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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)

. .

O

. .

O. .

O

. .

O. .

O

. .

O

. .

O

. .

O. .

O

. .

O. .

O. .

O

. .

O

. .

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