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Behaviour of fibroblast-like cells on grooved surfaces
Experimental Cell Research 65 (1971) 1 9 3 - 2 0 1
BEHAVIOUR
OF FIBROBLAST-LIKE
ON
GROOVED
Y. A. R O V E N S K Y ,
CELLS
SURFACES
I. L. SLAVNAJA and J. M. VASILIEV
Institute of Experimental and Clinical Ontology, Academy of Medical Sciences, Moscow, USSR
SUMMARY Behaviour of mouse and chick embryo fibroblast-like cells and of neoplastic mouse fibroblasts of L strain on surfaces with orderly distributed grooves of various depth (from 5 to 65/zm) was studied. A few hours after seeding, normal embryo cells migrated from the grooves of a certain depth (more than 15/~m) and spread over the surface between the grooves. At 24-48 h, cell density in the areas near the bottom of the grooves was several times lower than that in the intervals between the grooves. Most cells on these surfaces acquired an orientation parallel to that of the grooves. Various modifications of the conditions of culture (rotation of cultures, addition of colcemid, etc.) had no effect upon cell migration from the grooves. Migration of L cells was considerably less pronounced than that of normal cells. It is suggested that cell migration from the grooves can be regarded as a result of differences in the attachment of cells to surfaces with various geometrical configurations. Certain types of neoplastic cells may have decreased selectivity of reactions to the geometry of the underlying surface.
The
oriented
structure
s u b s t r a t e is k n o w n
of
the
underlying
to be one of the main
factors controlling locomotion
and orienta-
t i o n o f f i b r o b l a s t s c u l t u r e d i n v i t r o [9, 10]. To understand
better the nature of this con-
t r o l it is e s s e n t i a l t o l e a r n m o r e
about
the
e f f e c t s of v a r i o u s c h a r a c t e r i s t i c s of t h e s u b s t r a t e o n cell b e h a v i o u r . T h e a i m of t h e e x p e r i m e n t s d e s c r i b e d i n this paper
was to study the behaviour
of
cells s e e d e d u p o n s u r f a c e s w i t h o r d e r l y distributed
grooves
of v a r i o u s
depth.
It was
found that normal mouse and chick embryo fibroblast-like
cells
rapidly
migrate
from
grooves of a certain depth. We discuss the possible mechanisms.
MATERIALS
AND
METHODS
Characteristics of the surfaces of the substrates used are given in table 1 (see also fig. l). Polyvinylchloride 13 - 711804
plates were prepared to our order at the Recording Studios of the USSR; they were fragments of modified discs used for sound recording. In the course of preparation of these discs the grooves were cut mechanically at the original surface; then several copies of this surface were made on nickel plates by the galvanoplastic method; and the final copy was made on the polyvinylchloride plate by a stamping procedure. Grooved nickel plates obtained at the intermediate stage of the production of polyvinylchloride plates were also used as substrates. Polyethylene and polymethylmethacrylate plates were prepared in the Scientific Institute of Cinematography and Photography (NIKFI, Moscow); grooves were made by a photomechanical method. These were three varieties of lenticular plates used in photography. Plates of each type with smooth surfaces were used as controls. All plates were washed in water and in 96~ alcohol, then dried and sterilized by UV rays. Embryo fibroblast-like cells were obtained by trypsinization of 15-16-day-old mouse embryos or of 7-8-day-old chick embryos. Ceils were cultivated 3-4 days in large flasks, then removed from the glass with 0.25 % trypsin and resuspended in culture medium. 30 x 5 m m ~ plates of the tested substrates were placed in penicillin flasks. Two ml of cell suspension (105 cells/ml) were seeded in each flask. Flasks were stoppered and placed in the thermostat (37~ horizontally with grooved surfaces oriented upwards. Culture medium 199 with 10% bovine serum was
Exptl Cell Res 65
Y. A. Rovensky et al.
194 r
D
Table 1. Characteristics of grooved surfaces
a
5. Material Plates with parallel grooves Polyvinylchloride
Fig. 1. Side views of several types of grooved surface used in the experiments. H, depth of groove; D, interval between grooves. (a) Polyvinylchloride plate; H, 40 /~m; D, 200 /zm. (b)Polyvinylchloride plate; H, 5 /zm; D, 115/zm. (c) Nickel plate; H, 3 0 # m ; D, 120 /~m. Microphotographs, (a, b) x 220, (c) x 160.
used in all the experiments. In certain experiments medium pre-incubated with cultures of mouse embryo cells for 24 h ("conditioned" medium) or for 96 h ("exhausted" medium) was used. Mouse fibroblasts of L strain were seeded in identical fashion. Plates with the attached cells were fixed in Bouin mixture at various times after seeding; cells were stained with 0.1% methylene blue. Cultures were examined and photographed without embedding. To assess quantitatively the degree of cell migration from the grooves, a "migration index" was calculated for some cultures. This was the relation between average cell densities in two groups of parallel surface strips in the same plate: (a) 30 /~m wide strips in the middle of the interval between the grooves; (b) strips of the same width which contained the bottoms of the grooves along their mid-line. Number of cells per standard area of these strips was counted with the aid of an ocular micrometric grid; the cells crossing the margin of the strips were included in the count.
Nickel Polyethylene Polymethylmethacrylate
Depth of groovesa (/zm)
Interval between the ~ grooves (/zm)
5 15 25 30 40 30 65 65
115 80 155 120 200 120 300 310
Plates with hexagonal grooves Polymethylmethacrylate 45
440 e
a H, see fig. 1. D, see fig. 1. c Distance between two parallel grooves forming two opposite sides of one hexagon.
RESULTS
Experiments with normal embryo cells Results of all types of experiment with mouse cells and with chick cells were similar and will be described together. In control experiments the cells were spread on the smooth surfaces of the plates of all types during the first 3 h after seeding. At that time most cells acquired a normal elongated fibroblast-like form. Cells were
Table 2. Index of migration of fibroblast-like cells on polyvinylchloride surfaces with grooves
of varying deptha Depth of groove (/~m) Cell type
40
25
15
5
Normal mouse embryo cells Colcemid-treated mouse embryo cells Fibroblasts of L strain
5.9+1.32
4.6_+1.22
2.5_+1.41
1.0+0.04
18.2-t-3.87 2.1 •
10.0_+1.14 2.0+0.28
-1.8 -+0.62
-1.0+0.2
a Index of migration = Cell density in the interval between grooves/Cell density near the bottom of the groove. See text for details of counting procedure. All the indices were counted in cultures fixed 24 h after seeding of cells. Average indices for each group _+S.E. are given. Exptl Cell Res 65
Cell behaviour on grooved surfaces
195
Fig. 2. Mouse embryo cells on the polyvinylchloride plate with 40/~m deep grooves (a) 30 min after seeding, cells in the grooves; (b) 60 min after seeding. The cells begin to migrate from the grooves and to spread themselves on the side surfaces of the grooves. Microphotograph, x 100.
Exptl Cell Res 65
196
u A. Rovensky et al.
evenly distributed on these surfaces and had no preferential orientation. Polyvinylchloride plates with 40 # m deep grooves: At 30 min after seeding most cells were located in the grooves. These cells were not spread (fig. 2a). At 1-3 h the cells gradually migrated f r o m the grooves and spread themselves upon the side surfaces of the grooves (fig. 2b). Migrating cells gradually oriented themselves parallel with the grooves. At 3-6 h most cells were already localized in the intervals between the grooves. Later (at 10-18 h) the cell density in the intervals between grooves gradually increased, but the cell distribution was unchanged. The number of cells per unit area near the bottom of the grooves was several times lower than that in the middle of the interval between the grooves (table 2, fig. 3). The width of the zone with low cell density was about 40-60 #m. The few cells remaining in these zones were often attached with their processes at the two opposite sides of the groove. These groove-spanning cells often appeared less spread than other cells in the same culture: they had thinner cytoplasmatic processes and more intensely stained cell bodies. In several experiments, the surface of the polyvinylchloride plates was covered with a few microns thick layer of collodion before seeding of the cells. This coating did not alter considerably the form of the surface. Cell distribution on these collodion-coated plates was similar to that observed in the experiments with non-coated plates. Polyvinylchloride plates with 30, 25, 15 and 5 # m deep grooves: Cell behaviour on plates with 30 and 25/zm deep grooves was similar to that on 40 # m grooves. On plates with 15 # m deep grooves, migration of cells from the grooves was somewhat less pronounced and more variable (table 2). Cell orientation paExptl Cell Res 65
rallel to the grooves was observed on all these types of plates (fig. 4). Five micron deep grooves had no effect upon the distribution of cells on the surface. At 24 h after seeding the cell density near the grooves and between the grooves was equal (table 2); the cells had no orientation in relation to the grooves. Nickel, polyethylene and polymethylmethacrylate plates: Cell behaviour on these plates was similar to that on polyvinylchloride plates with 40 # m deep grooves: the cells migrated from the grooves and later remained located on the intervals between the grooves (fig. 5) Cells on the surfaces with parallel grooves became oriented parallel with these grooves. On surfaces with hexagonal grooves only those cells located near to the groove became oriented; the cells in the central parts of the intervals had no definite orientation (fig. 5). Modifications of the conditions of cultivation: The following variants of experiments with mouse embryo cells seeded upon polyvinylchloride plates with 40 # m grooves were made in order to assess the effects of conditions of cultivation upon the migration from the grooves: (a) The initial concentration of cells in suspension was decreased to 2.5 • 104 cells/ ml. In these experiments the cells were suspended and cultivated in conditioned medium. (b) Flasks with the cells were rotated at a speed of one rotation/ 5 min during the entire period of cultivation. (c) At 20 min after seeding, plates in the flasks were turned over so that the grooved surface with the attached cells faced downward during the entire period of cultivation. (d) At 30 min after seeding the culture medium in the flasks was replaced with "exhausted" medium.
Cell behaviour on grooved surfaces
197
Fig. 3. Mouse embryo cells on the polyvinylchloride plate with 40 #m deep grooves 24 h after seeding. Areas of the substrate near the bottoms of the grooves contain very few cells. Microphotograph, x 100. Fig. 4. Mouse embryo cells on polyvinylchloride plate with 15/~m deep grooves 24 h after seeding. Most cells are oriented parallel to the direction of the grooves. Microphotograph, x 120.
Exptl Cell Res 65
198
Y. A. Rovensky et al.
Fig. 5. Mouse embryo cells on the polymethylmethacrylate plate with hexagonal grooves 24 h after seeding. Areas near the bottoms of the grooves contain very few cells. Microphotograph, x 100. Fig. 6. Cells of L strain on polyvinylchloride plate with 40/zm grooves 24 h after seeding. Numerous cells near the bottoms of the grooves. The cells are not oriented. Microphotograph, x 150.
Exptl Cell Res 65
Cell behaviour on grooved surfaces These modifications had no effect upon cell migration and orientation on grooved plates. Colcemid was found to change the locomotory activities of fibroblasts considerably [8]. This was the reason for testing the effect of Colcemid upon cell migration from the grooves. Colcemid (0.1 #g/ml; Ciba) was added to the medium of cells grown in large flasks 8 h before these cells were re-suspended and seeded upon the grooved surfaces. The same concentration of Colcemid was added again to the medium of cell suspensions at the moment of seeding. Colcemid-treated cells grown on the grooved and smooth surfaces acquired an irregular polygonal form and had no orientation. However, cell migration from the grooves was not inhibited. In fact, the "migration index" at 24 h after seeding in Colcemid-treated cultures had higher values than in control ones (table 2). This increase can be explained by the change of cell form: Colcemid-treated cells in contrast to normal ones did not have long cytoplasmatic processes and therefore more rarely crossed the margin of the 30 # m wide zone around the bottom of the groove (see Methods) This decreased the number of cells counted in this zone. If Colcemid-containing medium was replaced with fresh medium without the drug, normal cell form and orientation were completely restored 24 h later.
Experiments with cells of L strain At 30-60 rain after seeding the distribution of L cells on the grooved surfaces was no different from that of normal cells. Later, at 3-24 h many cells had migrated from the grooves, though numerous cells remained (fig. 6). At 24 h the index of migration was lower than that obtained in the experiments with normal cells (table 2). Cells in the grooves appeared less spread than those located
199
on the areas between the grooves. L cells had no definite orientation on grooved surfaces. DISCUSSION Two main processes determine the distribution of normal fibroblast-like cells upon the surfaces with deep grooves: (a) such cells migrate from the grooves; (b) they orient themselves with regard to the direction of the grooves. We will discuss the possible mechanisms of these processes. Migration from the grooves could be due to some chemotactic reaction: the cells concentrated in the grooves shortly after seeding might repel each other by changing, in some way, the culture medium around them. This could lead to cell migration into areas with lower population density. However, results of a number of experiments render this mechanism improbable: migration was not affected by a decrease of the initial cell population density in the grooves, by increased circulation of the culture medium (experiments with rotation of cultures), or by changed composition of the medium (experiments with "exhausted" medium). It seems more probable that migration is due to differences in the interaction of cells with various parts of the grooved surface. Possibly, cells attach to the surface at the bottom of the grooves less readily than to other parts of the surface. This suggestion is in agreement with the morphological observations: at various times after seeding, cells located in t h e grooves appeared less spread than those on other parts of the surface. Carter [4] demonstrated oriented movement of the ceils in the direction of increasing adhesion to the substrate; a gradient of adhesion was produced by graded deposition of palladium upon the surface. Exptl Cell Res 65
200
Y. A. Rovensky et al.
In our experiments, in contrast to those of Carter, all parts of each substrate were chemically identical. What then could be the cause of decreased attachment of cells to the bottom of the grooves? There are two possibilities: (a) decreased attachment is due to some peculiarities of the microstructure of the surface in the grooves; (b) cell attachment depends on the gross geometrical configuration of the surface. One cannot exclude that certain differences in the microstructure of various parts of the surface may arise in the course of manufacture. However, if the cell behaviour were determined by these hypothetical heterogeneities of the surface microstructure, then one could expect to find considerable differences in cell distribution on the plates consisting of various materials and containing grooves made by various methods. This was not the case: on plates of all types the ceils migrated in a similar fashion f r o m grooves of similar depth. Pre-coating of the grooved surface with thin collodion film obviously changed its microstructure, but did not affect cell migration. These facts justify the conclusion that cell migration can be regarded as a reaction to a certain geometrical configuration of the substrate surface. More specifically, one can conclude that this migration occurs because the ceils cannot attach themselves to the surface at the bottom of grooves having a certain depth (more than 15 #m) and a certain f o r m (an angle between the sides of about 90~ These results as well as some other facts [3, 5, 10] show that not only adhesive properties of the substrate, but also its geometry are essential for cell attachment and m a y determine the direction of cell locomotion. The factors responsible for decreased cell attachment in the grooves remain unknown. One possible explanation is as follows. It has been suggestedthat some of the protruExptl Cell Res 64
sions formed at the leading edge of the fibroblast bend towards the substrate and attach themselves to the surface of this substrate [6]. Protrusions are formed independently in various parts of the leading edge [2]. Now, if the planar leading edge of the fibroblast happens to be located across the groove, then its central and lateral parts are at various distance from the substrate. Therefore, protrusions of equal length formed in the central and lateral parts of the edge have an unequal probability of touching the substrate when curling downwards. Protrusions formed near the lateral side of the groove will attach themselves to the substrate more often than those formed above the central part of the groove. This difference eventually would lead to displacement of the whole leading edge to one or the other side of the groove, that is, to cell migration from the groove. Colcemid did not inhibit cell migration from the grooves. Earlier it was found [7, 8] that Colcemid profoundly disturbs the polarity of locomotion: inactive parts of the surface disappear in Colcemid-treated cells and all the cell edge is seen to form protrusions. Thus, polar distribution of active and inactive parts of the surface is essential for directional cell migration on the smooth substrate [7], but obviously is not necessary for migration from the grooves. This result is compatible with the mechanism of migration discussed above: unequal probability of attachment of the surface protrusions formed in various parts of the cells located above the groove may be sufficient to cause displacement of these cells both in normal and in Colcemid-treated cultures. Orientation of cells on grooved surfaces m a y be related to decreased attachment in the grooves. Cells located upon the side surface of the groove cannot effectively spread themselves across the groove; this may lead to preferential spreading in the direction pa-
Cell behaviour on grooved surfaces rallel to that of the groove. However, not only the cells on the sides of the grooves, but also those located in the central parts of the wide intervals between parallel grooves often became oriented a few hours after seeding. This observation gives one reason to think that cell orientation m a y be caused not only by the presence of grooves, but also by some other factor. Possibly, the orientation may be a reaction to the curvature of the underlying cylindrical surface [5, 10]. Migration of L cells from the grooves was considerably less pronounced than that of normal fibroblast-like cells. These results as well as those obtained by Ambrose & Ellison [3] seem to indicate that certain types of neoplastic cells have decreased the selectivity of reactions to the geometry of the underlying substrate. Further experiments are necessary to find out whether this de-
201
creased reactivity is typical for many lines of neoplastic cells and whether it is correlated with certain characteristic changes of the locomotory behaviour of these cells. REFERENCES 1. Abercrombie, M, Exptl cell res, suppl. 8 (1961) 188. 2. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exptl cell res 59 (1970) 393. 3. Ambrose, E J & Ellison, M, Europ j cancer 4 (1968) 459. 4. Carter, S B, Nature 208 (1965) 1183. 5. Curtis, A S & Varde, M, J natl cancer inst 33 (1964) 15. 6. Ingram, V M, Nature 222 (1969) 641. 7. Vasiliev, Ju M, Gelfand, I M, Domnina, L V & Rappoport, R I, Exptl cell res 54 (1969) 83. 8. Vasiliev, Ju M, Gelfand, I M, Domnina, L V Ivanova, O Y, Komm, S G & Olshevskaja, L V, J embryol exptl morphol (1970). To be published. 9. Weiss, P, Intern rev cytol 7 (1958) 391. 10. Weiss, P, Garber, B, Proc natI acad sci US 38 (1952) 264. Received August 19, 1970
Exptl Cell Res 65
BEHAVIOUR
OF FIBROBLAST-LIKE
ON
GROOVED
Y. A. R O V E N S K Y ,
CELLS
SURFACES
I. L. SLAVNAJA and J. M. VASILIEV
Institute of Experimental and Clinical Ontology, Academy of Medical Sciences, Moscow, USSR
SUMMARY Behaviour of mouse and chick embryo fibroblast-like cells and of neoplastic mouse fibroblasts of L strain on surfaces with orderly distributed grooves of various depth (from 5 to 65/zm) was studied. A few hours after seeding, normal embryo cells migrated from the grooves of a certain depth (more than 15/~m) and spread over the surface between the grooves. At 24-48 h, cell density in the areas near the bottom of the grooves was several times lower than that in the intervals between the grooves. Most cells on these surfaces acquired an orientation parallel to that of the grooves. Various modifications of the conditions of culture (rotation of cultures, addition of colcemid, etc.) had no effect upon cell migration from the grooves. Migration of L cells was considerably less pronounced than that of normal cells. It is suggested that cell migration from the grooves can be regarded as a result of differences in the attachment of cells to surfaces with various geometrical configurations. Certain types of neoplastic cells may have decreased selectivity of reactions to the geometry of the underlying surface.
The
oriented
structure
s u b s t r a t e is k n o w n
of
the
underlying
to be one of the main
factors controlling locomotion
and orienta-
t i o n o f f i b r o b l a s t s c u l t u r e d i n v i t r o [9, 10]. To understand
better the nature of this con-
t r o l it is e s s e n t i a l t o l e a r n m o r e
about
the
e f f e c t s of v a r i o u s c h a r a c t e r i s t i c s of t h e s u b s t r a t e o n cell b e h a v i o u r . T h e a i m of t h e e x p e r i m e n t s d e s c r i b e d i n this paper
was to study the behaviour
of
cells s e e d e d u p o n s u r f a c e s w i t h o r d e r l y distributed
grooves
of v a r i o u s
depth.
It was
found that normal mouse and chick embryo fibroblast-like
cells
rapidly
migrate
from
grooves of a certain depth. We discuss the possible mechanisms.
MATERIALS
AND
METHODS
Characteristics of the surfaces of the substrates used are given in table 1 (see also fig. l). Polyvinylchloride 13 - 711804
plates were prepared to our order at the Recording Studios of the USSR; they were fragments of modified discs used for sound recording. In the course of preparation of these discs the grooves were cut mechanically at the original surface; then several copies of this surface were made on nickel plates by the galvanoplastic method; and the final copy was made on the polyvinylchloride plate by a stamping procedure. Grooved nickel plates obtained at the intermediate stage of the production of polyvinylchloride plates were also used as substrates. Polyethylene and polymethylmethacrylate plates were prepared in the Scientific Institute of Cinematography and Photography (NIKFI, Moscow); grooves were made by a photomechanical method. These were three varieties of lenticular plates used in photography. Plates of each type with smooth surfaces were used as controls. All plates were washed in water and in 96~ alcohol, then dried and sterilized by UV rays. Embryo fibroblast-like cells were obtained by trypsinization of 15-16-day-old mouse embryos or of 7-8-day-old chick embryos. Ceils were cultivated 3-4 days in large flasks, then removed from the glass with 0.25 % trypsin and resuspended in culture medium. 30 x 5 m m ~ plates of the tested substrates were placed in penicillin flasks. Two ml of cell suspension (105 cells/ml) were seeded in each flask. Flasks were stoppered and placed in the thermostat (37~ horizontally with grooved surfaces oriented upwards. Culture medium 199 with 10% bovine serum was
Exptl Cell Res 65
Y. A. Rovensky et al.
194 r
D
Table 1. Characteristics of grooved surfaces
a
5. Material Plates with parallel grooves Polyvinylchloride
Fig. 1. Side views of several types of grooved surface used in the experiments. H, depth of groove; D, interval between grooves. (a) Polyvinylchloride plate; H, 40 /~m; D, 200 /zm. (b)Polyvinylchloride plate; H, 5 /zm; D, 115/zm. (c) Nickel plate; H, 3 0 # m ; D, 120 /~m. Microphotographs, (a, b) x 220, (c) x 160.
used in all the experiments. In certain experiments medium pre-incubated with cultures of mouse embryo cells for 24 h ("conditioned" medium) or for 96 h ("exhausted" medium) was used. Mouse fibroblasts of L strain were seeded in identical fashion. Plates with the attached cells were fixed in Bouin mixture at various times after seeding; cells were stained with 0.1% methylene blue. Cultures were examined and photographed without embedding. To assess quantitatively the degree of cell migration from the grooves, a "migration index" was calculated for some cultures. This was the relation between average cell densities in two groups of parallel surface strips in the same plate: (a) 30 /~m wide strips in the middle of the interval between the grooves; (b) strips of the same width which contained the bottoms of the grooves along their mid-line. Number of cells per standard area of these strips was counted with the aid of an ocular micrometric grid; the cells crossing the margin of the strips were included in the count.
Nickel Polyethylene Polymethylmethacrylate
Depth of groovesa (/zm)
Interval between the ~ grooves (/zm)
5 15 25 30 40 30 65 65
115 80 155 120 200 120 300 310
Plates with hexagonal grooves Polymethylmethacrylate 45
440 e
a H, see fig. 1. D, see fig. 1. c Distance between two parallel grooves forming two opposite sides of one hexagon.
RESULTS
Experiments with normal embryo cells Results of all types of experiment with mouse cells and with chick cells were similar and will be described together. In control experiments the cells were spread on the smooth surfaces of the plates of all types during the first 3 h after seeding. At that time most cells acquired a normal elongated fibroblast-like form. Cells were
Table 2. Index of migration of fibroblast-like cells on polyvinylchloride surfaces with grooves
of varying deptha Depth of groove (/~m) Cell type
40
25
15
5
Normal mouse embryo cells Colcemid-treated mouse embryo cells Fibroblasts of L strain
5.9+1.32
4.6_+1.22
2.5_+1.41
1.0+0.04
18.2-t-3.87 2.1 •
10.0_+1.14 2.0+0.28
-1.8 -+0.62
-1.0+0.2
a Index of migration = Cell density in the interval between grooves/Cell density near the bottom of the groove. See text for details of counting procedure. All the indices were counted in cultures fixed 24 h after seeding of cells. Average indices for each group _+S.E. are given. Exptl Cell Res 65
Cell behaviour on grooved surfaces
195
Fig. 2. Mouse embryo cells on the polyvinylchloride plate with 40/~m deep grooves (a) 30 min after seeding, cells in the grooves; (b) 60 min after seeding. The cells begin to migrate from the grooves and to spread themselves on the side surfaces of the grooves. Microphotograph, x 100.
Exptl Cell Res 65
196
u A. Rovensky et al.
evenly distributed on these surfaces and had no preferential orientation. Polyvinylchloride plates with 40 # m deep grooves: At 30 min after seeding most cells were located in the grooves. These cells were not spread (fig. 2a). At 1-3 h the cells gradually migrated f r o m the grooves and spread themselves upon the side surfaces of the grooves (fig. 2b). Migrating cells gradually oriented themselves parallel with the grooves. At 3-6 h most cells were already localized in the intervals between the grooves. Later (at 10-18 h) the cell density in the intervals between grooves gradually increased, but the cell distribution was unchanged. The number of cells per unit area near the bottom of the grooves was several times lower than that in the middle of the interval between the grooves (table 2, fig. 3). The width of the zone with low cell density was about 40-60 #m. The few cells remaining in these zones were often attached with their processes at the two opposite sides of the groove. These groove-spanning cells often appeared less spread than other cells in the same culture: they had thinner cytoplasmatic processes and more intensely stained cell bodies. In several experiments, the surface of the polyvinylchloride plates was covered with a few microns thick layer of collodion before seeding of the cells. This coating did not alter considerably the form of the surface. Cell distribution on these collodion-coated plates was similar to that observed in the experiments with non-coated plates. Polyvinylchloride plates with 30, 25, 15 and 5 # m deep grooves: Cell behaviour on plates with 30 and 25/zm deep grooves was similar to that on 40 # m grooves. On plates with 15 # m deep grooves, migration of cells from the grooves was somewhat less pronounced and more variable (table 2). Cell orientation paExptl Cell Res 65
rallel to the grooves was observed on all these types of plates (fig. 4). Five micron deep grooves had no effect upon the distribution of cells on the surface. At 24 h after seeding the cell density near the grooves and between the grooves was equal (table 2); the cells had no orientation in relation to the grooves. Nickel, polyethylene and polymethylmethacrylate plates: Cell behaviour on these plates was similar to that on polyvinylchloride plates with 40 # m deep grooves: the cells migrated from the grooves and later remained located on the intervals between the grooves (fig. 5) Cells on the surfaces with parallel grooves became oriented parallel with these grooves. On surfaces with hexagonal grooves only those cells located near to the groove became oriented; the cells in the central parts of the intervals had no definite orientation (fig. 5). Modifications of the conditions of cultivation: The following variants of experiments with mouse embryo cells seeded upon polyvinylchloride plates with 40 # m grooves were made in order to assess the effects of conditions of cultivation upon the migration from the grooves: (a) The initial concentration of cells in suspension was decreased to 2.5 • 104 cells/ ml. In these experiments the cells were suspended and cultivated in conditioned medium. (b) Flasks with the cells were rotated at a speed of one rotation/ 5 min during the entire period of cultivation. (c) At 20 min after seeding, plates in the flasks were turned over so that the grooved surface with the attached cells faced downward during the entire period of cultivation. (d) At 30 min after seeding the culture medium in the flasks was replaced with "exhausted" medium.
Cell behaviour on grooved surfaces
197
Fig. 3. Mouse embryo cells on the polyvinylchloride plate with 40 #m deep grooves 24 h after seeding. Areas of the substrate near the bottoms of the grooves contain very few cells. Microphotograph, x 100. Fig. 4. Mouse embryo cells on polyvinylchloride plate with 15/~m deep grooves 24 h after seeding. Most cells are oriented parallel to the direction of the grooves. Microphotograph, x 120.
Exptl Cell Res 65
198
Y. A. Rovensky et al.
Fig. 5. Mouse embryo cells on the polymethylmethacrylate plate with hexagonal grooves 24 h after seeding. Areas near the bottoms of the grooves contain very few cells. Microphotograph, x 100. Fig. 6. Cells of L strain on polyvinylchloride plate with 40/zm grooves 24 h after seeding. Numerous cells near the bottoms of the grooves. The cells are not oriented. Microphotograph, x 150.
Exptl Cell Res 65
Cell behaviour on grooved surfaces These modifications had no effect upon cell migration and orientation on grooved plates. Colcemid was found to change the locomotory activities of fibroblasts considerably [8]. This was the reason for testing the effect of Colcemid upon cell migration from the grooves. Colcemid (0.1 #g/ml; Ciba) was added to the medium of cells grown in large flasks 8 h before these cells were re-suspended and seeded upon the grooved surfaces. The same concentration of Colcemid was added again to the medium of cell suspensions at the moment of seeding. Colcemid-treated cells grown on the grooved and smooth surfaces acquired an irregular polygonal form and had no orientation. However, cell migration from the grooves was not inhibited. In fact, the "migration index" at 24 h after seeding in Colcemid-treated cultures had higher values than in control ones (table 2). This increase can be explained by the change of cell form: Colcemid-treated cells in contrast to normal ones did not have long cytoplasmatic processes and therefore more rarely crossed the margin of the 30 # m wide zone around the bottom of the groove (see Methods) This decreased the number of cells counted in this zone. If Colcemid-containing medium was replaced with fresh medium without the drug, normal cell form and orientation were completely restored 24 h later.
Experiments with cells of L strain At 30-60 rain after seeding the distribution of L cells on the grooved surfaces was no different from that of normal cells. Later, at 3-24 h many cells had migrated from the grooves, though numerous cells remained (fig. 6). At 24 h the index of migration was lower than that obtained in the experiments with normal cells (table 2). Cells in the grooves appeared less spread than those located
199
on the areas between the grooves. L cells had no definite orientation on grooved surfaces. DISCUSSION Two main processes determine the distribution of normal fibroblast-like cells upon the surfaces with deep grooves: (a) such cells migrate from the grooves; (b) they orient themselves with regard to the direction of the grooves. We will discuss the possible mechanisms of these processes. Migration from the grooves could be due to some chemotactic reaction: the cells concentrated in the grooves shortly after seeding might repel each other by changing, in some way, the culture medium around them. This could lead to cell migration into areas with lower population density. However, results of a number of experiments render this mechanism improbable: migration was not affected by a decrease of the initial cell population density in the grooves, by increased circulation of the culture medium (experiments with rotation of cultures), or by changed composition of the medium (experiments with "exhausted" medium). It seems more probable that migration is due to differences in the interaction of cells with various parts of the grooved surface. Possibly, cells attach to the surface at the bottom of the grooves less readily than to other parts of the surface. This suggestion is in agreement with the morphological observations: at various times after seeding, cells located in t h e grooves appeared less spread than those on other parts of the surface. Carter [4] demonstrated oriented movement of the ceils in the direction of increasing adhesion to the substrate; a gradient of adhesion was produced by graded deposition of palladium upon the surface. Exptl Cell Res 65
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In our experiments, in contrast to those of Carter, all parts of each substrate were chemically identical. What then could be the cause of decreased attachment of cells to the bottom of the grooves? There are two possibilities: (a) decreased attachment is due to some peculiarities of the microstructure of the surface in the grooves; (b) cell attachment depends on the gross geometrical configuration of the surface. One cannot exclude that certain differences in the microstructure of various parts of the surface may arise in the course of manufacture. However, if the cell behaviour were determined by these hypothetical heterogeneities of the surface microstructure, then one could expect to find considerable differences in cell distribution on the plates consisting of various materials and containing grooves made by various methods. This was not the case: on plates of all types the ceils migrated in a similar fashion f r o m grooves of similar depth. Pre-coating of the grooved surface with thin collodion film obviously changed its microstructure, but did not affect cell migration. These facts justify the conclusion that cell migration can be regarded as a reaction to a certain geometrical configuration of the substrate surface. More specifically, one can conclude that this migration occurs because the ceils cannot attach themselves to the surface at the bottom of grooves having a certain depth (more than 15 #m) and a certain f o r m (an angle between the sides of about 90~ These results as well as some other facts [3, 5, 10] show that not only adhesive properties of the substrate, but also its geometry are essential for cell attachment and m a y determine the direction of cell locomotion. The factors responsible for decreased cell attachment in the grooves remain unknown. One possible explanation is as follows. It has been suggestedthat some of the protruExptl Cell Res 64
sions formed at the leading edge of the fibroblast bend towards the substrate and attach themselves to the surface of this substrate [6]. Protrusions are formed independently in various parts of the leading edge [2]. Now, if the planar leading edge of the fibroblast happens to be located across the groove, then its central and lateral parts are at various distance from the substrate. Therefore, protrusions of equal length formed in the central and lateral parts of the edge have an unequal probability of touching the substrate when curling downwards. Protrusions formed near the lateral side of the groove will attach themselves to the substrate more often than those formed above the central part of the groove. This difference eventually would lead to displacement of the whole leading edge to one or the other side of the groove, that is, to cell migration from the groove. Colcemid did not inhibit cell migration from the grooves. Earlier it was found [7, 8] that Colcemid profoundly disturbs the polarity of locomotion: inactive parts of the surface disappear in Colcemid-treated cells and all the cell edge is seen to form protrusions. Thus, polar distribution of active and inactive parts of the surface is essential for directional cell migration on the smooth substrate [7], but obviously is not necessary for migration from the grooves. This result is compatible with the mechanism of migration discussed above: unequal probability of attachment of the surface protrusions formed in various parts of the cells located above the groove may be sufficient to cause displacement of these cells both in normal and in Colcemid-treated cultures. Orientation of cells on grooved surfaces m a y be related to decreased attachment in the grooves. Cells located upon the side surface of the groove cannot effectively spread themselves across the groove; this may lead to preferential spreading in the direction pa-
Cell behaviour on grooved surfaces rallel to that of the groove. However, not only the cells on the sides of the grooves, but also those located in the central parts of the wide intervals between parallel grooves often became oriented a few hours after seeding. This observation gives one reason to think that cell orientation m a y be caused not only by the presence of grooves, but also by some other factor. Possibly, the orientation may be a reaction to the curvature of the underlying cylindrical surface [5, 10]. Migration of L cells from the grooves was considerably less pronounced than that of normal fibroblast-like cells. These results as well as those obtained by Ambrose & Ellison [3] seem to indicate that certain types of neoplastic cells have decreased the selectivity of reactions to the geometry of the underlying substrate. Further experiments are necessary to find out whether this de-
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creased reactivity is typical for many lines of neoplastic cells and whether it is correlated with certain characteristic changes of the locomotory behaviour of these cells. REFERENCES 1. Abercrombie, M, Exptl cell res, suppl. 8 (1961) 188. 2. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exptl cell res 59 (1970) 393. 3. Ambrose, E J & Ellison, M, Europ j cancer 4 (1968) 459. 4. Carter, S B, Nature 208 (1965) 1183. 5. Curtis, A S & Varde, M, J natl cancer inst 33 (1964) 15. 6. Ingram, V M, Nature 222 (1969) 641. 7. Vasiliev, Ju M, Gelfand, I M, Domnina, L V & Rappoport, R I, Exptl cell res 54 (1969) 83. 8. Vasiliev, Ju M, Gelfand, I M, Domnina, L V Ivanova, O Y, Komm, S G & Olshevskaja, L V, J embryol exptl morphol (1970). To be published. 9. Weiss, P, Intern rev cytol 7 (1958) 391. 10. Weiss, P, Garber, B, Proc natI acad sci US 38 (1952) 264. Received August 19, 1970
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