measuring cell contraction cell force monitor · 2019. 9. 12. · 2/16/2005 b. harley measuring...
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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
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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.
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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