Supplementary Information (SI)
Design of the stress field
As seen in Fig. 1S, the aspect ratio between the ellipse long axes to the short axes
determines the stress level (the relevant decay distance can also be easily deduced),
allowing the design of the required gradient.
Polydimethylsiloxane (PDMS) preparation was done using the Sylgard 184 Silicone
Elastomer Kit (Dow Corning). The elastomer and curing agent were thoroughly
mixed for 5 minutes at a 20 parts to 1 part ratio (20:1) in order to obtain a PDMS with
Young’s modulus of ~500 kPa. PDMS was then poured into a special designed mold
and degassed for 30 minutes to remove bubbles. The PDMS was cured at 80 C for 1
hr. The designed geometry of the PDMS included a well in which the cells could be
plated and maintained in medium throughout the experiment (Fig. 2S). Relaxation of
PDMS films during application of a constant strain (FF=10%) for three hours was
shown to be very mild and insignificant. To introduce a controlled varying stress field
in the matrix an elliptical shaped hole was created in the PDMS film. The elliptical
hole was created with a PALM Microbeam System (Zeiss) using a pulsed ultra-violet
(UV-A) laser beam of high quality (which is interfaced into the microscope). During
the cutting process the specimen was kept in water to prevent heat damage. The
cutting process is illustrated in Fig. 3S. The films were then sterilized by UV for 60
min, washed extensively with Phosphate buffered saline (PBS) and coated with
fibronectin (FN) (5g/ml, 4 C, overnight). Before plating the cells, the films were
washed three times with culturing medium.
FN density did not change and remained constant before and after the PDMS
substrate was strained (as observed using fluorescent FN coating) in all regions.
Changes of protein conformation and ligand spacing could not be detected by using
fluorescently labeled FN due to limited resolution.
Tensile tester apparatus for conducting real time lapse movies
The Petri dish, which was cut and replaced by a #1 glass slide in the middle, is
placed on an inverted microscope. The film on which the cells were plated is held
between two clamps, designed to be as close as possible to the glass slide. The
distance between the objective and the sample allows the use of 10X/0.3 and 20X/0.7
objectives. The objective power can be increased if a water objective is used in an
upright microscope. The apparatus presented here allows the cells to be kept in cell
medium throughout the entire length of the experiment. The film is stretched using the
two screws located outside the Petri dish (Fig. 4S). The applied strain is determined
by following the change in location of two tiny spots in the sample. Time-lapse
movies were recorded using the Real Time (RT) DeltaVision System (Applied
Precision Inc., Issaquah, WA, USA), consisting of an Olympus IX71 inverted
microscope equipped with a CoolSnap HQ camera (Photometrics, Tucson, AZ, USA)
and a weather station CO2 and temperature controller, operated by SoftWoRx and
Resolve3D software (Applied Precision Inc.). Cell response to the applied stress was
monitored for up to 6 hours with either 2 or 3 minutes time intervals between images.
Cell culture
Two different cell types were used:
1. A cell line of rat embryo fibroblasts (REF 52) were plated on PDMS films coated
with FN (5g/ml, 4 C, overnight) and incubated for 24hr at 37°C in Dulbecco’s
modified eagle’s medium (DMEM) supplemented with antibiotics and 10% fetal calf
serum.
2. Primary Human foreskin fibroblasts (HFF) (passages 17-23) were plated on PDMS
films coated with FN (5g/ml, 4 C, overnight) and incubated for 24hr at 37°C in
DMEM supplemented with antibiotics and 10% fetal calf serum.
Analysis of cell migration
Migration path was calculated for a stack of time-lapse phase contrast images
captured at 2 or 3 minute intervals, using ImageJ (National Institute of Health)
software with a manual tracking plug-in. Using Matlab, the migration direction of a
cell, α, was determined to be the angle between the linear migration distance vector,
B, and A=(1,0,0) (Fig.5S) as follows:
cos  
A B
A B
In both the compression and high tension analysis, angles between 0-90 and 270-360
indicate that the cells migrate toward higher compression stress or lower tensile stress
respectively. To generate histograms we calculated cos(α) since its value is between 0
and 1 for 0<α<90 and 270<α<360. If cells in a sample migrate in a particular direction
rather than a random direction it will be reflected in an increased frequency of a
particular cos() value leading to a mean value different than 0. A random directional
migration will lead to a mean value of cos() near 0.
In total, two samples of low confluent (minimum cell-cell contact) and one sample of
high confluence REF52 cells is shown here. For each condition the total number of
cells analyzed, n, is indicated in table 1.
Note that all figures in throughout the text except for Fig. 9S are for the HFF cells
Finite elements method (FEM)
The stress field profiles induced by a far-field strain in a PDMS film containing an
elliptical discontinuity were generated by means of the MSC Nastran FEM program.
To correlate between the stress gradient and cell response, using Matlab, different
stress zones of interest were chosen, plotted and superimposed on phase contrast
images as seen for example in Fig. 6S.
CAPTIONS TO FIGURES:
Figure 1S: Local stress concentration at the ellipse tip as a function of the elliptical
sharpness. For example, an elliptical hole 10 times longer than wide (axial ratio a/b
= 10) the local stress at the tip is 21 times the far field applied stress.
Figure 2S: PDMS film geometry; film width: 10 mm; inner well diameter: 8 mm; film
thickness: 0.3 mm. Arrows indicate the direction of applied load.
Figure 3S: Elliptical hole drilled in PDMS film using a PALM Microbeam system
(Zeiss), essentially a precise non-contact micro-dissection technique. (a) The contour
of the ellipse is cutout; (b) the interior is removed; (c) final elliptical hole.
Figure 4S: Tensile tester in a Petri dish: (1) glass slide; (2) clamps; (3) tensile
screws.
Figure 5S: Cell migration path is shown in a dotted line. B is the vector of the linear
migration distance (the start point, indicated by the red circle, to the final position).
The migration angle, α, is in correspondence to the defined vector A=(1,0,0). The
angle was calculated in the opposite clockwise direction.
Figure 6S: (a) Using Matlab different stress levels of interest were chosen and
plotted. Here (+) -30kPa to -23kPa, (+) -22kPa to -15kPa, and (+) -15kPa to 0kPa.
(b) The zone edges were outlined and superimposed on a phase contrast image
allowing correlation between cell respond to the induced stress level.
Figure 7S:
Cell migration under no load – (a) A representative migration paths of cells under no
load is shown. Red circle represents the starting point for overlaid paths. (b)
Histogram of cos () is shown.
Figure 8S: Cell migration in region C: (a) Representative cell migration paths in
region C. Black paths indicate cells that were initially positioned where
 = -27
kPa, red path indicate cells that were initially positioned where  =-10 kPa. The
blue dot indicates cell position after strain was applied. The X mark indicates the
final cell position three hours after strain was applied. The * mark points to cells
which passed from  =-10 kPa to  = -27 kPa compression stress level. The dashed
line indicated the position of the elliptical hole. The increased gradient of the
compression stress is indicated below the x-axis of the plot. (b) cos(a) frequency is
higher for values between 0 and 1, with a mean of cos()=0.26, indicating that the
migration direction is not random but in the direction of increased compression
stress. Cell migration in region T: (c) cos(a) frequency is higher for values between 0
and 1, with a mean of cos()=0.50, indicating that the migration direction is not
random but in the direction of decreased tensile stress.
Figure 9S: Qualitative observation of cell response in (a) region C (average  = -27
kPa) and (b) region T (average  = 230 kPa) at increasing times following straining
of the specimen (F=10%). (c) Number of REF52 cells in regions C and T at
increasing times following straining of the specimen. The colored dots follow the
change in time of cell centroid location for specific cells.
HFF, low
REF 52, high
No load
n=26
n=43
Region U
n=15
n=29
Region C
n=23
n=20
Region T
n=9
n=16
Table 1
Figure 1S
Figure 2S
Figure 3S
Figure 4S
Figure 5S
(a)
(b)
Figure 6S
C
ell
nu
m
be
r
cos(
Figure 7S
(a)

C
ell
nu
m
be
r
(b)
(c)
cos(
Figure 8S
Figure 9S