- Email: [email protected]
Wound healing processes in cell cultures
WOUND
Experimental
Cell Research 54 (1969) 83-93
HEALING
PROCESSES
JU. M. VASILIEV,
I. M. GELFAND,
IN CELL
L. V. DQMNINA,
CULTURES
and R. I. RAPPQPGRT
Institute of Experimental and Clinical Oncology, Academy of Medical Sciences of USSR, Laboratory of Mathematical Biology, Moscow State University, and Institute of the Virus Preparations, Ministry of Pabiic Health of USSR, Moscow, USSR
SUMMARY Reactions of various types of normal and neoplastic cell cultures to the mechanical removal of a part of the culture were studied. 1. Normal cells actively migrated from the edge of the wound on cell-free glass. Usually this migration was limited: most migrating cells remained within a certain zone near the edge. 2. Cells that synthetized or did not synthetize DNA before the operation migrated into the wound in proportions similar to their proportions in other parts of the culture. 3. Migration of cells into the wound was inhibited by colcemide. 4. Most cells migrating into the wound were induced to begin DNA synthesis after a latent period of about 12-18 h. This activation of DNA synthesis was local: the cells in the other aarts of operated cultures were not activated. 5. Migration of neoplastic cells into the wound was very slow as compared with that of the normal cells. This migration was not followed by the activation of DNA synthesis in neoplastic cells.
Locomotory behaviour and rate of growth of normal cells in vitro are controlled by the local interactions between the cells as well as by the composition of the medium [l, 5, 10-13, 191. To learn more about the regulatorv processes in cell cultures it is essential to study in detail reactions of these primitive multicellular systems to various types of experimental interventions. Changes of the culture after mechanical removal of its part, in other words, processes of wound healing in vitro are of special interest in this respect. In these experiments one can study various types and degrees of local cell interactions in the same culture. This greatly facilitates all types of comparisons. Wound healing processes in explants of embryo cells cultivated upon plasma clots had been described long ago [7, 81; cell migration into the wound and active division of migrating cells were observed in these ‘experiments. The purpose of the experiments described in this paper was to study the time course of wound
healing in cultures of various and neoplastic mouse and hum glass. MATERIALS
es of normal ells grown on
AND ME-I
Two types of normal cells were used: (a) Fibroblast-like cells obtained by the trypsinization of mouse embryos. The cultures of these cells were in tbeir first passage. (b) Lines of human embryonic diploid fibroblast-like cells obtained by R. I. Rappoport by the cultivation of cells from the skin of human embryos; 8-23 passages af these cells were used. Neoplastic cell strains included (a) L strain of mouse fibroblast-like cells, (b) strain of human diploid cells transformed by SV-40 virus, The cells were cultivated upon coverslips in Petri dishes. Mouse cells were grown in medium 199 supplemented with 10 % bovine serum. Human cells were grown in Eagles medium supplemented with 30 % of ~actalb~min hydrolysate and 10% bovine serum. In certain experiments conditioned medium was used. i.e. the medium was pre-incubated for 24 h with a culture of growing mouse embryo cells. The cultures were used for experiments at 6-8 days after subcultivation, when a confluent cell layer was alreadv formed on the coverslios. At that time narts of the cuitures were removed with a safety razor Glade or with a scalnel. The cultures were then examined under the microscope in order to check that ah the cells were removed from certain area of the coverslips. Coverslips
84
Ju. M. Vasiliev et al.
with the cultures were not removed from the dishes with the medium during these operations. Operations of the following types were made (see, fig. 1): (a) Square central part of the culture was removed; the side of the square was about 5-6 mm (large wounds). (b) Very small pieces of the cultures, about 1 mm in diameter, were removed (small wounds). (c) All the peripheral part of the culture was removed and only its central fragment was left on the glass. This fragment had an approximately square form with side of about 3 mm (large fragment). (d) All the culture was removed from the glass except small fragment about 1 mm in diameter (small fragments). (e) Half of the culture was removed. (f) Half of the culture was dried for 2-3 min in the air while the other half remained in the medium. After the operation the cells were cultivated again in the same conditions. In certain experiments the culture medium was changed immediately after the operation. At various times before or after the operation labelled 3H-thymidine (3HTh, Radiochemical Center, Amersham, England; specific activity 8.6 CjmM) was added to the culture medium. The final concentration of 3HTh in the medium was 0.25 PC/ml. After the incubation with 3HTh, cultures were washed in Earles solution and fixed in a mixture of acetic acid and ethyl alcohol (1 : l-for 10 min, and then in 96% ethyl alcohol for 30 min). Coverslips with the cultures were covered with fluid emulsion of the M type (NIKFI, Moscow); the time of exposure was about 5-6 days. After development of emulsion cultures were stained with Mayer haematoxylin. The following schemes of labelling with 3HTh were used:
Pulse labelling 3HTh was added to the medium 30 min before fixation.
Continuous labelling 3HTh was added immediately after the operation. Cultures were fixed at various intervals after the operation.
Pre-labelling 3HTh was added to the medium at 1, 20 or 48 h before the operation and remained in the medium until the time of operation. Immediately after the operation the cultures were washed with several portions of Earles solution and then placed into the fresh medium containing 20 pg/ml of non-labelled thymidine. Cultures were fixed at various intervals after the operation. The % of labelled cells (LI) was counted separately in various parts of the operated cultures for cells that migrated into the wound and for cells that remained in the pre-existing part of the cultures. From 500 to 2000 cells of each group were counted in each culture. If the total number of cells in certain group of the culture was less than 500 (i.e., number of cells in the wound at early hours after the operation), figures for several identical cultures were pooled. Mitotic indices were counted in a similar way.
RESULTS DNA synthesis in control cultures of normal cells
Human diploid cells in confluent cultures had an elongated form and formed parallel cell Exptl
Cell Res 54
Fig. I. Types of wounds made in confluent cultures grown on the coverslips. Shaded areas-parts of coverslips covered by viable cells after the operation. (A) Control non-operated cultures, (B) large wounds, (C) small wounds, (D) large and small fragments of the culture left on the glass, (E) removal of one half of the culture, (F) drying of one half of the culture. Area covered with dead cells is shown by dots. The size of coverslip was 2x1 cm.
strands. In certain areas of these cultures the growth of one layer of cells over another layer was observed. Orientation of mouse cells in the cultures was less regular, these cells often partially overlapped each other. After pulse labelling LI in the cultures of both cell types varied from 0.8 to 7.0% at 2-3 days after medium change. Cultures that remained 24 h in conditioned medium had similar LI. At 24 h in fresh medium LI increased at 24 h up to 15-17 %. Corresponding figures for the experiments with continuous labelling (3HTh added for 24 h simultaneously with the medium change) were 15-20 % in the conditioned medium and 25-30 % in the fresh medium. In several pulse-labelled cultures the LI in areas with various local cell densities was counted. In these cultures number Table 1. Per cent of labelled cells in culture areas with various cell densitiesa % of labelled cells
Number of cells per field of view
Mouse fibroblasts
Human diploid cells
I
II
III
IV
11-15 16-20 21-25 26-30 31-35 36-40
5.2t0.7 2.5 50.2 2.OiO.l 1.8i-0.1 1.6kO.4 1.5iO.l
5.8kO.5 4.0 +0.3 4.2f0.3 4.9kO.4 2X+0.6 2.3kO.6
7.3kO.5 6.2kO.2 5.1 kO.2 4.8i0.2 4.6kO.4 3.OkO.4
10.720.5 7.7kO.4 6.5kO.5 5.9k1.2 -
.
a Each vertical column contains the results of counts made in one non-operated culture fixed after 8 days of cultivation and 2 days after medium change. Pulse labelling with 3HTh.
Wound healing processes in cell
Fig. 2. Small wound in the culture of mouse embryonic fibroblast-like cells in the wound 24 h after the operation. Haematoxylin, x 120.
cells. Migrating
3. Edge of the large wound in the culture of mouse embryo fibroblast-like cells. Migrating cells are oriented perpendicularly to the edge. Twenty-four hours after the operation. Haematoxylin, x 120.
Fig.
cultures
85
86
Ju. M. Vasiliev et al.
of cells and number of labelled cells were counted separately in each of 800 fields at a magnification of 15 x 90. Results of these counts are given in table 1. As seen from table 1, LI was somewhat lower in the areas with higher local cell densities. Changes in the cultures of normal cells after operations Immediately after the operation margins of the large wounds were contracted so that the area of the wound somewhat increased and its form became round. At the same time cells at the edge of the wound often became oriented in such a way that they were approximately parallel to this edge. Large and especially small fragments of the cultures left on the glass were somewhat contracted immediately after the operation. All these early changes of the wound form were more prominent in the experiments with mouse cells than in those with human cells. When large wounds were made in mouse cultures the cells began to migrate from the margin of the wound about l-2 h after the operation. At 3 h a fringe consisting of one two rows of sparsely distributed cells was seen near the margin of the wound. At 24 h this fringe had about 10 rows of cells. At the early stages of migration most cells were oriented perpendicularly to the edge of the wound (fig. 3). A few degenerating cells with pycnotic nuclei were seen among the migrating cells. At 24 h a few cells were seen already near the center of the wound, but most cells at that time and during 2 following days remained still in the zone not farther than 0.5-0.8 mm from the margin of the wound. The general pattern of cell migration after the operations of other types was similar to that observed after the operation with central large wounds. Almost all the area of a small wound was already filled with cells at 24 h after the operation (fig. 3), although the cell density in this area was considerably lower than in the surrounding culture. The cells from large and small culture fragments usually did not migrate Exptl
Cell Res 54
farther than 1.0 mm from the edge of the fragment. The cell density in the central parts of these fragments remained similar to that in control cultures. Similar limited migration was observed after removal of the half of the culture. In these experiments the cells never migrated on the whole cell-free area in the first days after the operation, but instead formed a zone of growth near the edge of non-removed part of the culture. When half of the culture was dried, viable cells from the non-dried parts of the culture migrated over the dead cells. In the stained preparations migrating viable cells were easily distinguished from dead cells with pycnotic nuclei. The rate of cell migration upon the dead cells and upon the free glass was approximately similar. In the experiments with human cells the general sequence of changes was similar to that observed in the experiments with mouse cells. However, here migrating cells were more often connected with each other by their processes, as well as by their lateral surfaces. The rate of migration of human cells depended considerably on their previous orientation in the culture. If the margin of the wound happened to be perpendicular to the direction of the long axes of cells in the stream, then these cells migrated most rapidly into the wound (fig. 4). The rate of migration was much slower if the margin was parallel to the long axis of the cells (fig. 5). The cells in these areas slowly slid into the wound and then gradually changed their orientation. Colcemide was found to inhibit considerably migration of mouse and human cells into the wounds. If colcemide (0.1 pg/ml) was added to the medium immediately after the operation, 24 h later the wound contained only a few non-oriented cells. In the other areas of operated cultures as well as in control cultures, colcemide somewhat distorted cell orientation so that the cells in colcemide-treated cultures often looked polygonal instead of spindle-shaped. No manifestations of non-specific toxicity of colcemide were observed.
Wound healing processes in cell cultures
Fig. 4. Migration of human diploid cells into the wound 24 h after the operation. Area where the wound edge was approx. perpendicular to the pre-existing orientation of cells. Haematoxylin, ;: 120.
Fig. 5. Migration of human diploid cells into the wound. Area where the wound edge was parallel to the pre-existing orientation of cells. Twenty-four hours after the operation. Raematoxylin, x 120.
87
88
01
Ju. hf. Vasiliev et al.
. 3
. 6
9
,2
. ,5
18
. 7.1
24
7.7
30
Fig. 6. Activation of DNA synthesis in mouse fibroblastlike cells migrating into the wound. Medium was changed immediately after the operation. Pulse labelling with 3HTh. Figs 6-8. Abscissa: hours after the operation; ordinate: % of labelled nuclei. --, cells in the wound; ----, cells in the culture near the wound. Vertical bars-95 % confidence interval.
Changesof the numbersof DNA-synthetising cells in woundedculturesof normal cells Pulse labelling experiments
At any time after the operation the LI of the parts of cultures located far from the wounds remained similar to that of control non-operated cultures. The LI of cells located near the margin of the wound in most experiments remained as low as that of the cells located far from the wound. During the first 3-10 h after the operation the LI of the cells that migrated in the wound remained as low as that of the other parts of the same cultures. Later (at 12-18 h) the LI of the cells in the wound began to increase and remained high for several days thereafter (fig. 6). At 24-28 h after the operation the LI
01
3
. 6
9
. 12
. 15
18
21
11
Fig. 8. Activation of DNA synthesis in human diploid cells migrating into the wound. Continuous labelling with 3 HTh. Medium was changed 24 h before the operation.
of the cells in the wound was always much higher than the LI of the other parts of the culture, although both indices varied considerably from culture to culture in relation to the interval after medium change (table 2). The degree of increase of LI in the wounds at 24 h was similar in the cultures grown in fresh and conditioned medium. In the experiments with large and small culture fragments the LI of the cells that migrated on the glass were much higher than those of the cells remaining in the fragment. These latter indices were similar to those of the control cultures (table 3). The LI of migrating cells was increased to a similar degree in the experiments with mechanical removal of the half of the culture and in the experiments where half of the culture had been dried (table 4). At 2-3 days after removal of the half of the culture a wide zone of growing cells was formed near the edge of the “old” culture. Cell density in this zone gradually decreased with an increasing distance from the margin of the old culture. Counts made on various parts of these cultures had shown that LI decreased as cell density increased (table 5). Continuous labelling
Fig. 7. Activation of DNA synthesis in mouse fibroblastlike cells migrating into the wound. Medium was changed 24 h before the operations. Continuous labelling with 3HTh. Exptl Cell Res 54
Results of these experiments were similar to those obtained in the experiments with pulse labelling. The LI of the cells of the wound and in other parts of the culture remained similar
Wound healing processes in cell cultures
Table 2. Per cent of Iabelled cells in cultures with central woundsa % of labelled cells Time (days) _. After medium change Mouse 1
-..
Large wounds
After the operation
Cells in the wound
fibroblast-like
Cells around the wound
Small wounds Cells in the wound
50.0% .2 21.211.3 21.0~1.5 27.3 +2.1 10.6i 1.5 -
19.4& 1.3
cells
1 2 2 2 3
1 1 1 2 1 1
41.0i1.5 37.6k1.5 32.0~1.5 37.6k1.5 25.2 * 1.6 15.1 k 1.6
:
21
10.0 kO.9
23.111.3 21.6k1.3 1.8iO.4 1.7kO.6 4.8kl.O 1.2kO.5 2.7 iO.6
42.5 & 1.5 14.1 +1.1 43.6k2.1 25.6+ 1.6
14.0 f 1.2 2.8 +0.5 15.6k3.7 3.OiO.8
16.0+1.0 14.0&1.1
20.2il.l 2o.oi1.2
34.4k2.1 27.3t1.3
32.2 k2.0 23.Ok1.3
Human
diploid
1 2 1 2
cells
1 1 1 2
Mouse L cells 2 1 2 1 Human
transformed
1 1
Cells around the wound
Non-operated culture
2:.6+1.3 1.6zO.4
3‘610.6 3.2kO.9 3.6k6.8 2.0&0.6 2.3kO.7 1.8+0.6
1.610.6 0.8 zO.5 1.1 io.5 -
-
16.02 1.3 3.2=0.6 -
6.00.8
cells
1 1
’ Each horizontal row of figures contains the results of one experiment; coverslips with operated and non-operated cultures remained in the same Petri dish throughout the whole experiment. LI in the wound and around the wound were counted in various parts of the same culture. Pulse labelling with 3HTh was made in all the experiments.
during the first 10-14 h after the operation. Later the LI of the cells in the wound rapidly increased and reached 50-60 % at 24 h (figs 8-9).
what higher than those of the other parts of the cultures.
Experiments with prelabelling of cells before the operation
Mitotic indices of the cells located far from wounds in the operated cultures remained as low as those of control cultures (0.05-0.2 %). No striking increases of the MI of the cells in
As seen from table 6, the LI of the cells in the wound in these experiments was similar or some-
Mitotic counts
Table 3. Per cent of Iabelled cells in the experiments with large and small c~ltl~re~ragmentsa % of labelled cells Time (days) p After After medium the operchange ation Mouse
: Human
Jbroblast-like 21 diploid
2 a Conditions
1
Large fragments Cells migrating from the fragment
Small fragments Cells remaining in the fragment
Cells migrating from the fragment
Cells remaining in the fragment
Nonoperated culture
16.Oil.3 11.8k1.2
31.7k1.5 29.0 f 1.4
11.0+1.0 3.5 20.6
17.82 8.150.9i.2
-
-
cells
38.4il.5 25.8k1.4
cells
21.3k1.8
7.OkO.8
of experiments and designations are’the same as in table 2.
3.6-M
90
Ju. M. Vasiliev et al.
Table 4. Per cent of labelled cells in the experiments with mechanical removal or drying of one half of the culture of mouseJibroblast-like cells % of labelled cells Time (days)
Drying
Mechanical removal
After medium change
After the operation
Cells migrating above the dried cells
Cells remaining in the not-dried half of the culture
Cells migrating on the glass
Cells remaining in the not-removed half of the culture
Nonoperated cultures
2 1
1 1
37.7F2.2 28.0f2.0
3X10.8 13.0* 1.6
32.3 +2.0 29.0f2.0
7.0+ 1.0 5.8111
3.6kO.9 10.0+ 1.3
the wound were observed during the first 12 h after the operation. However, it was difficult to obtain reliable figures at these stages, as total number of cells in the wound was too small. At 24-30 h after the operation MI in the wound increased up to l.O-2.0%. In the experiments with continuous 3HTh labelling first labelled mitotic figures were found at 6-8 h after addition of 3HTh to the medium, both in the nonoperated and in the wounded cultures. At about 9-10 h 50 % of mitoses became labelled.
the migrating cells were not oriented in any definite way with regard to the wound edge. At 24 h after the operation the LI of the cells in the wound and in other parts of the culture remained identical (see table 2). DISCUSSION Each normal cell in the confluent culture is firmly linked with other cells and stretched from all sides by its neighbours. These mechanical interactions are most obvious in the cultures of mouse cells; they lead to contraction ‘of the wound and to orientation of cells at the margin of the wound. The nature of forces responsible for this stretching is not clear. Possibly, contractile structures in the cells and/or intercellular substances produced by these cells are of some~
Experiments with neoplastic cells In the experiments with all types of transformed cells no changes of the wound form and cell orientation were observed immediately after the operation. Cells migrated into the wounds, but the rate of this migration was slow and most of
Table 5. Per cent of labelled cells in the areas of wounded cultures with various cell densitiesa Cells remaining in the part that was not removed near its edge
I
II-III
IV-VI
VII-IX
X-XII
Mouse jibvoblast-like cells Mean number of cells per field % of labelled cells
31 15.5i1.1
17 21.4f1.8
13 28.421.2
7 31.5k1.2
3 31.611.2
2 36.6k2.8
Human diploid cells Mean number of cells per field % of labelled cells
17 8.OkO.6
12 12.0&1.0
9 12.311.0
5 15.121.2
4 20.711.5
2 2O.Ok2.2
Cells migrating on the glass
a Results of counts made in 2 pulse-labelled cultures of mouse and human cells after mechanical removal of one half of culture. Mouse culture was fixed 6 days after the operation and 1 day after medium change; human cell culture 3 days after the operation and after medium change. Increasing roman figures designate rows of fields of view that were approximately at the same distance from the edge of the half of the culture that was not removed. Number of labelled cells and total number of cells were counted in each field of view at the magnification 15 x 90. About 60-66 fields of view were counted in each row. Exptl Cell Res 54
Wound healing processes
importance. At the edge of the wound one side of the cell surface is released from contact inhibition of movement while cell contacts on the other side of the same cell remain intact. This gradient of contact inhibition may explain oriented cell movement into the wound. Preestablished orientation of cells in the culture determines to a considerable degree the initial rate of migration as change of cell orientation takes considerable time. Probably this change of orientation involves some structural reorganization of the cell. In most experiments the cells that migrated into the wound tended to remain in a certain zone near the edge of pre-existing culture and did not move farther on the glass. This limited character of migration was most evident in the experiments with culture fragment left on the glass: migration of cells usually did not lead to total “‘disintegration” of these fragments. Limited outward migration of fibroblasts from culture fragment containing small number of cells was observed earlier by Abercrombie & Gitlin [2]. Many facts indicate that a certain minimal local cell density is optimal for the proliferation of- these cells in culture [S, 13, 151. Possibly, fibroblasts tend to remain in the zones of optimal cell density and do not migrate farther into the areas where cell density is too low. The nature of the processes essential for these preferences is not clear. Mutual adhesion of neighbour cells may play a certain role in this process: migrating cells do not break all their adhesions with each other and this limits their migration. Probably most cells that happen to be located near the edge of the wound would migrate into that wound. Experiments with prelabelling of cells before the operation indicate that there is no or little selectivity in migration of labelled or non-labelled cells into the wound. Colcemide strikingly inhibited cell movement into the wound. In the concentrations used in our experiments colcemide had no obvious nonspecific toxic effect upon the cells. Probably this drug selectively interfered with the ability of cells to perform oriented movements. This sug-
in cell cultures
9
Table 6. Per cent of labelled cells in wo~~~de cultures labelled with 3NTh before the o~e~~t~o~ 76 of labelled
Time Time of incubation with 3HTh W Mouse 48 48 48 48 48 48
jibroblast-like 0 6 18 24 30
1 1 Diploid 1 1 20 20 20
after the operation (h)
0 20 human
cells 0 24 12 24 30
cells
Cells migrating into the wound
Celk ar0LKld
0petW3d
the wound
cu!t-xes
40.4To.9 41.2kO.7 32.8 +0.6 35.5 * 1 .o 30.5 -IQ.7
26.5+1.4 28.5~1.6 27.5kl.l 29.81-1.2 27.9kI.4 25.9k1.1
28.O:k16 28.8kI.4 24.8k1.3 -
Non-
cells
9.3-i-0.9
13.21.1.1 9.4+ 1.7 9.3il.O 7.6kO.8
11.0+1.0 12.2+ I .o
9.4 i-o.9 12.3+1.0 5.8 i-O.8 5.3 *o.a 5.6kO.7
4.8 kO.7
gestion is in good agreement with the ~reIimi~ary results of our experiments with cultures of mouse embryo fibroblast-like cells grown on an oriented substrate (Vasiliev & Domnina, unpub lished). As suggested by Weiss [19], tish scale was used as an oriented substrate. hen cells were attached to this substrate, they immediately acquired elongated, spindle-like shape, their long axis being oriented in parallel with the ridges of the substrate. Addition of colcemide (0.1 pug/ml) to the medium did not inhibit cell attachment to the substrate, but almost completely prevented their orientation. In colcemidecontaining medium cells attached to the scale had an irregular polygonal shape. When these cultures were transferred into the medium without colcemide, the cells gradually acquired spindle-like shape. Colchicine was found to inhibit oriented growth of plant roots and chemotactic migration of human leucocytes (see review in 16, 141). Colcemide altered polarity in Hydra [lS]. Perhaps all these effects, as well as metaphase arrest, have something to do with the changes of cytoplasmic viscosity produced by these alkoloids (see review in [14]). Only a small fraction of cells in confluent cul-
92
Ju. M. Vasiliev et al.
tures is synthetizing DNA at any moment. In the experiments described above two types of factors increased considerably this fraction: (a) medium changes, and (b) cell migration into the wound. The latent periods after the action of both these factors were of the same order: LI in the cultures began to increase at 15-18 h after medium change. Increased LI in the wound was observed not earlier than at 12-18 h after the operation. However, the efficiency of these two factors was different. One may try to estimate the fraction of cells induced to enter S-phase in the wound in the following way. Let us assume that:
(4 an interval
between the migration of the cell into the wound and activation of DNA synthesis is t, h and fraction of the cells activated after this interval is Y; n-number of cells migrating into the wound per hour is constant during the first 24 h after the operation; among the cells remaining in the wound less than t, h fraction of labelled cells is the same as that among the cells in other parts of the culture (I,>.
(b)
cc>
Then fraction of labelled cells & = LI/ 100) in the wound at T h after the operation (TG 24) is
y=L*
T-1,. T-t,,
t,, -
Values I, and I, were obtained in the experiments with continuous labelling. t, was assumed to be 15 h. Calculations showed that Y in most experiments varied from 0.7 to 1.0. Although calculations of this type are inevitably very crude, they show that almost all cells migrating into the wound are induced to enter S-phase. Thus, migration into the wound was much more efficient growth inducing factor than medium change. Experiments of Todaro and collaborators [16] suggest that fresh serum contains a factor inducing DNA synthesis and division Exptl
Cell
Res 54
in cells that are in close contact, but is not necessary for the growth of dispersed cells in the wound. Further work is needed to elucidate these questions. Data of Nilausen & Green [12] indicate that 3T3 mouse cells in saturation density cultures are blocked in Gl phase of the mitotic cycle, while the experiments of Maciera-Coelho et al. [l I] with human diploid cells suggest that these cells are blocked in G2 phase. Experiments now in progress in this laboratory should show whether mouse and human cells migrating into the wound enter S phase from some point in Gl phase or from G2 phase. Results of wound healing experiments described above show that activation of DNAsynthesis is a local phenomenon: only the cells that leave the confluent culture and migrate on the free glass are stimulated, but not the cells remaining in the culture even if they are located near the edge of the wound. Cells remaining in the small fragment of the culture were not stimulated. On the other hand, cells in the small wound were activated although they were surrounded by a large area of confluent culture. Similar local stimulation of DNA-synthesis in the wound were observed in our earlier experiments [17] and in the experiments of Todaro et al. [16]. Fisher & Yeh [9] had shown that DNA synthesis in large colonies of 3T3 cells is confined mainly to cells at the periphery of each colony. Obviously the cell is activated or not activated to begin DNA synthesis depending on the state of its immediate neighbourhood. It is irrelevant for activation of this cell what happens at distances exceeding a few cell lengths, whether there is free glass or confluent culture. Role of the local factors in the regulation of cell growth is suggested also by the results of the counts made in control cultures and in the experiments with the removal of one half of the culture. These counts had shown that % of DNAsynthetizing cells decrease with an increasing local cell density. Dead dried cells in contrast to viable cells have no effect upon the growth of their neighbours. Thus, the growth inhibiting effects of neigh-
bour cells has a very short range of action. It seems improbable that these effects can be explained by the changes of concentration of low molecular substances in the cell environment [I3]. These effects may be due either to the contact of cell surfaces or to the accumulation of some high molecular substance (e.g. some polysaccharide) near the surface of these cells [3, 41, Difference between these 2 explanations seems to be hardly significant, as carbohydrate-containing macromolecular substances seem to be essential components of the external surface coats of the cells of various types. DNA synthesis in neoplastic cells is not stimulated by their migration into the wound. These experiments confirm that these cells become insensitive to the local factors regulating growth in non-neoplastic cultures [l]. Migration of neoplastic cells into the wound was unoriented and, probably, is due mostly to the random movements of cells near the edge of the wound. This inability to perform oriented movements, possibly, is a manifestation of some deficiency of locomotory behaviour of neoplastic cells [I]. It is not clear at present how this inability is correlated with other manifestations of abnormal locomotory behavior of ncoplastic cells, especially with the loss of contact inhibition of movement.
REFERENCES 1. Abercrombie. M & Ambrose. , E J.I Cancer res 22 (1962) 525. ’ 2. Abcrcrombie, M & Gitlin, G, Proc royal sot B 162 (1965) 988. 3. Btirk, R R, Growth-regulating substances for animal cells in culture (ed V Defendi & M Stoker) pi 39. The Wistar Univ. Press, Philadelphia (1967). 4. Cox, R P & Gesner, B M, Cancer res 27 (1967) 974. 5. Eagle, H, General physiology of cell specialization (ed D Mazia & A Taylor) p. 151. Academic Press, New York (1963). 6. Eigsti, 0 J & Dustin, R, Jr, Colchicine-in agriculture, medicine, biology, and chemistry. Iowa State College Press, Ames,-Iowa (1955). I. Ephrussi, B, PhCnom&es dint&ration dans les cultures des t&us. Hermann, Paris-(1935). 8. Fischer, A, Biology of tissue cells. Cambridge Univ. Press (1946). 9. Fischer. M W & Yell. J. Science 155 11967) 581. 10. Lcvinc,‘E N, Becker,’ G, Boone, C W &‘Eagle, H, Proc natl acad sci 53 119651 350. 11. Ma&h-a-Coelho, A, Ponten, J & Philipson, L, Exptl cell res 43 (1966) 20. 12. Nilauscn, K & Green, H, Expti cell res 40 (1965) 166. 13. Rubin, H & Rein, A, Growth regulating substances for animal cells in culture (cd V Defendi & M Stoker) p. 51. The Wistar Univ. Press, Philadelphia (1967).’ 14. Savel, II. Progr exptl tumor research 8 (1966) _ , 189. Karger, Base1 & New York. 15. Stoker, M & Sussman, M, Exptl cell rcs 38 (1965) 645. 16. Todaro, G, Matsuya, Y, Bloom, S, Robbins, A & Green, I-I, Growth regulating substances for aniinal cells in culture (cd V Defendi & M Stoker) p. 87. The Wistar Univ Press, Philadelphia (1967). 17. Vasiliev, Ju M, Gelfand, I M & Erofeeva, L V. Dokl akad nauk SSSR 171 (1966) 721. (In Russian.) 18. Webster, G, J embryo1 exptl morphol 18 (1967) 181. 19. Weiss, P, Intern rev cytol 7 (1958) 391. Received May 20, 1968
Experimental
Cell Research 54 (1969) 83-93
HEALING
PROCESSES
JU. M. VASILIEV,
I. M. GELFAND,
IN CELL
L. V. DQMNINA,
CULTURES
and R. I. RAPPQPGRT
Institute of Experimental and Clinical Oncology, Academy of Medical Sciences of USSR, Laboratory of Mathematical Biology, Moscow State University, and Institute of the Virus Preparations, Ministry of Pabiic Health of USSR, Moscow, USSR
SUMMARY Reactions of various types of normal and neoplastic cell cultures to the mechanical removal of a part of the culture were studied. 1. Normal cells actively migrated from the edge of the wound on cell-free glass. Usually this migration was limited: most migrating cells remained within a certain zone near the edge. 2. Cells that synthetized or did not synthetize DNA before the operation migrated into the wound in proportions similar to their proportions in other parts of the culture. 3. Migration of cells into the wound was inhibited by colcemide. 4. Most cells migrating into the wound were induced to begin DNA synthesis after a latent period of about 12-18 h. This activation of DNA synthesis was local: the cells in the other aarts of operated cultures were not activated. 5. Migration of neoplastic cells into the wound was very slow as compared with that of the normal cells. This migration was not followed by the activation of DNA synthesis in neoplastic cells.
Locomotory behaviour and rate of growth of normal cells in vitro are controlled by the local interactions between the cells as well as by the composition of the medium [l, 5, 10-13, 191. To learn more about the regulatorv processes in cell cultures it is essential to study in detail reactions of these primitive multicellular systems to various types of experimental interventions. Changes of the culture after mechanical removal of its part, in other words, processes of wound healing in vitro are of special interest in this respect. In these experiments one can study various types and degrees of local cell interactions in the same culture. This greatly facilitates all types of comparisons. Wound healing processes in explants of embryo cells cultivated upon plasma clots had been described long ago [7, 81; cell migration into the wound and active division of migrating cells were observed in these ‘experiments. The purpose of the experiments described in this paper was to study the time course of wound
healing in cultures of various and neoplastic mouse and hum glass. MATERIALS
es of normal ells grown on
AND ME-I
Two types of normal cells were used: (a) Fibroblast-like cells obtained by the trypsinization of mouse embryos. The cultures of these cells were in tbeir first passage. (b) Lines of human embryonic diploid fibroblast-like cells obtained by R. I. Rappoport by the cultivation of cells from the skin of human embryos; 8-23 passages af these cells were used. Neoplastic cell strains included (a) L strain of mouse fibroblast-like cells, (b) strain of human diploid cells transformed by SV-40 virus, The cells were cultivated upon coverslips in Petri dishes. Mouse cells were grown in medium 199 supplemented with 10 % bovine serum. Human cells were grown in Eagles medium supplemented with 30 % of ~actalb~min hydrolysate and 10% bovine serum. In certain experiments conditioned medium was used. i.e. the medium was pre-incubated for 24 h with a culture of growing mouse embryo cells. The cultures were used for experiments at 6-8 days after subcultivation, when a confluent cell layer was alreadv formed on the coverslios. At that time narts of the cuitures were removed with a safety razor Glade or with a scalnel. The cultures were then examined under the microscope in order to check that ah the cells were removed from certain area of the coverslips. Coverslips
84
Ju. M. Vasiliev et al.
with the cultures were not removed from the dishes with the medium during these operations. Operations of the following types were made (see, fig. 1): (a) Square central part of the culture was removed; the side of the square was about 5-6 mm (large wounds). (b) Very small pieces of the cultures, about 1 mm in diameter, were removed (small wounds). (c) All the peripheral part of the culture was removed and only its central fragment was left on the glass. This fragment had an approximately square form with side of about 3 mm (large fragment). (d) All the culture was removed from the glass except small fragment about 1 mm in diameter (small fragments). (e) Half of the culture was removed. (f) Half of the culture was dried for 2-3 min in the air while the other half remained in the medium. After the operation the cells were cultivated again in the same conditions. In certain experiments the culture medium was changed immediately after the operation. At various times before or after the operation labelled 3H-thymidine (3HTh, Radiochemical Center, Amersham, England; specific activity 8.6 CjmM) was added to the culture medium. The final concentration of 3HTh in the medium was 0.25 PC/ml. After the incubation with 3HTh, cultures were washed in Earles solution and fixed in a mixture of acetic acid and ethyl alcohol (1 : l-for 10 min, and then in 96% ethyl alcohol for 30 min). Coverslips with the cultures were covered with fluid emulsion of the M type (NIKFI, Moscow); the time of exposure was about 5-6 days. After development of emulsion cultures were stained with Mayer haematoxylin. The following schemes of labelling with 3HTh were used:
Pulse labelling 3HTh was added to the medium 30 min before fixation.
Continuous labelling 3HTh was added immediately after the operation. Cultures were fixed at various intervals after the operation.
Pre-labelling 3HTh was added to the medium at 1, 20 or 48 h before the operation and remained in the medium until the time of operation. Immediately after the operation the cultures were washed with several portions of Earles solution and then placed into the fresh medium containing 20 pg/ml of non-labelled thymidine. Cultures were fixed at various intervals after the operation. The % of labelled cells (LI) was counted separately in various parts of the operated cultures for cells that migrated into the wound and for cells that remained in the pre-existing part of the cultures. From 500 to 2000 cells of each group were counted in each culture. If the total number of cells in certain group of the culture was less than 500 (i.e., number of cells in the wound at early hours after the operation), figures for several identical cultures were pooled. Mitotic indices were counted in a similar way.
RESULTS DNA synthesis in control cultures of normal cells
Human diploid cells in confluent cultures had an elongated form and formed parallel cell Exptl
Cell Res 54
Fig. I. Types of wounds made in confluent cultures grown on the coverslips. Shaded areas-parts of coverslips covered by viable cells after the operation. (A) Control non-operated cultures, (B) large wounds, (C) small wounds, (D) large and small fragments of the culture left on the glass, (E) removal of one half of the culture, (F) drying of one half of the culture. Area covered with dead cells is shown by dots. The size of coverslip was 2x1 cm.
strands. In certain areas of these cultures the growth of one layer of cells over another layer was observed. Orientation of mouse cells in the cultures was less regular, these cells often partially overlapped each other. After pulse labelling LI in the cultures of both cell types varied from 0.8 to 7.0% at 2-3 days after medium change. Cultures that remained 24 h in conditioned medium had similar LI. At 24 h in fresh medium LI increased at 24 h up to 15-17 %. Corresponding figures for the experiments with continuous labelling (3HTh added for 24 h simultaneously with the medium change) were 15-20 % in the conditioned medium and 25-30 % in the fresh medium. In several pulse-labelled cultures the LI in areas with various local cell densities was counted. In these cultures number Table 1. Per cent of labelled cells in culture areas with various cell densitiesa % of labelled cells
Number of cells per field of view
Mouse fibroblasts
Human diploid cells
I
II
III
IV
11-15 16-20 21-25 26-30 31-35 36-40
5.2t0.7 2.5 50.2 2.OiO.l 1.8i-0.1 1.6kO.4 1.5iO.l
5.8kO.5 4.0 +0.3 4.2f0.3 4.9kO.4 2X+0.6 2.3kO.6
7.3kO.5 6.2kO.2 5.1 kO.2 4.8i0.2 4.6kO.4 3.OkO.4
10.720.5 7.7kO.4 6.5kO.5 5.9k1.2 -
.
a Each vertical column contains the results of counts made in one non-operated culture fixed after 8 days of cultivation and 2 days after medium change. Pulse labelling with 3HTh.
Wound healing processes in cell
Fig. 2. Small wound in the culture of mouse embryonic fibroblast-like cells in the wound 24 h after the operation. Haematoxylin, x 120.
cells. Migrating
3. Edge of the large wound in the culture of mouse embryo fibroblast-like cells. Migrating cells are oriented perpendicularly to the edge. Twenty-four hours after the operation. Haematoxylin, x 120.
Fig.
cultures
85
86
Ju. M. Vasiliev et al.
of cells and number of labelled cells were counted separately in each of 800 fields at a magnification of 15 x 90. Results of these counts are given in table 1. As seen from table 1, LI was somewhat lower in the areas with higher local cell densities. Changes in the cultures of normal cells after operations Immediately after the operation margins of the large wounds were contracted so that the area of the wound somewhat increased and its form became round. At the same time cells at the edge of the wound often became oriented in such a way that they were approximately parallel to this edge. Large and especially small fragments of the cultures left on the glass were somewhat contracted immediately after the operation. All these early changes of the wound form were more prominent in the experiments with mouse cells than in those with human cells. When large wounds were made in mouse cultures the cells began to migrate from the margin of the wound about l-2 h after the operation. At 3 h a fringe consisting of one two rows of sparsely distributed cells was seen near the margin of the wound. At 24 h this fringe had about 10 rows of cells. At the early stages of migration most cells were oriented perpendicularly to the edge of the wound (fig. 3). A few degenerating cells with pycnotic nuclei were seen among the migrating cells. At 24 h a few cells were seen already near the center of the wound, but most cells at that time and during 2 following days remained still in the zone not farther than 0.5-0.8 mm from the margin of the wound. The general pattern of cell migration after the operations of other types was similar to that observed after the operation with central large wounds. Almost all the area of a small wound was already filled with cells at 24 h after the operation (fig. 3), although the cell density in this area was considerably lower than in the surrounding culture. The cells from large and small culture fragments usually did not migrate Exptl
Cell Res 54
farther than 1.0 mm from the edge of the fragment. The cell density in the central parts of these fragments remained similar to that in control cultures. Similar limited migration was observed after removal of the half of the culture. In these experiments the cells never migrated on the whole cell-free area in the first days after the operation, but instead formed a zone of growth near the edge of non-removed part of the culture. When half of the culture was dried, viable cells from the non-dried parts of the culture migrated over the dead cells. In the stained preparations migrating viable cells were easily distinguished from dead cells with pycnotic nuclei. The rate of cell migration upon the dead cells and upon the free glass was approximately similar. In the experiments with human cells the general sequence of changes was similar to that observed in the experiments with mouse cells. However, here migrating cells were more often connected with each other by their processes, as well as by their lateral surfaces. The rate of migration of human cells depended considerably on their previous orientation in the culture. If the margin of the wound happened to be perpendicular to the direction of the long axes of cells in the stream, then these cells migrated most rapidly into the wound (fig. 4). The rate of migration was much slower if the margin was parallel to the long axis of the cells (fig. 5). The cells in these areas slowly slid into the wound and then gradually changed their orientation. Colcemide was found to inhibit considerably migration of mouse and human cells into the wounds. If colcemide (0.1 pg/ml) was added to the medium immediately after the operation, 24 h later the wound contained only a few non-oriented cells. In the other areas of operated cultures as well as in control cultures, colcemide somewhat distorted cell orientation so that the cells in colcemide-treated cultures often looked polygonal instead of spindle-shaped. No manifestations of non-specific toxicity of colcemide were observed.
Wound healing processes in cell cultures
Fig. 4. Migration of human diploid cells into the wound 24 h after the operation. Area where the wound edge was approx. perpendicular to the pre-existing orientation of cells. Haematoxylin, ;: 120.
Fig. 5. Migration of human diploid cells into the wound. Area where the wound edge was parallel to the pre-existing orientation of cells. Twenty-four hours after the operation. Raematoxylin, x 120.
87
88
01
Ju. hf. Vasiliev et al.
. 3
. 6
9
,2
. ,5
18
. 7.1
24
7.7
30
Fig. 6. Activation of DNA synthesis in mouse fibroblastlike cells migrating into the wound. Medium was changed immediately after the operation. Pulse labelling with 3HTh. Figs 6-8. Abscissa: hours after the operation; ordinate: % of labelled nuclei. --, cells in the wound; ----, cells in the culture near the wound. Vertical bars-95 % confidence interval.
Changesof the numbersof DNA-synthetising cells in woundedculturesof normal cells Pulse labelling experiments
At any time after the operation the LI of the parts of cultures located far from the wounds remained similar to that of control non-operated cultures. The LI of cells located near the margin of the wound in most experiments remained as low as that of the cells located far from the wound. During the first 3-10 h after the operation the LI of the cells that migrated in the wound remained as low as that of the other parts of the same cultures. Later (at 12-18 h) the LI of the cells in the wound began to increase and remained high for several days thereafter (fig. 6). At 24-28 h after the operation the LI
01
3
. 6
9
. 12
. 15
18
21
11
Fig. 8. Activation of DNA synthesis in human diploid cells migrating into the wound. Continuous labelling with 3 HTh. Medium was changed 24 h before the operation.
of the cells in the wound was always much higher than the LI of the other parts of the culture, although both indices varied considerably from culture to culture in relation to the interval after medium change (table 2). The degree of increase of LI in the wounds at 24 h was similar in the cultures grown in fresh and conditioned medium. In the experiments with large and small culture fragments the LI of the cells that migrated on the glass were much higher than those of the cells remaining in the fragment. These latter indices were similar to those of the control cultures (table 3). The LI of migrating cells was increased to a similar degree in the experiments with mechanical removal of the half of the culture and in the experiments where half of the culture had been dried (table 4). At 2-3 days after removal of the half of the culture a wide zone of growing cells was formed near the edge of the “old” culture. Cell density in this zone gradually decreased with an increasing distance from the margin of the old culture. Counts made on various parts of these cultures had shown that LI decreased as cell density increased (table 5). Continuous labelling
Fig. 7. Activation of DNA synthesis in mouse fibroblastlike cells migrating into the wound. Medium was changed 24 h before the operations. Continuous labelling with 3HTh. Exptl Cell Res 54
Results of these experiments were similar to those obtained in the experiments with pulse labelling. The LI of the cells of the wound and in other parts of the culture remained similar
Wound healing processes in cell cultures
Table 2. Per cent of Iabelled cells in cultures with central woundsa % of labelled cells Time (days) _. After medium change Mouse 1
-..
Large wounds
After the operation
Cells in the wound
fibroblast-like
Cells around the wound
Small wounds Cells in the wound
50.0% .2 21.211.3 21.0~1.5 27.3 +2.1 10.6i 1.5 -
19.4& 1.3
cells
1 2 2 2 3
1 1 1 2 1 1
41.0i1.5 37.6k1.5 32.0~1.5 37.6k1.5 25.2 * 1.6 15.1 k 1.6
:
21
10.0 kO.9
23.111.3 21.6k1.3 1.8iO.4 1.7kO.6 4.8kl.O 1.2kO.5 2.7 iO.6
42.5 & 1.5 14.1 +1.1 43.6k2.1 25.6+ 1.6
14.0 f 1.2 2.8 +0.5 15.6k3.7 3.OiO.8
16.0+1.0 14.0&1.1
20.2il.l 2o.oi1.2
34.4k2.1 27.3t1.3
32.2 k2.0 23.Ok1.3
Human
diploid
1 2 1 2
cells
1 1 1 2
Mouse L cells 2 1 2 1 Human
transformed
1 1
Cells around the wound
Non-operated culture
2:.6+1.3 1.6zO.4
3‘610.6 3.2kO.9 3.6k6.8 2.0&0.6 2.3kO.7 1.8+0.6
1.610.6 0.8 zO.5 1.1 io.5 -
-
16.02 1.3 3.2=0.6 -
6.00.8
cells
1 1
’ Each horizontal row of figures contains the results of one experiment; coverslips with operated and non-operated cultures remained in the same Petri dish throughout the whole experiment. LI in the wound and around the wound were counted in various parts of the same culture. Pulse labelling with 3HTh was made in all the experiments.
during the first 10-14 h after the operation. Later the LI of the cells in the wound rapidly increased and reached 50-60 % at 24 h (figs 8-9).
what higher than those of the other parts of the cultures.
Experiments with prelabelling of cells before the operation
Mitotic indices of the cells located far from wounds in the operated cultures remained as low as those of control cultures (0.05-0.2 %). No striking increases of the MI of the cells in
As seen from table 6, the LI of the cells in the wound in these experiments was similar or some-
Mitotic counts
Table 3. Per cent of Iabelled cells in the experiments with large and small c~ltl~re~ragmentsa % of labelled cells Time (days) p After After medium the operchange ation Mouse
: Human
Jbroblast-like 21 diploid
2 a Conditions
1
Large fragments Cells migrating from the fragment
Small fragments Cells remaining in the fragment
Cells migrating from the fragment
Cells remaining in the fragment
Nonoperated culture
16.Oil.3 11.8k1.2
31.7k1.5 29.0 f 1.4
11.0+1.0 3.5 20.6
17.82 8.150.9i.2
-
-
cells
38.4il.5 25.8k1.4
cells
21.3k1.8
7.OkO.8
of experiments and designations are’the same as in table 2.
3.6-M
90
Ju. M. Vasiliev et al.
Table 4. Per cent of labelled cells in the experiments with mechanical removal or drying of one half of the culture of mouseJibroblast-like cells % of labelled cells Time (days)
Drying
Mechanical removal
After medium change
After the operation
Cells migrating above the dried cells
Cells remaining in the not-dried half of the culture
Cells migrating on the glass
Cells remaining in the not-removed half of the culture
Nonoperated cultures
2 1
1 1
37.7F2.2 28.0f2.0
3X10.8 13.0* 1.6
32.3 +2.0 29.0f2.0
7.0+ 1.0 5.8111
3.6kO.9 10.0+ 1.3
the wound were observed during the first 12 h after the operation. However, it was difficult to obtain reliable figures at these stages, as total number of cells in the wound was too small. At 24-30 h after the operation MI in the wound increased up to l.O-2.0%. In the experiments with continuous 3HTh labelling first labelled mitotic figures were found at 6-8 h after addition of 3HTh to the medium, both in the nonoperated and in the wounded cultures. At about 9-10 h 50 % of mitoses became labelled.
the migrating cells were not oriented in any definite way with regard to the wound edge. At 24 h after the operation the LI of the cells in the wound and in other parts of the culture remained identical (see table 2). DISCUSSION Each normal cell in the confluent culture is firmly linked with other cells and stretched from all sides by its neighbours. These mechanical interactions are most obvious in the cultures of mouse cells; they lead to contraction ‘of the wound and to orientation of cells at the margin of the wound. The nature of forces responsible for this stretching is not clear. Possibly, contractile structures in the cells and/or intercellular substances produced by these cells are of some~
Experiments with neoplastic cells In the experiments with all types of transformed cells no changes of the wound form and cell orientation were observed immediately after the operation. Cells migrated into the wounds, but the rate of this migration was slow and most of
Table 5. Per cent of labelled cells in the areas of wounded cultures with various cell densitiesa Cells remaining in the part that was not removed near its edge
I
II-III
IV-VI
VII-IX
X-XII
Mouse jibvoblast-like cells Mean number of cells per field % of labelled cells
31 15.5i1.1
17 21.4f1.8
13 28.421.2
7 31.5k1.2
3 31.611.2
2 36.6k2.8
Human diploid cells Mean number of cells per field % of labelled cells
17 8.OkO.6
12 12.0&1.0
9 12.311.0
5 15.121.2
4 20.711.5
2 2O.Ok2.2
Cells migrating on the glass
a Results of counts made in 2 pulse-labelled cultures of mouse and human cells after mechanical removal of one half of culture. Mouse culture was fixed 6 days after the operation and 1 day after medium change; human cell culture 3 days after the operation and after medium change. Increasing roman figures designate rows of fields of view that were approximately at the same distance from the edge of the half of the culture that was not removed. Number of labelled cells and total number of cells were counted in each field of view at the magnification 15 x 90. About 60-66 fields of view were counted in each row. Exptl Cell Res 54
Wound healing processes
importance. At the edge of the wound one side of the cell surface is released from contact inhibition of movement while cell contacts on the other side of the same cell remain intact. This gradient of contact inhibition may explain oriented cell movement into the wound. Preestablished orientation of cells in the culture determines to a considerable degree the initial rate of migration as change of cell orientation takes considerable time. Probably this change of orientation involves some structural reorganization of the cell. In most experiments the cells that migrated into the wound tended to remain in a certain zone near the edge of pre-existing culture and did not move farther on the glass. This limited character of migration was most evident in the experiments with culture fragment left on the glass: migration of cells usually did not lead to total “‘disintegration” of these fragments. Limited outward migration of fibroblasts from culture fragment containing small number of cells was observed earlier by Abercrombie & Gitlin [2]. Many facts indicate that a certain minimal local cell density is optimal for the proliferation of- these cells in culture [S, 13, 151. Possibly, fibroblasts tend to remain in the zones of optimal cell density and do not migrate farther into the areas where cell density is too low. The nature of the processes essential for these preferences is not clear. Mutual adhesion of neighbour cells may play a certain role in this process: migrating cells do not break all their adhesions with each other and this limits their migration. Probably most cells that happen to be located near the edge of the wound would migrate into that wound. Experiments with prelabelling of cells before the operation indicate that there is no or little selectivity in migration of labelled or non-labelled cells into the wound. Colcemide strikingly inhibited cell movement into the wound. In the concentrations used in our experiments colcemide had no obvious nonspecific toxic effect upon the cells. Probably this drug selectively interfered with the ability of cells to perform oriented movements. This sug-
in cell cultures
9
Table 6. Per cent of labelled cells in wo~~~de cultures labelled with 3NTh before the o~e~~t~o~ 76 of labelled
Time Time of incubation with 3HTh W Mouse 48 48 48 48 48 48
jibroblast-like 0 6 18 24 30
1 1 Diploid 1 1 20 20 20
after the operation (h)
0 20 human
cells 0 24 12 24 30
cells
Cells migrating into the wound
Celk ar0LKld
0petW3d
the wound
cu!t-xes
40.4To.9 41.2kO.7 32.8 +0.6 35.5 * 1 .o 30.5 -IQ.7
26.5+1.4 28.5~1.6 27.5kl.l 29.81-1.2 27.9kI.4 25.9k1.1
28.O:k16 28.8kI.4 24.8k1.3 -
Non-
cells
9.3-i-0.9
13.21.1.1 9.4+ 1.7 9.3il.O 7.6kO.8
11.0+1.0 12.2+ I .o
9.4 i-o.9 12.3+1.0 5.8 i-O.8 5.3 *o.a 5.6kO.7
4.8 kO.7
gestion is in good agreement with the ~reIimi~ary results of our experiments with cultures of mouse embryo fibroblast-like cells grown on an oriented substrate (Vasiliev & Domnina, unpub lished). As suggested by Weiss [19], tish scale was used as an oriented substrate. hen cells were attached to this substrate, they immediately acquired elongated, spindle-like shape, their long axis being oriented in parallel with the ridges of the substrate. Addition of colcemide (0.1 pug/ml) to the medium did not inhibit cell attachment to the substrate, but almost completely prevented their orientation. In colcemidecontaining medium cells attached to the scale had an irregular polygonal shape. When these cultures were transferred into the medium without colcemide, the cells gradually acquired spindle-like shape. Colchicine was found to inhibit oriented growth of plant roots and chemotactic migration of human leucocytes (see review in 16, 141). Colcemide altered polarity in Hydra [lS]. Perhaps all these effects, as well as metaphase arrest, have something to do with the changes of cytoplasmic viscosity produced by these alkoloids (see review in [14]). Only a small fraction of cells in confluent cul-
92
Ju. M. Vasiliev et al.
tures is synthetizing DNA at any moment. In the experiments described above two types of factors increased considerably this fraction: (a) medium changes, and (b) cell migration into the wound. The latent periods after the action of both these factors were of the same order: LI in the cultures began to increase at 15-18 h after medium change. Increased LI in the wound was observed not earlier than at 12-18 h after the operation. However, the efficiency of these two factors was different. One may try to estimate the fraction of cells induced to enter S-phase in the wound in the following way. Let us assume that:
(4 an interval
between the migration of the cell into the wound and activation of DNA synthesis is t, h and fraction of the cells activated after this interval is Y; n-number of cells migrating into the wound per hour is constant during the first 24 h after the operation; among the cells remaining in the wound less than t, h fraction of labelled cells is the same as that among the cells in other parts of the culture (I,>.
(b)
cc>
Then fraction of labelled cells & = LI/ 100) in the wound at T h after the operation (TG 24) is
y=L*
T-1,. T-t,,
t,, -
Values I, and I, were obtained in the experiments with continuous labelling. t, was assumed to be 15 h. Calculations showed that Y in most experiments varied from 0.7 to 1.0. Although calculations of this type are inevitably very crude, they show that almost all cells migrating into the wound are induced to enter S-phase. Thus, migration into the wound was much more efficient growth inducing factor than medium change. Experiments of Todaro and collaborators [16] suggest that fresh serum contains a factor inducing DNA synthesis and division Exptl
Cell
Res 54
in cells that are in close contact, but is not necessary for the growth of dispersed cells in the wound. Further work is needed to elucidate these questions. Data of Nilausen & Green [12] indicate that 3T3 mouse cells in saturation density cultures are blocked in Gl phase of the mitotic cycle, while the experiments of Maciera-Coelho et al. [l I] with human diploid cells suggest that these cells are blocked in G2 phase. Experiments now in progress in this laboratory should show whether mouse and human cells migrating into the wound enter S phase from some point in Gl phase or from G2 phase. Results of wound healing experiments described above show that activation of DNAsynthesis is a local phenomenon: only the cells that leave the confluent culture and migrate on the free glass are stimulated, but not the cells remaining in the culture even if they are located near the edge of the wound. Cells remaining in the small fragment of the culture were not stimulated. On the other hand, cells in the small wound were activated although they were surrounded by a large area of confluent culture. Similar local stimulation of DNA-synthesis in the wound were observed in our earlier experiments [17] and in the experiments of Todaro et al. [16]. Fisher & Yeh [9] had shown that DNA synthesis in large colonies of 3T3 cells is confined mainly to cells at the periphery of each colony. Obviously the cell is activated or not activated to begin DNA synthesis depending on the state of its immediate neighbourhood. It is irrelevant for activation of this cell what happens at distances exceeding a few cell lengths, whether there is free glass or confluent culture. Role of the local factors in the regulation of cell growth is suggested also by the results of the counts made in control cultures and in the experiments with the removal of one half of the culture. These counts had shown that % of DNAsynthetizing cells decrease with an increasing local cell density. Dead dried cells in contrast to viable cells have no effect upon the growth of their neighbours. Thus, the growth inhibiting effects of neigh-
bour cells has a very short range of action. It seems improbable that these effects can be explained by the changes of concentration of low molecular substances in the cell environment [I3]. These effects may be due either to the contact of cell surfaces or to the accumulation of some high molecular substance (e.g. some polysaccharide) near the surface of these cells [3, 41, Difference between these 2 explanations seems to be hardly significant, as carbohydrate-containing macromolecular substances seem to be essential components of the external surface coats of the cells of various types. DNA synthesis in neoplastic cells is not stimulated by their migration into the wound. These experiments confirm that these cells become insensitive to the local factors regulating growth in non-neoplastic cultures [l]. Migration of neoplastic cells into the wound was unoriented and, probably, is due mostly to the random movements of cells near the edge of the wound. This inability to perform oriented movements, possibly, is a manifestation of some deficiency of locomotory behaviour of neoplastic cells [I]. It is not clear at present how this inability is correlated with other manifestations of abnormal locomotory behavior of ncoplastic cells, especially with the loss of contact inhibition of movement.
REFERENCES 1. Abercrombie. M & Ambrose. , E J.I Cancer res 22 (1962) 525. ’ 2. Abcrcrombie, M & Gitlin, G, Proc royal sot B 162 (1965) 988. 3. Btirk, R R, Growth-regulating substances for animal cells in culture (ed V Defendi & M Stoker) pi 39. The Wistar Univ. Press, Philadelphia (1967). 4. Cox, R P & Gesner, B M, Cancer res 27 (1967) 974. 5. Eagle, H, General physiology of cell specialization (ed D Mazia & A Taylor) p. 151. Academic Press, New York (1963). 6. Eigsti, 0 J & Dustin, R, Jr, Colchicine-in agriculture, medicine, biology, and chemistry. Iowa State College Press, Ames,-Iowa (1955). I. Ephrussi, B, PhCnom&es dint&ration dans les cultures des t&us. Hermann, Paris-(1935). 8. Fischer, A, Biology of tissue cells. Cambridge Univ. Press (1946). 9. Fischer. M W & Yell. J. Science 155 11967) 581. 10. Lcvinc,‘E N, Becker,’ G, Boone, C W &‘Eagle, H, Proc natl acad sci 53 119651 350. 11. Ma&h-a-Coelho, A, Ponten, J & Philipson, L, Exptl cell res 43 (1966) 20. 12. Nilauscn, K & Green, H, Expti cell res 40 (1965) 166. 13. Rubin, H & Rein, A, Growth regulating substances for animal cells in culture (cd V Defendi & M Stoker) p. 51. The Wistar Univ. Press, Philadelphia (1967).’ 14. Savel, II. Progr exptl tumor research 8 (1966) _ , 189. Karger, Base1 & New York. 15. Stoker, M & Sussman, M, Exptl cell rcs 38 (1965) 645. 16. Todaro, G, Matsuya, Y, Bloom, S, Robbins, A & Green, I-I, Growth regulating substances for aniinal cells in culture (cd V Defendi & M Stoker) p. 87. The Wistar Univ Press, Philadelphia (1967). 17. Vasiliev, Ju M, Gelfand, I M & Erofeeva, L V. Dokl akad nauk SSSR 171 (1966) 721. (In Russian.) 18. Webster, G, J embryo1 exptl morphol 18 (1967) 181. 19. Weiss, P, Intern rev cytol 7 (1958) 391. Received May 20, 1968