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Centrifugal assessment of cell adhesion
Journal of Biochemical and Biophysical Methods, 4 (1981) 29--38
29
© Elsevier/North-Holland Biomedical Press
CENTRIFUGAL ASSESSMENT OF CELL ADHESION
W.D. CORRY * and V. DEFENDI
New York University Medical School, Department of Pathology, 550 Ist Avenue, New York, N Y 10016, U.S.A. (Received 14 May 1980; accepted 28 July 1980)
The design of a chamber for determining the centrifugal force necessary to detach cells from various substrates is presented. Cells from an SV40-transformed murine peritoneal macrophage line and human erythrocytes were used to assess the feasibility of using the chamber for studies of cell adhesion. This work was confirmed the usefulness of the chamber and provided data concerning the force necessary to detach the cells. These data indicated that the percentage of cells detached from a glass substrate was not a function of force alone. The number of cells detaching increased with the impulse applied to the cells when they were exposed to a constant force. Similarly, when the impulse applied to the IC21 cells was maintained at a constant level, the percentage of cells detached by a centrifugal force increased with the magnitude of the force. Key words: macrophage; erythrocyte, centrifugation; adhesion.
INTRODUCTION
An improved centrifugal technique for measuring the force required to detach cells from various substrates is presented in this paper. Cells are allowed to attach to a transparent substrate and then placed in a special chamber. The chamber and cells are then subjected to a preselected centrifugal force and the percentage of cells that detach measured. The primary advantages of this technique are that it allows the investigator access to centrifugal fields up to 114 000 × g and enables him to measure the impulse imparted to the cells. The utility of this technique was assessed by measuring the force required to detach human erythrocytes (RBC) and IC21 cells, an SV40-transformed murine peritoneal macrophage [1], from a glass substrate. These cells were chosen as they represent opposite extremes in adhesive behavior. The RBC adhere weakly while the IC21 cells adhere with a tenacity that exceeds the capacity of most techniques for measuring the force necessary to detach
* Present address: University of Oregon Health Sciences Center, Department of Neurology, 3181 SW Sam Jackson Park Rd., Portland, OR 97201, U.S.A.
30 them. Although techniques for measuring the force necessary to separate tightly bound objects [2,3] exist, they have serious drawbacks. The centrifugal m e t h o d presented here was developed to circumvent these drawbacks. This technique was chosen for three reasons. First, by using a Beckman SW27 rotor, centrifugal fields in excess of 100 000 × g can be attained. Second, the force generated by this field is uniform and therefore suitable for an analysis of the distribution of force over the attached cell. Finally, this technique offers a method of monitoring the impulse applied to the cells. Impulse is usually defined as the amount of m o m e n t u m transferred to an object when it is subjected to a force, F, over a period of time 0 to t. In mathematical terrq.s, impulse, I, can be defined by the equation t
I = f Fdt
(1)
0
When a particle is attached to a substrate in a centrifugal rotor at a distance r from its center of rotation, the impulse applied to the particle is given by t
I = rm f co2dt
(2)
0
where co is the angular velocity of the rotor and m the b u o y a n t mass of the cell. The rationale for measuring impulse is derived from several sources. A number of studies [4,5] of cell detachment have shown that the percentage of cells detaching from a given substrate depends on the length of time they were stressed as well as the magnitude of the force to which they were subjected. These observations are in agreement with studies by Halpin and Polley [6] on the fracture of viscoelastic bodies. In this work Halpin and Polley [6] indicated that when a viscoelastic solid is subjected to a stress its likelihood of rupturing will depend not only on the magnitude of the force to which it is subjected, b u t also its duration. The applicability of this theory to the present situation is suggested by studies that indicate that the cell membrane behaves as a viscoelastic solid [7] and that cell detachment is a process which usually involves the rupture of the cell membrane [8] rather than the cell--substrate adhesive joint. Finally, the use of the concept of impulse is dictated for centrifugal techniques by the fact that much of the cell detachm e n t process may be accomplished by forces which are less than maximal. Measurement of the impulse applied to these cells is one step towards placing the study of cell detachment on a quantitative basis. MATERIALS
AND METHODS
All studies of cell adhesion were carried o u t with Coming No. 1 thickness coverslips. They were cleaned by sequentially immersing them for 12 h in
31
distilled de-ionized water; 8 h in an ethanolic-KOH solution (3 M KOH/15 M ethanol); five times in distilled de-ionized water; 12 h in acid (85% H2SO4/ 5% HNO3/10% H20 (v/v}); and five times in distilled de-ionized water. Each distilled water rinse utilized a minimum of 500 ml of fluid. After this cleaning procedure the coverslips were heat sterilized. All studies of the adhesion of cells to a glass substrate were done with either human erythrocytes or IC21 cells. The IC21 cell line is an established tissue culture line [ 1 ] which had been obtained by transforming murine peritoneal macrophages with SV40 virus. The IC21 line was grown in minimal essential medium (MEM; GIBCO) supplemented with 1× antibiotic/antimycotic (GIBCO) and 10% fetal bovine serum ( F B S ) i n an injection incubator with a 5% CO2 atmosphere. Subconfluent cultures were fed between 18 and 24 h before harvest to ensure that they would be in a log phase growth at harvest. The cells were detached by exposing them to a 37°C PBS/EDTA solution (137 mM NaC1/3 mM KC1/8 mM Na2HPO,/1 mM K H 2 P O J 5 mM Na2EDTA) for 5 min and tapping the T-75 flask (Falcon Plastics) in which they were grown to dislodge them. The decanted cell suspension was then promptly mixed with an equal volume of MEM, centrifuged 5 min at 160 × g, and the supernatant decanted. They were then suspended in the MEM containing 10% FBS, plated onto coverslips at a density of 1--5(~. 104 cells/cm 2 and then incubated as before. Erythrocytes were prepared for adhesion assays from blood drawn from t h e antecubital vein into a heparinized syringe (100 units/10 ml blood) and by centrifuging the blood for 10 min at 650 X g, room temperature (23 ± 2°C). The plasma and b u f f y coat were drawn off and discarded. The red cells were suspended in phosphatebuffered saline (PBS: 4.8 mM K H 2 P O J 2 5 . 2 mM N a 2 H P O J 1 2 1 . 4 mM NaC1; pH 7.42 ± 0.02, 292 t 2 mOsm) to a RBC : PBS ratio of approximately 1 : 4 and centrifuged as before. After centrifugation any remaining white cells were removed. This washing procedure was done a total of three times. The cells were suspended in PBS to a concentration of 1 . 8 . 1 0 4 cells/ml, and plated onto coverslips. The settling RBC attained a concentration of 5--6 •
Fig. 1. E x p l o d e d view o f a c e n t r i f u g a l c h a m b e r . F r o m t h e l e f t the pieces o f t h e c h a m b e r are t h e u p p e r h a l f of t h e c h a m b e r , a 6 m m i.d. t e f l o n disk, a n 18 m m glass coverslip, a 2 m m i.d. t e f l o n disk, a n 18 m m glass coverslip and the lower h a l f o f t h e c h a m b e r . The t h i n b r o k e n line passing t h r o u g h t h e c e n t e r o f the chamber represents the path of any i l l u m i n a t i n g light t o enter the c h a m b e r . T h e o p e n circle o n t h e upper coverslip indicates t h e r e g i o n t h a t is o b s e r v e d w h e n the percentage of cells detaching is measured.
32
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103 cells/cm 2 on the coverslips and were allowed to remain on the coverslips a minimum o f 2 h before being assembled in the centrifugal chamber. All assays of red cell adhesion were done at 3 9 0 0 X g. All cell detachment assays were done with an aluminum chamber (Velotron Machine Co., Brooklyn, N.Y.) adapted for use in a Beckman SW27 rotor. Exploded and cross-sectional views of the chamber can be seen in Figs. 1--3. The chamber and coverslip containing the cells were assembled by the
33
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SIDE CUSHION MATL: DELRIN Fig. 3. Machinist's drawing of chamber cushion and chamber assembly wrench. The chamber assembly wrench also serves as a platform for viewing cells in the assembled chamber with a microscope. The chamber cushion is placed beneath the chamber in the b o t t o m of the bucket for the centrifuge rotor to distribute evenly the load placed on the bucket by the chamber. Unless otherwise stated, all dimensions are in inches.
following procedure. The lower half of the chamber was immobilized on the chamber assembly wrench pictured in Fig. 3 and a clean coverslip, which had been rinsed in MEM or PBS, was placed on top of it. The fluid used to rinse the coverslip and assemble the chamber was chosen to keep the environment o f the test cells as constant as possible. Whenever MEM was used in the assembly of the chamber it was taken from the same petri dish in which the test cells (IC21 cells) had been cultured. PBS was used t h r o u g h o u t when red
34
cell detachment was being examined. After a coverslip had been placed on top of the lower chamber half, MEM was placed on top of it until it was about to run o f f the coverslip. The lower teflon spacer, pictured in Fig. 1, was then rinsed in MEM, dried and quickly pushed directly down on the coverslip in a manner that allowed some of the MEM to fill up the hole through the middle of the spacer. Approximately 0.5 ml of MEM was placed on top of the spacer with a pasteur pipette. This was done by directing the MEM into the hole in the spacer in a manner that would dislodge any bubbles which had attached to the spacer in this region. The coverslip containing the cells was then floated onto the MEM so that the side of the coverslip to which the cells were attached faced the teflon spacer. The upper chamber housing containing the upper teflon spacer was then partially tightened down and the region between the upper coverslip and upper chamber half flooded with MEM. The chamber halves were then tightened down but only to finger tightness. Great care was taken to prevent any bubbles from being trapped between the two coverslips during the assembly of the chamber. All cells on the b o t t o m of the upper coverslip, in the area {Fig. 1) illuminated by light passing through the hole in the lower chamber half, were counted on a microscope equipped with an 8× eyepiece and a 40× long working distance objective. When the centrifugal chambers were loaded into the buckets of the SW27 rotor they were immersed in PBS (~1 cm) to prevent the introduction of bubbles into the chamber. All cells were accelerated to terminal speed at a maximal rate. The rotor was always precooled to 22 ± 2°C. The percentage of cells detached from the glass coverslip by the centrifugation process was calculated from measurements of the total number of cells adhering to the illuminated area of the upper glass slide before and after centrifugation. The impulse, I, imparted to the cells during a centrifugal assay was calculated from measurements of the rotor's speed at 5-s intervals as the rotor came to terminal speed at maximal acceleration, and then was either immediately decelerated at a constant rate or kept at terminal speed for a fixed period of time before being decelerated. Under the former conditions a cell of mass m would be subjected to an impulse of 3.15 • 101° m • dyne • s when the rotor was brought to a speed to 27 000 rev./min and then decelerated. When the cells were held at a constant speed of 27 000 rev./min for a period of t seconds they were exposed to an impulse of (3.15 • 10 l° + 1.12 • 10st) m • dyne • s. RESULTS AND DISCUSSION
RBS and cells from the IC21 line respond similarly to a detaching force when attached to a glass substrate. The percentage of cells detached depends on the impulse to which the cells are exposed as well as the magnitude of the force they experience. This was demonstrated most clearly with the IC21 line. When IC21 cells were exposed to a constant force of 0.0056 dyne
35
90 80 70 60 50 40 30 20 I0
2
4
I 6
B
I0
I 12
I
I
I
14
16
t8
Impulse (dyne seconds} Fig. 4. IC21 d e t a c h m e n t as a f u n c t i o n o f impulse. All cells are e x p o s e d t o a m a x i m a l centrifugal field o f 114 0 0 0 × g for varying l e n g t h s o f t i m e to investigate t h e e f f e c t of impulse o n cell d e t a c h m e n t . T h e d o t t e d line s e g m e n t re.presents t h e a n t i c i p a t e d b e h a v i o r o f t h e cells.
(assumed b u o y a n t cell mass 50 pg) the percent of cells detaching increased linearly with the magnitude of the impulse to which they were exposed (Fig. 4). Assuming a linear relationship between the percentage of cells detached and impulse, 50% of the cells were detached by an impulse of 8 dyne • s. However, detachment does n o t seem to be a function of impulse alone. When IC21 cells were exposed to an impulse of 11.7 dyne • s, the percentage of cells detaching from the substrate increased with the magnitude of the force they experienced (Fig. 5). One shortcoming of this analysis is that it ignores the fact that forces below a certain critical level will have little or no effect on the detachment of cells. Thus, it is possible that cell detachm e n t m a y be quite heavily dependent on an 'effective impulse'. This 'effective impulse' would be the impulse delivered to the cells by forces in excess of a certain critical level. In any event, the present study and others [4,5] have indicated that the detachment of cells depends on both the detaching force to which the cells are exposed as well as the impulse associated with it. This fact makes any sort of detailed comparison of the present study, with other studies of the mean force necessary ~o detach macrophages, quite difficult. To date no other' studies concerning macrophages have stated or given sufficient data to calculate the impulse associated with their measurements. Assessment of the adhesion of RBC t o bare glass also demonstrated that
36 90 80 ¸ 70
60
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50
"6
40
g ~
30 20 ////
I0 0
" I
I
I
i
I
I
2
3
4
5
~
6
Force (millidynes) Fig. 5. IC21 d e t a c h m e n t as a f u n c t i o n o f c e n t r i f u g a l force. A l l cells were e x p o s e d t o an impulse of 11.7 dyne • s which was delivered by forces of varying magnitude. Force was c a l c u l a t e d f r o m t h e a p p l i e d c e n t r i f u g a l field a n d a n a s s u m e d b u o y a n t cell m a s s o f 5 0 pg. T h e d o t t e d line r e p r e s e n t s t h e a n t i c i p a t e d b e h a v i o r o f t h e cells.
the percent of RBC detached from a substrate depends on the impulse they experience. When RBC were exposed to a force of 30 #dyne/cell, approximately 50% of the cells could be detached at an impulse of 0.0018 dyne • s and 75% of the cells at an impulse of 0.017 dyne • s. Although a large coefficient of variation {45%) was associated with these measurements, they agree well with those of Mohandas and coworkers [4], who, using a h y d r o d y n a m i c technique, found that the percentage of the RBC removed by a given force depended on the impulse associated with it. Approximately 50% of the attached RBC could be removed by force of 65 #dyne/cell at an impulse of 0.003 d y n e ' s or by a force of 10 #dyne/cell at an impulse of 0.012 d y n e • s. Their data and the present data conflict with the results of George et al. [9], who found that no RBC were detached from a bare glass substrate by a centrifugal field that generated forces up to 48 #dyne/cell. This discrepancy has been attributed [4] to differences in the manner in which the two techniques distributed detaching forces over the membrane of the adherent RBC. However, this explanation cannot account for the differences between the present work and that of George et al. [9] as a centrifugal technique was used in both experiments. It is therefore probable that the observed differences can be attributed to the washing procedures [10] and types of glass [11] used in the two experiments. No attempts were made to show that the
37
percentage of erythrocytes detaching from glass increased with increasing force at a constant impulse. In summary, the data show that for both RBC and cells from the IC21 line the extent of detachment from a substrate depends on the impulse as well as the force to which they are exposed. These observations, when coupled with the work of others [4,5], suggest that this type of behavior may be a universal property of cells. If the dependence of cell detachment on impulse is a universal property of cells, all future measurements of the force necessary to detach a cell from its substrate must be accompanied by estimates of the impulse associated with them. SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS The present technique should facilitate further studies on the relationship between applied impulse and the force necessary to detach cells as well as studies on chemotaxis, locomotion, metastasis, phagocytosis and other phenomena involving cell adhesiveness. In essence, the method is one which allows the investigator to attach cells to a transparent substrate under defined conditions and then to determine whether individual cells have detached after being subjected to a preselected force and impulse regimen. The primary advantages of this technique are as follows: The properties of individual cells (e.g. surface area, morphology), rather than the average value of populations, can be correlated with a measure of the force or applied impulse required to detach them. By generating centrifugal fields of up to 114 000 X g, studies of the detachment of many different cell types are possible. The effect of cell substrate and the surrounding medium on cell detachment can be readily assessed. Cell residue and non-detached cells can be cultured or studied after the centrifugation process. ACKNOWLEDGMENTS
We would like to thank Dr. R. Skalak for helpful discussion. This work was supported in part by the NIH grants 5-T32-GMO7552 and CA 16247. REFERENCES 1 Mauel, J. and Defendi, V. (1971) Infection and transformation of mouse peritoneal macrophages by simian virus 40. J. Exp. Med. 1 3 4 , 3 3 5 - - 3 5 0 2 McKeever, P.E. (1974) Methods to study pulmonary alveolar macrophage adherence: Micromanipulation and quantitation. J. Reticuloendothel. Soc. 16, 313--317 3 Krupp, H. (1967) Particle adhesion. Theory and experiment. Adv. Colloid Interface Sci. 1 , 1 1 1 - - 2 3 9 4 Mohandas, N., Hochmuth, R.M. and Spaeth, E.E. (1974) Adhesion of red cells to foreign surfaces in the presence of flow. J. Biomed. Mater. Res. 8, 119--136 5 Weiss, L. (1961) The measurement of cell adhesion. Exp. Cell Res., Suppl. 8, 141-153 6 Halpin, J.C. and Polley, H.W. (1967) Observations on the fracture of viscoelastic bodies. J. Compos. Mater. 1, 64--81. 7 Jay, A.W.L. (1973} Viscoelastic properties of the human red blood cell membrane I. Deformation, volume loss and rupture of red cells in micropipettes. Biophys. J. 13, 1166--1182.
38
8 Rosen, J.J. and Culp, L.A. (1977) Morphology and cellular origins of substrateattached material from mouse fibroblasts. Exp. Cell Res. 107, 139--149 9 George, J.N., Weed, R.I. and Reed, C.F. (1970) Adhesion of human erythroeytes to glass: The nature of the interaction on the effect of serum and plasma. J. Cell. Physiol. 77, 51--59 10 Nordlings, S. (1967) Adhesiveness, growth behavior and charge density of cultured cells. Acta Pathol. Microbiol. Scand., Suppl. 192, 1--100 11 Rappaport, C., Poole, J.P. and Rappaport, H.P. (1960) Studies on properties of surfaces required for growth of mammalian cells in synthetic medium. Exp. Cell Res. 20, 465--510
29
© Elsevier/North-Holland Biomedical Press
CENTRIFUGAL ASSESSMENT OF CELL ADHESION
W.D. CORRY * and V. DEFENDI
New York University Medical School, Department of Pathology, 550 Ist Avenue, New York, N Y 10016, U.S.A. (Received 14 May 1980; accepted 28 July 1980)
The design of a chamber for determining the centrifugal force necessary to detach cells from various substrates is presented. Cells from an SV40-transformed murine peritoneal macrophage line and human erythrocytes were used to assess the feasibility of using the chamber for studies of cell adhesion. This work was confirmed the usefulness of the chamber and provided data concerning the force necessary to detach the cells. These data indicated that the percentage of cells detached from a glass substrate was not a function of force alone. The number of cells detaching increased with the impulse applied to the cells when they were exposed to a constant force. Similarly, when the impulse applied to the IC21 cells was maintained at a constant level, the percentage of cells detached by a centrifugal force increased with the magnitude of the force. Key words: macrophage; erythrocyte, centrifugation; adhesion.
INTRODUCTION
An improved centrifugal technique for measuring the force required to detach cells from various substrates is presented in this paper. Cells are allowed to attach to a transparent substrate and then placed in a special chamber. The chamber and cells are then subjected to a preselected centrifugal force and the percentage of cells that detach measured. The primary advantages of this technique are that it allows the investigator access to centrifugal fields up to 114 000 × g and enables him to measure the impulse imparted to the cells. The utility of this technique was assessed by measuring the force required to detach human erythrocytes (RBC) and IC21 cells, an SV40-transformed murine peritoneal macrophage [1], from a glass substrate. These cells were chosen as they represent opposite extremes in adhesive behavior. The RBC adhere weakly while the IC21 cells adhere with a tenacity that exceeds the capacity of most techniques for measuring the force necessary to detach
* Present address: University of Oregon Health Sciences Center, Department of Neurology, 3181 SW Sam Jackson Park Rd., Portland, OR 97201, U.S.A.
30 them. Although techniques for measuring the force necessary to separate tightly bound objects [2,3] exist, they have serious drawbacks. The centrifugal m e t h o d presented here was developed to circumvent these drawbacks. This technique was chosen for three reasons. First, by using a Beckman SW27 rotor, centrifugal fields in excess of 100 000 × g can be attained. Second, the force generated by this field is uniform and therefore suitable for an analysis of the distribution of force over the attached cell. Finally, this technique offers a method of monitoring the impulse applied to the cells. Impulse is usually defined as the amount of m o m e n t u m transferred to an object when it is subjected to a force, F, over a period of time 0 to t. In mathematical terrq.s, impulse, I, can be defined by the equation t
I = f Fdt
(1)
0
When a particle is attached to a substrate in a centrifugal rotor at a distance r from its center of rotation, the impulse applied to the particle is given by t
I = rm f co2dt
(2)
0
where co is the angular velocity of the rotor and m the b u o y a n t mass of the cell. The rationale for measuring impulse is derived from several sources. A number of studies [4,5] of cell detachment have shown that the percentage of cells detaching from a given substrate depends on the length of time they were stressed as well as the magnitude of the force to which they were subjected. These observations are in agreement with studies by Halpin and Polley [6] on the fracture of viscoelastic bodies. In this work Halpin and Polley [6] indicated that when a viscoelastic solid is subjected to a stress its likelihood of rupturing will depend not only on the magnitude of the force to which it is subjected, b u t also its duration. The applicability of this theory to the present situation is suggested by studies that indicate that the cell membrane behaves as a viscoelastic solid [7] and that cell detachment is a process which usually involves the rupture of the cell membrane [8] rather than the cell--substrate adhesive joint. Finally, the use of the concept of impulse is dictated for centrifugal techniques by the fact that much of the cell detachm e n t process may be accomplished by forces which are less than maximal. Measurement of the impulse applied to these cells is one step towards placing the study of cell detachment on a quantitative basis. MATERIALS
AND METHODS
All studies of cell adhesion were carried o u t with Coming No. 1 thickness coverslips. They were cleaned by sequentially immersing them for 12 h in
31
distilled de-ionized water; 8 h in an ethanolic-KOH solution (3 M KOH/15 M ethanol); five times in distilled de-ionized water; 12 h in acid (85% H2SO4/ 5% HNO3/10% H20 (v/v}); and five times in distilled de-ionized water. Each distilled water rinse utilized a minimum of 500 ml of fluid. After this cleaning procedure the coverslips were heat sterilized. All studies of the adhesion of cells to a glass substrate were done with either human erythrocytes or IC21 cells. The IC21 cell line is an established tissue culture line [ 1 ] which had been obtained by transforming murine peritoneal macrophages with SV40 virus. The IC21 line was grown in minimal essential medium (MEM; GIBCO) supplemented with 1× antibiotic/antimycotic (GIBCO) and 10% fetal bovine serum ( F B S ) i n an injection incubator with a 5% CO2 atmosphere. Subconfluent cultures were fed between 18 and 24 h before harvest to ensure that they would be in a log phase growth at harvest. The cells were detached by exposing them to a 37°C PBS/EDTA solution (137 mM NaC1/3 mM KC1/8 mM Na2HPO,/1 mM K H 2 P O J 5 mM Na2EDTA) for 5 min and tapping the T-75 flask (Falcon Plastics) in which they were grown to dislodge them. The decanted cell suspension was then promptly mixed with an equal volume of MEM, centrifuged 5 min at 160 × g, and the supernatant decanted. They were then suspended in the MEM containing 10% FBS, plated onto coverslips at a density of 1--5(~. 104 cells/cm 2 and then incubated as before. Erythrocytes were prepared for adhesion assays from blood drawn from t h e antecubital vein into a heparinized syringe (100 units/10 ml blood) and by centrifuging the blood for 10 min at 650 X g, room temperature (23 ± 2°C). The plasma and b u f f y coat were drawn off and discarded. The red cells were suspended in phosphatebuffered saline (PBS: 4.8 mM K H 2 P O J 2 5 . 2 mM N a 2 H P O J 1 2 1 . 4 mM NaC1; pH 7.42 ± 0.02, 292 t 2 mOsm) to a RBC : PBS ratio of approximately 1 : 4 and centrifuged as before. After centrifugation any remaining white cells were removed. This washing procedure was done a total of three times. The cells were suspended in PBS to a concentration of 1 . 8 . 1 0 4 cells/ml, and plated onto coverslips. The settling RBC attained a concentration of 5--6 •
Fig. 1. E x p l o d e d view o f a c e n t r i f u g a l c h a m b e r . F r o m t h e l e f t the pieces o f t h e c h a m b e r are t h e u p p e r h a l f of t h e c h a m b e r , a 6 m m i.d. t e f l o n disk, a n 18 m m glass coverslip, a 2 m m i.d. t e f l o n disk, a n 18 m m glass coverslip and the lower h a l f o f t h e c h a m b e r . The t h i n b r o k e n line passing t h r o u g h t h e c e n t e r o f the chamber represents the path of any i l l u m i n a t i n g light t o enter the c h a m b e r . T h e o p e n circle o n t h e upper coverslip indicates t h e r e g i o n t h a t is o b s e r v e d w h e n the percentage of cells detaching is measured.
32
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Fig. 2. Machinist's drawing o f the upper and l o w e r halves o f the chamber and the t e f l o n spacers. Unless o t h e r w i s e stated, all d i m e n s i o n s are in inches.
103 cells/cm 2 on the coverslips and were allowed to remain on the coverslips a minimum o f 2 h before being assembled in the centrifugal chamber. All assays of red cell adhesion were done at 3 9 0 0 X g. All cell detachment assays were done with an aluminum chamber (Velotron Machine Co., Brooklyn, N.Y.) adapted for use in a Beckman SW27 rotor. Exploded and cross-sectional views of the chamber can be seen in Figs. 1--3. The chamber and coverslip containing the cells were assembled by the
33
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SIDE CUSHION MATL: DELRIN Fig. 3. Machinist's drawing of chamber cushion and chamber assembly wrench. The chamber assembly wrench also serves as a platform for viewing cells in the assembled chamber with a microscope. The chamber cushion is placed beneath the chamber in the b o t t o m of the bucket for the centrifuge rotor to distribute evenly the load placed on the bucket by the chamber. Unless otherwise stated, all dimensions are in inches.
following procedure. The lower half of the chamber was immobilized on the chamber assembly wrench pictured in Fig. 3 and a clean coverslip, which had been rinsed in MEM or PBS, was placed on top of it. The fluid used to rinse the coverslip and assemble the chamber was chosen to keep the environment o f the test cells as constant as possible. Whenever MEM was used in the assembly of the chamber it was taken from the same petri dish in which the test cells (IC21 cells) had been cultured. PBS was used t h r o u g h o u t when red
34
cell detachment was being examined. After a coverslip had been placed on top of the lower chamber half, MEM was placed on top of it until it was about to run o f f the coverslip. The lower teflon spacer, pictured in Fig. 1, was then rinsed in MEM, dried and quickly pushed directly down on the coverslip in a manner that allowed some of the MEM to fill up the hole through the middle of the spacer. Approximately 0.5 ml of MEM was placed on top of the spacer with a pasteur pipette. This was done by directing the MEM into the hole in the spacer in a manner that would dislodge any bubbles which had attached to the spacer in this region. The coverslip containing the cells was then floated onto the MEM so that the side of the coverslip to which the cells were attached faced the teflon spacer. The upper chamber housing containing the upper teflon spacer was then partially tightened down and the region between the upper coverslip and upper chamber half flooded with MEM. The chamber halves were then tightened down but only to finger tightness. Great care was taken to prevent any bubbles from being trapped between the two coverslips during the assembly of the chamber. All cells on the b o t t o m of the upper coverslip, in the area {Fig. 1) illuminated by light passing through the hole in the lower chamber half, were counted on a microscope equipped with an 8× eyepiece and a 40× long working distance objective. When the centrifugal chambers were loaded into the buckets of the SW27 rotor they were immersed in PBS (~1 cm) to prevent the introduction of bubbles into the chamber. All cells were accelerated to terminal speed at a maximal rate. The rotor was always precooled to 22 ± 2°C. The percentage of cells detached from the glass coverslip by the centrifugation process was calculated from measurements of the total number of cells adhering to the illuminated area of the upper glass slide before and after centrifugation. The impulse, I, imparted to the cells during a centrifugal assay was calculated from measurements of the rotor's speed at 5-s intervals as the rotor came to terminal speed at maximal acceleration, and then was either immediately decelerated at a constant rate or kept at terminal speed for a fixed period of time before being decelerated. Under the former conditions a cell of mass m would be subjected to an impulse of 3.15 • 101° m • dyne • s when the rotor was brought to a speed to 27 000 rev./min and then decelerated. When the cells were held at a constant speed of 27 000 rev./min for a period of t seconds they were exposed to an impulse of (3.15 • 10 l° + 1.12 • 10st) m • dyne • s. RESULTS AND DISCUSSION
RBS and cells from the IC21 line respond similarly to a detaching force when attached to a glass substrate. The percentage of cells detached depends on the impulse to which the cells are exposed as well as the magnitude of the force they experience. This was demonstrated most clearly with the IC21 line. When IC21 cells were exposed to a constant force of 0.0056 dyne
35
90 80 70 60 50 40 30 20 I0
2
4
I 6
B
I0
I 12
I
I
I
14
16
t8
Impulse (dyne seconds} Fig. 4. IC21 d e t a c h m e n t as a f u n c t i o n o f impulse. All cells are e x p o s e d t o a m a x i m a l centrifugal field o f 114 0 0 0 × g for varying l e n g t h s o f t i m e to investigate t h e e f f e c t of impulse o n cell d e t a c h m e n t . T h e d o t t e d line s e g m e n t re.presents t h e a n t i c i p a t e d b e h a v i o r o f t h e cells.
(assumed b u o y a n t cell mass 50 pg) the percent of cells detaching increased linearly with the magnitude of the impulse to which they were exposed (Fig. 4). Assuming a linear relationship between the percentage of cells detached and impulse, 50% of the cells were detached by an impulse of 8 dyne • s. However, detachment does n o t seem to be a function of impulse alone. When IC21 cells were exposed to an impulse of 11.7 dyne • s, the percentage of cells detaching from the substrate increased with the magnitude of the force they experienced (Fig. 5). One shortcoming of this analysis is that it ignores the fact that forces below a certain critical level will have little or no effect on the detachment of cells. Thus, it is possible that cell detachm e n t m a y be quite heavily dependent on an 'effective impulse'. This 'effective impulse' would be the impulse delivered to the cells by forces in excess of a certain critical level. In any event, the present study and others [4,5] have indicated that the detachment of cells depends on both the detaching force to which the cells are exposed as well as the impulse associated with it. This fact makes any sort of detailed comparison of the present study, with other studies of the mean force necessary ~o detach macrophages, quite difficult. To date no other' studies concerning macrophages have stated or given sufficient data to calculate the impulse associated with their measurements. Assessment of the adhesion of RBC t o bare glass also demonstrated that
36 90 80 ¸ 70
60
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50
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40
g ~
30 20 ////
I0 0
" I
I
I
i
I
I
2
3
4
5
~
6
Force (millidynes) Fig. 5. IC21 d e t a c h m e n t as a f u n c t i o n o f c e n t r i f u g a l force. A l l cells were e x p o s e d t o an impulse of 11.7 dyne • s which was delivered by forces of varying magnitude. Force was c a l c u l a t e d f r o m t h e a p p l i e d c e n t r i f u g a l field a n d a n a s s u m e d b u o y a n t cell m a s s o f 5 0 pg. T h e d o t t e d line r e p r e s e n t s t h e a n t i c i p a t e d b e h a v i o r o f t h e cells.
the percent of RBC detached from a substrate depends on the impulse they experience. When RBC were exposed to a force of 30 #dyne/cell, approximately 50% of the cells could be detached at an impulse of 0.0018 dyne • s and 75% of the cells at an impulse of 0.017 dyne • s. Although a large coefficient of variation {45%) was associated with these measurements, they agree well with those of Mohandas and coworkers [4], who, using a h y d r o d y n a m i c technique, found that the percentage of the RBC removed by a given force depended on the impulse associated with it. Approximately 50% of the attached RBC could be removed by force of 65 #dyne/cell at an impulse of 0.003 d y n e ' s or by a force of 10 #dyne/cell at an impulse of 0.012 d y n e • s. Their data and the present data conflict with the results of George et al. [9], who found that no RBC were detached from a bare glass substrate by a centrifugal field that generated forces up to 48 #dyne/cell. This discrepancy has been attributed [4] to differences in the manner in which the two techniques distributed detaching forces over the membrane of the adherent RBC. However, this explanation cannot account for the differences between the present work and that of George et al. [9] as a centrifugal technique was used in both experiments. It is therefore probable that the observed differences can be attributed to the washing procedures [10] and types of glass [11] used in the two experiments. No attempts were made to show that the
37
percentage of erythrocytes detaching from glass increased with increasing force at a constant impulse. In summary, the data show that for both RBC and cells from the IC21 line the extent of detachment from a substrate depends on the impulse as well as the force to which they are exposed. These observations, when coupled with the work of others [4,5], suggest that this type of behavior may be a universal property of cells. If the dependence of cell detachment on impulse is a universal property of cells, all future measurements of the force necessary to detach a cell from its substrate must be accompanied by estimates of the impulse associated with them. SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS The present technique should facilitate further studies on the relationship between applied impulse and the force necessary to detach cells as well as studies on chemotaxis, locomotion, metastasis, phagocytosis and other phenomena involving cell adhesiveness. In essence, the method is one which allows the investigator to attach cells to a transparent substrate under defined conditions and then to determine whether individual cells have detached after being subjected to a preselected force and impulse regimen. The primary advantages of this technique are as follows: The properties of individual cells (e.g. surface area, morphology), rather than the average value of populations, can be correlated with a measure of the force or applied impulse required to detach them. By generating centrifugal fields of up to 114 000 X g, studies of the detachment of many different cell types are possible. The effect of cell substrate and the surrounding medium on cell detachment can be readily assessed. Cell residue and non-detached cells can be cultured or studied after the centrifugation process. ACKNOWLEDGMENTS
We would like to thank Dr. R. Skalak for helpful discussion. This work was supported in part by the NIH grants 5-T32-GMO7552 and CA 16247. REFERENCES 1 Mauel, J. and Defendi, V. (1971) Infection and transformation of mouse peritoneal macrophages by simian virus 40. J. Exp. Med. 1 3 4 , 3 3 5 - - 3 5 0 2 McKeever, P.E. (1974) Methods to study pulmonary alveolar macrophage adherence: Micromanipulation and quantitation. J. Reticuloendothel. Soc. 16, 313--317 3 Krupp, H. (1967) Particle adhesion. Theory and experiment. Adv. Colloid Interface Sci. 1 , 1 1 1 - - 2 3 9 4 Mohandas, N., Hochmuth, R.M. and Spaeth, E.E. (1974) Adhesion of red cells to foreign surfaces in the presence of flow. J. Biomed. Mater. Res. 8, 119--136 5 Weiss, L. (1961) The measurement of cell adhesion. Exp. Cell Res., Suppl. 8, 141-153 6 Halpin, J.C. and Polley, H.W. (1967) Observations on the fracture of viscoelastic bodies. J. Compos. Mater. 1, 64--81. 7 Jay, A.W.L. (1973} Viscoelastic properties of the human red blood cell membrane I. Deformation, volume loss and rupture of red cells in micropipettes. Biophys. J. 13, 1166--1182.
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8 Rosen, J.J. and Culp, L.A. (1977) Morphology and cellular origins of substrateattached material from mouse fibroblasts. Exp. Cell Res. 107, 139--149 9 George, J.N., Weed, R.I. and Reed, C.F. (1970) Adhesion of human erythroeytes to glass: The nature of the interaction on the effect of serum and plasma. J. Cell. Physiol. 77, 51--59 10 Nordlings, S. (1967) Adhesiveness, growth behavior and charge density of cultured cells. Acta Pathol. Microbiol. Scand., Suppl. 192, 1--100 11 Rappaport, C., Poole, J.P. and Rappaport, H.P. (1960) Studies on properties of surfaces required for growth of mammalian cells in synthetic medium. Exp. Cell Res. 20, 465--510