Measuring Cell Contraction

Cell Force Monitor

Brendan Harley

2.785J/3.97J/BEH.411J/HST.523J

Massachusetts Institute of Technology

March 11, 2004

Courtesy of Brendan Harley. Used with permission.

Harvard-MIT Division of Health Sciences and TechnologyHST.523J: Cell-Matrix MechanicsProf. Myron SpectorProf. Ioannis Yannas

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 2

Clinical Application

• Wound healing following severe injury: Repair

– Wound contraction

• Myofibroblasts ( -SMA)

– Synthesis of scar tissue

• Anisotropic tissue

• Significantly stiffer, reduced range of motion, pain, inferior functional

properties

• Bioactive scaffolds developed to induce regeneration

– Cell continuity is practically absent

– Contractile cells are randomly oriented within the bioactive scaffold

– Hypothesis: the structure of the bioactive ECM analog prevents the

coordinated cell contraction that results in wound contraction and scar

formation

Yannas, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 3

Development of Bioactive Scaffolds

• Bioactive scaffolds engineered to induce an appropriate or desired cell response in vitro or invivo

• Requires critical adjustment of four physical and structural properties :

1. Chemical composition (ligands)

2. Average pore size

3. Degradation rate

4. For induced regeneration: Collagen fiber structure

Yannas, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 4

Bioactive Scaffold: Pore Size

• Scaffold bioactivity is significantly

affected by pore structure

– Large enough to allow cells to migrate into

the structure

– Small enough to establish a sufficiently

high specific surface area

• Non-uniform scaffolds

– Patches of scaffold are inactive

• Fabrication of a uniform scaffold

– Uniform activity throughout scaffold

– Study cell-scaffold interactions at a

microscopic scale

Figure by MIT OCW. After Yannas

et al., 1989.

0.1 1

0

10

10

20

30

100 1000 1 cm

Average Pore Diameter, µm

WoundHalf-life,Days

ungraftedcontrol

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 5

Design of Cell/Tissue Specific Biomaterials

• Material and structural properties significantly affect cell behavior

– Different cell types respond differently to different scaffolds

• Architecture mediated?

• Stiffness mediated?

• Composition mediated?

• Degradation rate mediated?

• How do cells detect and respond to their surrounding environments (i.e., mechanical, structural, chemical) and what are the critical cues?

– Cell-mediated contraction

Nehrer et al., 1997

Yannas, 2001

Salem et al., 2002

Claase et al. 2003

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 6

Single Cell Mechanics on 2-D Substrates

• Study behavior of a single cell on a flat

membrane (i.e., silicone, polyacrylamide)

– Mechanical environment (i.e., system stiffness)

– Chemical environment (i.e., ligands, growth

factors, cytokines)

• Experimental tools:

– Microscopic analysis of membrane deflection

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 7

Single Cell Mechanics on 2-D Substrates

• Study cell-generated forces on flexible membranes

– Traction forces during migration

– Measure deflection of membranes

Photos removed for copyright reasons.

Silicone membrane with a regular dot

pattern

Polyacrylamide membrane

with fluorescent microspheresSilicone membrane

Beningo and Wang, 2002

Array of flexible

PDMS postsDiagrams and photo removed for copyright reasons.

Tan et al., 2003

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 8

Single Cell Mechanics on 2-D Substrates3T3 Fibroblast, Substrate Deformation:

Diagram showing states at t = 0 and t = 30 minremoved for copyright reasons.

Three diagramsremoved for copyright reasons.

Deformation vectors Field of traction stressesCell on membrane

Pelham et al., 1999

Munevar et al., 2000

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 9

Cell Mechano-SensitivityPolyacrylamide gel with two distinct regions of rigidity (14 vs. 20 kPa):

Stronger traction forces on

stiff substrate (1.1 vs. 0.6 Pa)

Faster migration on soft

substrate (0.44 vs. 0.23

µm/min)

Series of 16 photos (t=0:00 to t=3:50)removed for copyright reasons.

Lo et al., 2000

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 10

Single Cell Mechanics on 2-D Substrates

Cell deformation of an array of flexible posts:

Single cell Cell population Fibronectin on

post tips

Localization of

focal adhesion

protein vinculin

Force/post vs.

Adhesion area

Before and after treatment

with 2,3-butanedione

monoxime (inhibitor of

myosin contractility)

Source: Figures 2 and 3 in Tan, John L. et al. “Cells lying on a bed of microneedles: An approach to isolate mechanicalforce.” PNAS 100:4 (February 18, 2003) 1484-1489. Courtesy of the National Academy of Sciences. Used with permission.

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 11

Single Cell Mechanics: Results Summary

• Cells can sense and respond to the surrounding mechanical

environment

– Substrate flexibility/rigidity regulates cell migration and applied traction

forces

– Substrate flexibility/rigidity regulates cell growth and apoptosis

• Cells on more flexible (4.7 vs. 14 kPa) substrates show decreased rates of

DNA synthesis and increased rates of apoptosis

• Kinetics of contractile force development (smooth muscle cell):

Graph removed for copyright reasons.

Lo et al., 2000

Wang et al., 2000

Tan et al., 2003

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 12

Cell Population Mechanics in 3-D Substrates

• Study behavior of a population of cells in a scaffold

(i.e., collagen-GAG scaffold)

– Mechanical environment (i.e., system stiffness)

– Chemical environment (i.e., ligands, growth factors,

cytokines)

• Experimental tools:

– Live cell imaging

– Cell Force Monitor (CFM)

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 13

Collagen-GAG Scaffold Fabrication

Production of Collagen-GAG (CG) co-Polymer Suspension:

DegasP=50mtorr

Collagen:type I, bovine tendon,

microfibrillar

Integra LifeSciences

GAG:chondroitin 6-sulfate,

shark cartilage

Sigma-Aldrich

Acetic Acid:0.05M, pH ~3.2

Refrigerant-cooled

flask (4°C)

Store suspension

at 4oC

Mix CG Suspension:•Overhead blender

•15,000 rpm

Combine

Fabrication of CG Scaffolds with Different Pore Sizes:

Heat transfer

CG Suspension

PanFreeze-dryer

shelf held at

constant Tf

P

T0oC 20oCTf

S

L

V75mTorr

1 atm

Sublimation:P=75mTorr, T=0oC

Removes ice content

Freeze:T = Tf

Genesis Freeze-dryer

VirTis Co.

Place CG suspension

into stainless steel pan

Ice crystals

surrounded by

collagen and GAG

fibers

Porous, CG

scaffold

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 14

Collagen-GAG Pore Structure

Photo removed for copyright reasons.

Pek et al., 2003

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 15

Effect of Tf on Mean Pore Size

0.0

50.0

100.0

150.0

200.0

250.0

-10 -20 -30 -40

Final Temperature of Freezing,oC

Av

era

ge

Po

re S

ize,

um

Longitudinal

Transverse

*

**

**

****

**

* p > 0.05

** p < 0.05

Significant effect

(p<0.05) of Tf on

mean pore size

Tf, °C Mean Pore Size, µm Relative Density

-10ºC 150.5 ± 32.1 0.0062

-20ºC 121.0 ± 22.5 0.0061

-30ºC 109.5 ± 18.3 0.0059

-40ºC 95.9 ± 12.3 0.0058

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 16

Mechanical Model of Open-Cell Foams

• Open-cell tetrakaidecahedron

– Packs to fill space

– Approximates structural features of low-density foams

0.05 – 0.1

densification

E*

D

collapse plateau*rel. density

s

s

EE

2*

*

Strut bending dependence

Gibson and Ashby, 1997

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 17

CG Scaffold Mechanics: Results

densification

E*

D

collapse plateau*

Cellular Solids Model Prediction:

Experimental Results:

Collagen-GAG Scaffold Compressive Test

0

2000

4000

6000

8000

10000

12000

14000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Strain

Str

es

s, P

a

densification

E*

Hydrated Collagen-GAG Scaffold Compressive Test

-10

0

10

20

30

40

50

60

70

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Strain

Str

es

s,

Pa

densification

E*

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 18

CG Scaffold Mechanics: Results

Non-hydrated Scaffold Mechanical Properties:

Tf,oC E, kPa

-10 36.2 ± 3.8

-20 28.0 ± 3.5

-30 28.1 ± 2.4

-40 30.3 ± 3.3

Compressive Modulus, CG Scaffolds, 8mm Punch

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

-10 -20 -30 -40

Final Temperature of Freezing,oC

E, P

a

**

** p < 0.05

****

Modulus determined from linear fit of data between 0.05 and 0.12 strain

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 19

CG Scaffold Mechanics: Results

Non-hydrated Scaffold Modulus determined through Experiment:

Tf,oC Enon-hydrated, kPa Ehydrated, Pa

-10 36.2 ± 3.8

28.0 ± 3.5

28.1 ± 2.4

30.3 ± 3.3

-20

200

175

175-30

-40 175

*/ s = 0.006

Cellular Solids Model Predictions:

s

s

EE

2*

*

Relative density = 0.006

Non-hydrated Collagen modulus:

(Es) ~ 1GPa

E ~ 36kPa

No dependence on mean pore size

Agreement between cellular

solids model estimated and

experimentally measured

mechanical properties

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 20

Live Cell Imaging

Cell Seeded

Matrix

Cover Slip

Heated Stage

Infinity Focus

Objective

Thick Microscope

Slide with Well

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 21

Live Cell Imaging

Freyman, 2001

Photos removed for copyright reasons.

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 22

Fibroblast Morphology in CG Scaffold

Photos removed for copyright reasons.

Cell Aspect Ratio:

w

lARFreyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 23

Fibroblast Aspect Ratio vs. Time in CG Scaffold

1.0

1.5

2.0

2.5

3.0

3.5

0 10 20 30 40 50 60Time (h)

Asp

ect

Rat

io

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 24

Fibroblast Aspect Ratio Frequency vs. Time

0

0.25

0.5

0.75

1

1 2 3 4 5 6 7

Aspect ratio

Nor

mal

ized

fre

qu

ency

0 h 4 h 8 h

22 h 15 h 48 h

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 25

Cell Force Monitor (CFM)

Adjustable

Height

Post

Rotation

Stage

Be-Cu

Beam

Base Plate

Adjustable

Horizontal Stage

Silicone

Well

To Amplifier / PC

Proximity

Sensor

Matrix

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 26

-0.005

0

0.005

0.01

0.015

0 12 24Time [Hours]

For

ce [

N]

Cell-Free

Corrected

Cells and BaselineResponse

Force Calculation

beam matrix force displ matrixF F F V C V C K

• Parallel - sum of forces

• Voltage, Cforce, Cdispl,, and Kmatrix

• Correct for matrix onlyFreyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 27

Cell Force Monitor: Interpretation of Data

1t

aF F e• Fit data for each sample to:

• Each data set defined by Nc , Fa , and

0

0.005

0.01

0 12 24

Time [h]

Forc

e [

N]

Fa

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 28

Effect of Cell Number on Contractile Force

-5

0

5

10

15

0 6 12 18 24

Time [Hours]

Forc

e [m

N]

10 Million Attached

Fibroblasts at 22h

7.2

6.0

4.4

2.3

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 29

Effect of Cell Number: Fitting Parameters

Number of Attached

Cells, Nc [x 106]

Time Constant,

[hr.]

Asymptotic Force, Fa

[mN]

2.3 ± 0.31 5.0 ± 1.3 3.7 ± 0.6

4.4 ± 0.21 4.0 ± 0.5 5.4 ± 1.4

6.0 ± 0.13 5.0 ± 0.4 8.1 ± 0.5

7.2 ± 0.05 7.0 ± 1.5 10.0 ± 1.9

10.0 ± 0.23 4.0 ± 0.5 12.0 ± 0.7

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 30

Asymptotic Force Generation vs. Cell Number

0

5

10

15

0 4 8 12

Number of Attached Fibroblasts at 22 Hours [Millions]

Asy

mp

toti

c F

orc

e [m

N]

(n=4)

(2)

(3)

(3)

(4)

Fa = 1.0·Nc + 1.2

R2 = 0.97

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 31

Asyptotic Force Generation vs. Cell Number, Time

F = 0.46 x Ncells - 0.2

R2 = 0.90

F = 0.9 x Ncells + 0.9

R2 = 1.0

-5

0

5

10

15

0 4 8 12

Number of Attached Fibroblasts at 22 Hours[Millions]

For

ce [

mN

]

10 Hours Post-Seeding

2 Hours Post-Seeding

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 32

Effect of System Stiffness on Contractile Force

-0.5

1.0

2.5

4.0

0 6 12 18 24

Time [Hours]

Dis

pla

cem

ent

per

Cel

l [n

m]

Stiffness = 10 N/m

1.4 N/m

0.7 N/mCell number not statistically

different (p = 0.4)

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 33

Effect of System Stiffness: Fitting Parameters

Curve Fit Parameters 10 N/m 1.4 N/m 0.7 N/m Trend

Mean Asymptotic

Force per Cell, Fcell

[nN]

3.2 ± 0.3 2.9 ± 0.2 2.7 ± 0.4

3.2 ± 0.6

5.1 ± 0.60

0.63 ± 0.06

Const.

Mean Asymptotic

Displacement per

Cell, dcell [nm]

0.32 ± 0.03 2.0 ± 0.2

Mean Time Constant,

[hr.]

7.9 ± 1.3 5.2 ± 0.85 Const.

Rate of Contraction

per Cell [nm/(hr cell)]

0.04 ± 0.004 0.38 ± 0.04

Total System Stiffness

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 34

Force Generation vs. System Stiffness

-1

0

1

2

3

4

0 6 12 18 24

Time [Hours]

Fo

rce p

er C

ell

[n

N]

10 N/m

0.7 N/m

Stiffness = 1.4 N/m

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 35

Cell Population Mechanics: Results Summary

• Cell-generated contractile forces– Defined by Fa , Nc, and

)1(t

eFF a

)1(t

e

Fa = ~1 nN/cell

= Cell aspect ratio

= 5.2 ± 0.5 hr

0

0.005

0.01

0 12 24

Time [h]

Forc

e [N

]

Nc*F

a

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 36

Fibroblast Model: Elongation and Contraction

MatrixStrutAdhesion Sites

Cell

(a)11

Increasing

Time

Post-

Seeding(b)

1 12 2

CytoplasmMotion

CytoplasmMotion

(c)

BuckledStrut

1 1 22 33 Adhesion SiteMotion

After Freyman, 2001

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 37

CFM: Conclusions

• Force determined at level of individual cells (i.e., not

cooperatively)

– Cell-generating contractile forces independent of

cell density (1200 – 5100 cells/mm3)

• Contraction was limited by the force which

developed; not displacement

• Force measured was that required to support cell

elongation

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 38

CFM: Conclusions

• Cells elongate (spread) on scaffold struts prior

to generating contractile forces

– Time for contraction to develop independent of

cell density and system stiffness

• Cell-generated contractile force independent of

system stiffness

2/16/2005 B. Harley Measuring Cell Contraction March 11, 2004 39

Conclusions

• Cell-mediated contraction

– Single cells on 2-D membranes

– Cell populations in 3-D scaffolds

– Cells are exquisite mechanotransducers

• Cells respond to mechanical environment

– Migration, Traction force, Apoptosis, Cell growth (2-D)

– Cell spreading, Migration, Contractile Force (3-D)

• CFM Demonstration