Download Direct measurement of cell detachment force on single cells using a

Direct measurement of cell detachment force on single cells using a
new electromechanical method
G. W. FRANCIS, L. R. FISHER, R. A. GAMBLE
CSIRO Division of Food Research, PO Box 52, North Ryde, NSW 2113, Australia
and D. GINGELL
Department of Anatomy and Biology as Applied to Medicine, Middlesex Hospital Medical School, Cleveland St, London W1P 6DB, UK
Summary
We describe a new device in which an accurately
measured force is applied to individual adherent
cells while the topography of the adhesion zone is
simultaneously monitored. The force is applied
via a flexible glass micropipette, attached by
suction to the cell under study, and is calculated
directly from the measured pipette deflection.
Regions of close contact in the adhesion zone
are observed using interference reflection microscopy. We have used the device to measure the
force required to detach human red blood cells
from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum
amoebae from a hydrophobic glass surface. The
measured forces per unit length of contact perimeter are within an order of magnitude of the
tensions required for membrane rupture.
Introduction
Gingell & Todd, 1980) can be combined with simultaneous optical assessment, they depend on assumptions about average cell surface properties.
Some methods of measuring the adhesion of single
cells permit an accurate measurement of the applied
force. Coman (1961), for example, inserted a fine glass
microneedle into cells sticking to glass coverslips, and
then used the needle to pull the cell off the substrate,
calculating the detachment force from the bending of
the microneedle. Evans (1984) has developed elegant
methods for sucking cells into micropipettes at known
pressures while simultaneously observing the cell profile. The mechanical properties of the cell membrane
can be calculated from these measurements, and for
adherent cells adhesion energies can be calculated from
the suction pressure and cell shape. The adhesion zone
is not directly observed in either Evans' or Coman's
experiments, although Evans was able to calculate its
area on the assumptions of cell axisymmetry and
uniform contact behaviour - assumptions that are likely
to be obeyed by the vesicles and red blood cells studied
by Evans, but which are probably not obeyed by most
cell types.
Experiments to assess the strength of cell adhesion,
either to other cells or to inanimate substrates, should
produce two basic data: the force being applied to the
cell, and the detailed response of the contact zone.
Unfortunately, these data are not usually available
simultaneously, and a knowledge of the applied force
alone tells us little unless we know the details of the area
over which it is being applied. Centrifugation, for
example, is a widely used direct method of applying a
known average removal force to cells attached to solid
surfaces (George et al. 1971; Tromm\e.retal. 1985) but
suffers from lack of visual observation of the removal
process as well as the limited removal-force range and
dependence of local force on local cell thickness. This
latter point is not generally appreciated. Studies using
viscous shearing in laminar flow conditions (Visser,
1978) can incorporate visual observation of the contact
zone (Mohandas et al. 1974), but the dependence of
calculated removal force on cell shape and hydrodynamic conditions is a severe limitation. While indirect
methods of applying forces (Gingell & Fornes, 1976;
Journal of Cell Science 87, 519-523 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Key words: adhesion, biomaterials, adhesion energy.
519
washed in redistilled chloroform and kept under chloroform
until used.
Cell contacts were observed with a Zeiss Universal microscope (Carl Zeiss Ltd, Welwyn Garden City, UK) fitted with
interference reflection facilities (Gingell & Todd, 1980). The
microscope was arranged for videomicroscopy, incorporating
a Falcon SIT camera (Custom Camera Devices, Wells,
Somerset, UK). Arlunya framestore (Agar Aids Ltd,
Stansted, UK) and JVC videotape recorder with stop-frame
facilities.
MO
4-!
AP
RS
M
I
,4
B
I
FA
Force measurement
Fig. 1. Top: schematic diagram of apparatus for force
measurements (see text). M, mirror; P, micropipette;
MO, microscope objective; S, substratum (cover slip);
RS, reflective sensor (emitter and detector); FA, feedback
amplifier; A, piezoelectric height controller;
B, piezoelectric height controller/sensor; F, force;
AP, pressure differential between ends of pipette.
Bottom: detail of pipette tip and adherent cell; C, cell.
We present here a new approach to the measurement
of cell adhesion, which includes elements of Evans' and
Coman's approaches but overcomes some disadvantages of those methods. The force is applied to the cell
by a fine glass micropipette, which is micromanipulated into a position so that local suction can be applied
to an adherent cell (Fig. 1). The pipette is then moved
away from the substratum, and (with adjustments to
the suction pressure) the cell is pulled off the surface.
The force applied to the cell is calculated from the
degree of bending of the pipette. The contact zone
between cell and substratum is continuously observed
by interference reflection microscopy during the detachment process, and the detailed behaviour of the
cell-substratum contact as a function of applied force
and suction pressure is recorded on videotape.
Materials and methods
Human red blood cells were obtained and washed as described by Trommler et al. (1985). The procedures for
culturing Dictyostelium discoideum amoebae (strain Ax2)
were according to Swann & Garrod (1975). Water was
redistilled from potassium permanganate in all-glass equipment and had a surface tension of 72-9 mNirT 1 . Glass
coverslips (Chance Propper Ltd, UK) were degreased and
exposed to 4 % hydrofluoric acid in 50 % nitric acid for a few
seconds, rinsed copiously with distilled water and allowed to
hydrate in water for 24 h before use. Air-dried glass was
rendered hydrophobic by immersion in chlorodimethylstearyl silane (Sigma Ltd, Poole, UK) for lOmin, then
520
G. W. Francis et al.
A diagram of the apparatus is given in Fig. 1. The adherent
cell selected for study is first partly drawn into a glass
micropipette (inner tip diameter = 3 urn for red cell work,
=6/im for other cells) by hydrostatic suction. The pipette
acts as a cantilever spring, the deflection being proportional
to the force exerted on the cell. In our experiments, a typical
pipette had a flexible shank of length 20 mm, with an outer
diameter of 48 /lm and an inner diameter of 27 ^m. The force
constant for such a pipette is 2-1X 10~8 N Jim"1 deflection of
the pipette tip. Deflections measured ranged from 0'05 fim to
1'5/im, with a measurement error of ±0-01 fim.
A novel optical system, based on a design described by
Petersen et al. (1982) was used to measure the pipette
deflection. The thick (undrawn) end of the pipette is clamped
to a piezoelectric height controller (A). At the knee bend near
the tip is a small mirror. Light from an optical reflective
sensor (as used in commercial bar code readers: HEDS-1000,
Hewlett-Packard Inc., USA) is focused onto the lower edge
of the mirror, and reflected from there into the sensor's
detector. In our adaptation of the original design, the unit is
mounted on a second piezoelectric crystal (B). Both crystals
were manufactured by Physik Instrumente and supplied by
Lambda Photometries Ltd, Harpenden, UK). Movement of
the pipette tip changes the intensity of the light reflected onto
the sensor. A feedback circuit incorporating the piezo controller re-positions the sensor so that the original light
intensity at the detector is restored. The movement of the
piezoelectric crystal (B), which is equal to that of the pipette
tip, is displayed on a monitor.
The difference between the movements of the height
controller (A) and the pipette tip gives the total deflection of
the glass shaft. Direct calibration with small weights then
gives the corresponding deflection force F acting on the cell.
In the experiments reported here, the movement of the
pipette base was less precisely known than that of the tip,
limiting the experiments to determinations of the change in
force on final detachment; that is, a jump of the tip (B) with
constant base position (A) when the cell finally separates from
the glass. The apparatus is currently being upgraded to
permit accurate measurements (±10nm) of the pipette
heights at both ends, when measurements of contact behaviour as a function of applied stress up to and including
detachment will become possible.
Results
We present preliminary measurements of the forces
required to detach cells from substrates for three
particular cell/substrate combinations: red blood cells
sticking to a hydrophilic glass surface, red blood cells
sticking to a glass surface that has been rendered
hydrophobic, and vegetative Dictyostelium discoideum
amoebae sticking to a glass surface that has been
rendered hydrophobic. These cases are presented as
illustrations of the use of the apparatus.
The net force (F) on the cell due to the bending of
the pipette is obtained directly from the change in
pipette tip height, and useful deductions can sometimes be made from this quantity alone. The difference
(P) between hydrostatic pressure at the pipette tip and
that in the bathing medium also enters into the force
calculation in many cases, and in general the relationship among F, P and the shape of the cell-substrate
contact area requires careful analysis. For the present,
we concentrate on conclusions that can be drawn from
measurements of F alone just before cell detachment.
Red blood cells
Our observations of a series of red cells adherent to
both hydrophobic and hydrophilic glass surfaces show
that, as a force tending to detach the cell is applied, the
area of the contact zone decreases monotonically until
the cell detaches (Fig. 2). Detachment may happen
smoothly or suddenly, giving a measurable jump of the
pipette tip in the latter case. Detachment forces calculated from such jumps, with corresponding contact
areas at detachment, are given in Table 1.
The interpretation of these data depends upon the
model adopted for the detachment process. In general
B
the total force on the cell can be divided into two
components: (1) a hydrostatic pressure component
(which can be either positive or negative) acting over
the area of contact; and (2) membrane tension, acting
at the contact perimeter and tending to detach the cell.
Both forces act whenever a cell is mechanically grabbed
and pulled, whether by hydrostatic suction as in our
experiments or by any other method. The values in
Table 1 represent the net removal force: that is, (1)
plus (2).
An approximate value for the membrane tension just
prior to cell detachment can be calculated by assuming
the contact area to be a circle, and neglecting the
contribution of the hydrostatic pressure to the total
force. The tension is then the (measured) applied force
divided by the length of the circle perimeter, assuming
that cos 9 = 1 (Fig. 1, bottom). Values of the tension
calculated in this way are given in Table 1. These
approximate values for the membrane tension can be
combined with the known cell diameters and areas of
contact to provide an estimate of the contribution of the
hydrostatic pressure to the total force. In all cases this
contribution is less than 20 % of the total force, so that
the original approximation is reasonable. In view of the
various uncertainties in the calculation of the hydrostatic pressure contribution, we have not attempted to
correct the values of the detachment tension in Table 1
for this contribution.
It is possible to compare the detachment tensions
with the tensile strength of the red blood cell membrane, although considerable caution is needed, since
Fig. 2. Red cell adhesion to hydrophilic glass.
A. Transmitted light. Left: pipette tip (upper right
of picture) and red cell (lower left of picture). The
horizontal shaft of the pipette appears as the dark
out-of-focus band. Right: the red cell has now been
moved to a position directly above the pipette. A
second red cell, also adhering to the coverslip, is
visible towards the bottom of the picture.
B. Reflected light. Interference reflection
microscopy (IRM). Left-hand panel: the areas of
close contact of the two red cells in the upper righthand panel now appear as dark areas. The
micropipette tip, positioned below the upper red cell
and in contact with its lower surface, does not show
up in IRM. Centre panel: suction has now been
applied and the pipette lowered to apply a
downward force F to the cell. This reduces the
cell-substratum contact area but the applied force is
not sufficient to detach the cell. Right-hand panel: a
slight increase in the applied force has been
sufficient to detach the cell from the substratum.
The pipette tip jumped on cell detachment, the
jump distance permitting calculation of the applied
force necessary for cell detachment.
Measurement of cell detachment force
521
Table 1. Detachment forces for cells on glass
Cell/surface
Detachment
force
(NxlO9)
(pirn2)
Detachment
tension
(mNm"1)
Area
Red cell/
hydrophilic
o-s
1-4
1-6
0-2
0-3
0-3
0-3
0-7
0-8
Red cell/
hydrophobic
1-3
1-7
2-4
0-4
1-9
0-8
0-5
(t, see text)
D. dhcoideumj
hydrophobic
9
11
15
1-8
1-5
3-7
1-9
2-5
2-2
Each set of readings is for a different cell. Errors in force (F)
are about ±10%; errors in area are about ±0'l^m z , and arise
mostly from the limits of resolution of the videomicroscope.
this quantity is strongly time-dependent. For stresses
applied over several minutes, a value of around
5-10 mNirT 1 seems sensible (Rand, 1964). Given that
the area of the contact zone is not necessarily equal to
the minimum cross-sectional area of the tether leading
from it, so that our values of the membrane tension at
detachment are lower bounds, it is possible that abrupt
detachments are due to the snapping of a single tether.
Against this is the fact that cellular material was not
usually visible on the glass surface after cell detachment. The simple model thus needs further testing.
Regardless of the contribution of membrane rupture to
red cell detachment, our results show that similar
forces are needed to pull red cells off both hydrophilic
and hydrophobic glass. Thus, hydrophobic glass does
not constitute a non-adhesive surface for red cells, a
fact that needs to be accounted for in considering the
physicochemical basis of cell-substratum interactions.
Our measured forces for the detachment of red cells
from clean glass can be compared with those reported
by George et al. (1971). They found that a centrifugal
force of 5XlO~'°N failed to remove cells from glass in
physiological saline. Trommler et al. (1985) found that
a centrifugal force of l-5xl0~ 1 0 N removed about 20 %
of cells in similar conditions. Our measured forces for
red cell removal are, as expected, in excess of these
values.
There appear to be no published detachment forces
for cells on hydrophobic glass. Weiss & Blumenson
(1967) showed that washed red cells in protein-free salt
solution are far more strongly retarded during passage
through a column of 250 fxva diameter glass beads than
in a similar column of siliconized beads, as determined
by Coulter counter analysis of the column effluent. The
reason for the marked discrepancy may be that our
protocol allowed for 20min settling time, whereas in
522
G. W. Francis et al.
the method of Weiss & Blumenson the settling time is
effectively zero.
We wish to emphasize that our calculation of membrane tension is order-of-magnitude only, and that
even such an apparently simple cell/substrate combination as red cells and glass can display considerable
complexities. For example, in the measurement
marked | in Table 1, the contact area first shrank to a
three-pointed star. Detachment occurred as two points
separated without leaving visible material behind,
whereupon the third snapped, leaving a ruptured
tether attached to the glass. Such observations
underline the fact that detachment forces cannot be
interpreted without simultaneous optical recording.
Especially important is the fact that, even for red cells,
tethers can form and perhaps play a role at any stage in
the detachment process, as observed both in these
experiments and in experiments where attached cells
are exposed to shearing forces in a laminar flow
chamber (Owens, Gingell & Trommler, unpublished
results).
Tether formation may also explain why, for the case
of red blood cells on glass in isotonic solutions of
reduced ionic strength, Trommler et al. (1985) found
that detachment force is not simply related to the
average contact area measured before centrifugation at
each ionic strength.
Dictyostelium discoideum amoebae
In the case of these cells, the mechanics of detachment
may be complicated by the transmission of stresses via
cytoskeletal elements within the cytoplasm. Nevertheless, we have calculated membrane tensions using the
same model as for red cells. The tensions are greater,
although the interpretation of this result is by no means
clear.
In summary, our equipment gives the first directly
measured detachment force for single cells on a substratum where the contact details can be observed
simultaneously by light microscopy. With tissue cells
this may enable us to distinguish between the relative
contributions of 'focal' and 'close' contacts and make it
possible to relate force, measured directly, with details
of the contact zone. We intend to apply our technique
to measure the forces needed to detach cells from wellcharacterized substrata of biological interest.
The position sensor is an adaptation of a device described
by Petersen et al. (1982), and we thank the authors for
generously communicating the details of their design and for
their helpful comments. Our particular thanks are also due to
Professor Evan Evans and Dr Joe Wolfe for helpful and
encouraging discussions. D.G. thanks the Science and
Engineering Research Council and the Wellcome Trust for
their support. L.R.F. thanks the Royal Society of London
and the Australian Academies of Science for a travelling
fellowship, the CSIRO and Macquarie University for individual support and for a Collaborative Research Grant, and
the Medical Engineering Research Association for support.
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Measurement of cell detachment force
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