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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. References COMAN, D. R. (1961). Adhesiveness and stickiness: two independent properties of cell surfaces. Cancer Res. 21, 1436-1438. EVANS, E. A. (1984). Energetics of red blood cell—lipid vesicle and lipid vesicle—lipid vesicle aggregation in glucose polymer (Dextran) solutions. Colloids & Surfaces 10, 133-141. GEORGE, J. N., WEED, R. I. & REED, C. F. (1971). Adhesion of human erythrocytes to glass: The nature of the interaction and the effect of serum and plasma, J. cell. Physiol. 77, 51-60. GINGELL, D. & FORNES, J. A. (1976). Interaction of red blood cells with a polarized electrode. Evidence of longrange intermolecular forces. Biophys.J. 16, 1131-1153. GINGELL, D. & TODD, I. (1980). Red blood cell adhesion. II. Interferometnc examination of the interaction of hydrocarbon oil and glass. J. Cell Sri. 41, 135-149. PETERSEN, N. O., MCCONNAUGHEY, W. B. & ELSON, E. L. (1982). Dependence of locally measured cellular deformability on position on the cell, temperature, and cytochalasin B. Proc. natn. Acad. Sri. U.SA. 79, 5327-5331. RAND, R. P. (1964). Mechanical properties of the red cell membrane. 2. Viscoelastic breakdown of the membrane. Biophys.J. 4, 303-316. SWANN, A. P. & GARROD, D. R. (1975). Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum. Expl Cell Res. 93, 479-484. TROMMLER, A., GINGELL, D. & WOLF, H. (1985). Red blood cells experience electrostatic repulsion but make molecular adhesions with glass. Biophys. J. 48, 835-841. VlSSER, J. (1978). Colloid and other forces in particle adhesion and particle removal (a review). Deposition Filtr. Part. Gases Liq. Symp., pp. 121-141. London: Soc. Chem. Ind. WEISS, L. & BLUMENSON, L. (1967). Dynamic adhesion and separation of cells in vitro: Interactions of cells with hydrophilic and hydrophobic surfaces. J. cell. Physiol. 70, 23-32. MOHANDAS, N., HOCHMUTH, R. M. & SPAETH, E. E. (1974). Adhesion of red cells to foreign surfaces in the presence of flow._7. biomed. mater. Res. 8, 119-136. (Recrived 24 October 1986 -Accepted, in revised form, 10 February 1987) Measurement of cell detachment force 523