.00
Distance from center um
250.00
300.00
10uL/min
6000.00
5000.00
Velocity um/s
4000.00
6um 10uL/min 120deg
6um 10uL/min 30deg
10um 10uL/min 120deg
3000.00
2000.00
1000.00
0.00
0.00
50.00
100.00
150.00
200.00
Distance from center um
250.00
300.00
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Future
• Look more closely at fluid dynamics in main
branch
• Run computational fluid dynamics for actual
pattern
• Analyze flow with beads at apex
• Run experiments with leukocytes
• Look into using pulsatile flow
Acknowledgments
• Dr. Schmidtke
• Travis Spain
• Phillip Coghill
• Alex
• Dr. Johnson
• Dr. Keay
Questions?
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Atherosclerosis
Disorder of the large
arteries.
Endothelial cells
Atheroma formation:
accumulation of fatty
substances, cholesterol and
cellular waste products.
Smooth muscle cells
Monocytes
Pool of fat
Major cause of morbidity in the United States:
Affects close to 60 million Americans
 Contributes to 33% of all deaths in U.S each year.
In vitro
• Advantages
–Watch experiment in real time
–Process has high degree repeatability
–Control over variables
• Disadvantages
–Geometric constraints
–Not an actual artery
–Different surfaces
How do monocytes penetrate
Ley et al…Immunology 2007
Predominant human anatomic sites for the
localization of atherosclerotic lesions
High Shear
Region
(15-50dyn/cm2)
Low Shear
Region
(0-4dyn/cm2)
Cross section of the
carotid sinus
Carotid artery
Stagnation Point
DeBakey et al. Ann. Surg. 1985
Ku et al. Atherosclerosis. 1985
Schematic sudden expansion flow chamber. H, chamber height
downstream of expansion; h chamber height upstream of expansion;
S, gasket thickness; x coordinate parallel to glass slide; y, coordinate
normal to glass slide.
Research Aim
Take advantage of the microfabrication technologies
to study the pattern of cell adhesion and rolling at
bifurcated geometries
 Techniques easy to implement
 Many different geometries are available
Previous Studies
 In vivo Models
Ramos et al. Circ Res. 1999
Westrick et al. Circulation 2001
 Computational simulation
Malek et al. J.A.M.A 1999
Berger et al. Ann. Rev. Fluid Mech. 2000
 Glass models of bifurcations
Malek et al. J.A.M.A 1999
Karino et al. J. Biomechanics 1990
Pedersen et al. Biomechanics 1992
Malinauskas et al. Atherosclerosis 1998
 Sudden Expansion Flow Chambers
Barber et al. Am. J. Physiology 1998
Skilbeck et al. Biorheology 2001
Skilbeck et al. Arterioscler Thromb Vasc Biol. 2001
Skilbeck et al. Biorheology 2001
In vivo
• Uses mice to study adhesion
• Advantages
–Genetically tailored
–Real results
• Disadvantages
–Cannot watch in real
time
–Fluid mechanics not
observed
–More variability