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TRANSCRIPT
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Reichert Technologies Webinar
September 20, 2016
Michael Hill
Debanjan Sarkar Lab: Laboratory of Biomaterials and Regenerative Therapeutics
State University of New York at Buffalo
Use of surface plasmon
resonance for probing
cell-matrix interactions
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• Introduction and Motivation
• Part I: Characterization of surface energy of immobilized
proteins using liquid contact angle studies
• Part II: Test of SPR for non-specific adhesion applications
using endothelial cells
• Part III: Test of SPR for specific adhesion applications using
white blood cells
• Summary and conclusions
Outline of the presentation
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Surface forces regulating cell adhesion
200 Å
10 Å
3 Å
o Van der Waals Forces:
Long range; decay as
1/R6
o H-bonding and double
layer: Strong but fall off
more quickly with
distance
- Non-specific longer range
forces are important during
cell adhesion
- Protein-coats present their
own surface field. Specific interactions overcome these shielding effects.
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• Specific forces: due to a pattern
of short-ranged hydrogen
bonding or electrostatic forces
(<1nm under physiological
conditions)
-Can be defined by their ability to
transmit across albumin blankets
• Arginylglycylaspartic acid
(RGD) peptides: bind
specifically with domains of
integrins
-“specific” arrangement of polar
and non-polar groups responsible
for such bonding
Specific vs. non-specific biological adhesion
Polar
Apolar
Polar
Receptor-Ligand
Interactions
Albumin
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• Evanescent wave: set up by shining
a light with resonant frequency of a
thin gold film
-Since wave is a near field-effect,
molecules interacting with surface are
detected by changing surface
refractive index (micro refractive index
units (µRIU))
• Range of signal: few 100 nm from
the surface
Surface Plasmon Resonance (SPR): A brief introduction
https://en.wikipedia.org/wiki/Surface_plasmon_resonance
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• Reichert SR7500DC: modular tubing
and flow chamber
-wide possible range of flow rates
• SPR gold chip has carboxyl/PEG
terminated SAM
-integrated with a flow chamber to enable
cell attachment under shear
• Controlled shear and real-time signal
is major advantage to cell adhesion
studies
SPR studies: experimental setup
A = EDC-NHS
B = Protein
C = Ethanol amine
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SPR versus previous cell adhesion metricsPassive Attach –
Manual Count
Cell Attachment
Under Defined
Shear
Atomic Force
Microscopy
SPR
Cost Low intermediate High Intermediate
Complexity Low intermediate High Intermediate
Challenge
Adhesion
No Yes Yes Yes
Resolution Low Intermediate High High
Natural No Yes No Yes
Real Time No No Yes Yes
Speed High High Low high
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• Part I: Surface energy of immobilized protein surface is
estimated for non-specific cell adhesion application
• Part II: SPR is used to test adhesive interaction of endothelial
cells to protein surfaces of differential surface energy
• Part III: Specific interaction between Immobilized P-selectin and
white blood cells (HL-60) across a blanket of albumin. Study
confirms use of SPR for measurements across multiple protein
interlayers
Summary of Experiments
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• In vivo Endothelial Cells (ECs): come into contact with two classes of
extracellular matrix proteins:
• Basement membrane (collagen IV/Laminin dominated)
• Stromal tissue (collagen I/III dominated)
• ECs are typically separated from stromal tissue by basement membrane
• When injury occurs, the cells contact and invade the stromal tissues, and this induces angiogenesis
Introduction
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Methods
-Collagen I (stromal protein), Matrigel® (basement membrane),
and serum (control protein) were immobilized
-Contact angle measurements using various diagnostic liquids
were performed for biosurface energy estimation
http://www.ramehart.com/images/advanced_goniometer_577_large.jpg
Low surface
energy
High surface
energy
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Surface energy theories and biological adhesion I. Critical Surface Tension (CST)
https://en.wikipedia.org/wiki/Sessile_drop_technique
(Weiss and Blumenson 1967)
No serum serum
Biomaterials CST (dynes/cm)
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Surface energy theories and biological adhesion II. Kaelble’s Method
γp = polar
γd = dispersive
- Multiple liquids used, similar to CST
- Equation is solved numerous times for each
liquid pair
- Arithmetic average is taken
- Values outside standard deviation rejected
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Surface energy theories and biological adhesion III. Van Oss-Good-Chaudhury Theory (vOGCT)
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O. .
O
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O
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O
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O
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O. .
O. .
O
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O
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O
γ+ = Lewis acid
γ- = Lewis base
- 2 polar and 1 apolar liquid are
measured
- 3 simultaneous linear equations
are solved
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• Collagen I: greater γp and γ+
than other proteins (greater
surface energy)
• Matrigel: lowest γp with lower γ+
(less surface energy)
SPR studies: Surface energy measurement of proteins on SPR chip
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SPR studies. cell attachment studies
Cells
Flow rate reduced
- Differential cell adhesion to immobilized
proteins can be correlated to protein
surface energy
- Proteins with higher surface energy have
greater tendency to bind cells
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SPR studies. Visualization of cell monolayers at 24h on three substrates
Collagen I Matrigel® Serum
200 µm
10x
20x
200 µm
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• Surface energy measurements may be a predictor of optimal
conditions for cell capture and adhesion. This can be used to
optimize cell binding conditions on the sensor chip.
• Protocols were established for the formation of confluent live
endothelial cell monolayers.
• Cell binding was most efficient when extra-cellular matrix
proteins were immobilized at physiological pH.
Discussion
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• Part I: Surface energy of immobilized protein surface is
estimated for non-specific cell adhesion application
• Part II: SPR is used to test adhesive interaction of endothelial
cells to protein surfaces of differential surface energy
• Part III: Specific interaction between Immobilized P-selectin and
white blood cells (HL-60) across a blanket of albumin. Study
confirms use of SPR for measurements across multiple protein
interlayers
Summary of Experiments
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SPR studies: Higher surface energies correlate with greater hyper-osmolar shock response
• 100 mM hyperosmolar
mannitol• Isotonic HEPES
Collagen I
Matrigel®
Serum
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SPR studies. Greater strength of binding on collagen I substrates
200 µm
200 µm
Collagen I Matrigel®
10x
20x
Serum
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• Collagen I: Higher γp and γ+ correlate with increased cell adhesion
strength. This is the condition for ECs on stromal tissue as they
undergo angiogensis.
• Matrigel®: Lower γp and γ+ correlates with reduced EC interaction.
This occurs when cells form monolayers on basement
membranes
• Young’s modulus of adhesion calculated via SPR
• Matrigel® ~0.5MPa & Collagen ~2MPa
• All results correlate well with AFM
Discussion
Collagen I: Immobilized arrays of
Lewis acid and Lewis base groups
Matrigel®: Cross-linked
structure causes shielding of
Lewis acid/base.
+ = Lewis acid
- = Lewis base
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• Part I: Surface energy of immobilized protein surface is
estimated for non-specific cell adhesion application
• Part II: SPR is used to test adhesive interaction of endothelial
cells to protein surfaces of differential surface energy
• Part III: Specific interaction between Immobilized P-selectin and
white blood cells (HL-60) across a blanket of albumin. Study
confirms use of SPR for measurements across multiple protein
interlayers
Summary of Experiments
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• White blood cells: recruited to specific tissue sites upon injury
or inflammation
• Endothelial cells: line the blood vessels, express “selectins” in
injured tissues
-White blood cells first tether onto selectins such as P-selectin on
the endothelium via glycoprotein ligands including PSGL-1 (P-
selectin glycoprotein ligand-1).
Introduction
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Non-specific adhesion of HL-60 A B C D
• Non-specific adhesion: overpowers the effects of specific
adhesion when the long-range forces are not blocked by a blanket
of albumin
A = EDC-NHS
B = IgG
C = Ethanol amine
D = P-selectin
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Isolation of specific effects of HL-60 recruitment• BSA blocking:
ensures
specific
interactions are
probed
• MAb KPL-1
(anti-PSGL-1
mAb) : confirms
specificity
- SPR response
across multiple
protein interlayers
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• Specific adhesion of HL-60: SPR confirms the specificity of cell
adhesion across multiple protein interlayers
- Distance of SPR signal is limited to few 100 nanometers
- Cells are large (10-100’s of µm) colloidal objects
- SPR can be used to closely monitor the nature by which cells
spread on ligand bearing substrates in a narrow distance scale.
Something that cannot be done using standard microscopy.
Discussion
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• SPR: Used to probe cell-matrix interactions in the context of both
specific (BSA blocked) and non-specific cell adhesion systems
• The two types of adhesion often co-exist in particular
biological contexts
• SPR is useful to dissect intermolecular force characteristics of
cell adhesion in different model systems, especially at short
length and time scales
• Hyperosmolar shock studies with SPR: can quantify strength of
cell interactions with proteins
• Cheaper, simpler, easier compared to past methods
• Similar quantitative results in comparison to Atomic Force Microscopy
Conclusions
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• Visualization of cells: It is useful to visualize cells during SPR
studies. This can enable:
- Correlation of SPR signal to kinetics of cell spreading
• Regeneration of the sensor chip: Removing cells while keeping
immobilized protein intact is not possible:
- High shear may cause cohesive rupture of cells
- Digestive enzymes may alter protein film
Methods to release the entire chemistry on SPR sensor chip would be helpful.
- Model development: SPR is typically used to measure monovalent
binding interactions. Estimation of kinetic on-off data for multivalent
cellular interactions requires more research.
Future Work
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Q & A
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Appendix
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• 19Fc : Control IgG fragment
- Slope of P-Selectin signal
increases > 3-fold compared
to 19Fc control substrate
- Cells continue to spread
during dissociation phase
- Further increase in signal
upon introduction of more
HL-60 cells.
- Cells do not bind the control
19Fc substrate efficiently
P-Selectin versus Control 19Fc Surfaces
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Methods
107 cells/ml
(Endothelial cells)
EDC-NHS80 µg/ml protein 1 M ethanol-
amine
12
3
4Cells
spread for
3 hours
5
Chip removed and
cells cultured
overnight
6
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Methods
107 cells/ml
(Endothelial cells)
EDC-NHS80 µg/ml protein 1 M ethanol-
amine
12
3
4Cells
spread for
3 hours
5100 mM
hyperosmolar
mannitol cycles
6
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Methods
107 cells/ml
(cell binding
facilitated by PSGL-1)
EDC-NHS80 µg/ml IgG antibody
immobilized1 M ethanol-
amine
5 µg/ml
P-selectin
2% BSA
12
3
4
5
6
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Human Embryonic Kidney
CELL-PROTEIN BINDING
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HEK Cells Capture
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200 nM Fibrinogen InjectionSurface Heterogeneity Model
ka1 = 2.40 e3 M-1s-1
kd1 = 8.51 e-3 s-1
KD1 = 3.54 mM
ka2 = 9.74 e3 M-1s-1
kd2 = 2.57 e-4 s-1
KD2 = 26.4 nM