/. Embryol. exp. Morph. Vol. 23, J, pp. 253-272, 1970
Printed in Great Britain
253
On the occurrence of specific adhesion
between cells
By A. S. G. C U R T I S 1
From the Department of Cell Biology,
University of Glasgow
The idea that cells adhere to one another in a specific manner, such that cells
of one type stick only to cells of the same type, appears to have had its origin
from the work of Wilson (1907). He found that when cell suspensions from two
species of marine sponge were mixed and allowed to aggregate, each individual
aggregate body was composed of cells of one species alone. This conclusion has
been supported by the results obtained by Humphreys (1963) amongst others,
though some workers, who have used different species of sponge, have failed to
detect signs of specific adhesion of the cells (Sara, Liaci & Melone, 1966). Until
recently there has been little evidence in favour or against the idea that specific
adhesion occurs between the cells of higher animals. Clearly it is of great interest
to discover whether or not specific adhesion occurs, not only because such a
phenomenon might be of great importance in the control of morphogenesis but
also because its occurrence would have an important bearing on the mechanisms
by which cells adhere to one another.
Recently Roth & Weston (1967) and Roth (1968) have produced experimental
evidence for the occurrence of specific adhesion in embryonic chick cells. In this
experiment they prepared aggregates composed of one cell type and then placed
these aggregates in suspensions of cells either of the same or a different type.
The cells in these suspensions were labelled with tritiated thymidine. They
found that about twenty times as many cells adhered to aggregates of their own
cell type as adhered to aggregates of a different type. This experiment appears to
be convincing evidence for the occurrence of specific adhesion, although only
about 0-1 % of the cells in the suspensions adhered to the aggregates even
when they were of the same type. It is conceivable that these cells are not
representative of the majority. A new technique for testing whether or not
specific adhesion of cells occurs has been introduced (Curtis, 1969a); since this
technique reveals the behaviour of the majority of the cells it seems appropriate
to reinvestigate the system used by Roth & Weston. During the course of this
work evidence emerged which suggested that Roth & Westons' result might be
1
Author's address: Department of Cell Biology, The University, Glasgow, W2.
254
A. S. G. CURTIS
due to a certain sequence of changes in adhesiveness in the cells rather than to
specific adhesion; consequently further experiments were carried out to test
this hypothesis.
METHODS
(a) Theory
One of the main technical problems in the investigation of the occurrence of
specific adhesion arises from the difficulty of accurate recognition of the type of
every cell in an aggregate. If it is impossible to identify every cell in an aggregate
it is impossible to claim that complete specificity of cell adhesion takes place or
even to make an accurate estimate of the extent of any specificity which is claimed.
Although the use of tritiated thymidine to label one cell type is clearly a valuable
technique, it is impossible to label all cells of one type with this marker. Obviously
a technique for the detection of specific adhesion is required which does not
involve the identification of the type of each cell. Such a technique has been
introduced by Curtis (1969a).
In essence the method is based on the theory of collision of particles in a
laminar shear flow. If a suspension of cells is aggregated in a laminar shear flow
the instantaneous collision rate is given by the relationship
b=^Gn*(2rf,
(1)
where G is the shear rate, and n the concentration of cells of radius r per unit
volume (Curtis, 1969b). The proportion of collisions which result in permanent
adhesions is given by a factor a, the collision efficiency or stability ratio. The
collision efficiency can be used to derive a direct measure of the adhesiveness of
the cells (Curtis, 1969&). The factor a is measured by comparing the rate of
formation of adhesions with the total rate of collision. This can be carried out
by making use of the relationship obtained by Swift & Friedlander (1964)
AU
TT '
yA)
where Nm0 is the total concentration of single cells at time 0, Nmt the total concentration of aggregates and single cells at time t, $ the volume fraction of all the
cells and G the shear rate. If cells are aggregated in a Couette viscometer a shear
rate of known value of G can be applied to the cell suspension.
If a cell suspension of two types o and/?, of the same adhesiveness (and thus of
the same value of collision efficiency) and no specificity of adhesion, is aggregated then the instantaneous rate of aggregation between the cell types is given
by
bns=*«Gn%r0 + rPY,
(3)
Specific adhesion between cells
255
where n = n0 + np and n0 and np are the concentrations of cells of types o and/?
of radii r0 and rp respectively. Thus by application of this equation, it can be
stated that if two cell types of equal collision efficiency (and thus of equal
adhesiveness) are mixed in any proportion the adhesiveness of the mixture will
be of the same value as for the cell types singly, if there is no specificity in their
adhesion. Similarly if two cell types of differing adhesiveness are mixed in any
proportion, the adhesiveness of the mixture will be the mean of the two separate
adhesiveness weighted for the proportion of each cell type in the mixture.
However, if the cells show specificity of adhesion such that they will adhere
only to their own type then the aggregation rate for each cell type is proportional
to «o and n\ and the combined aggregation rate for a mixture is proportional to
nl + n\. By comparison the aggregation rate when there is no specificity of
adhesion is proportional to (n0 + np)2. Thus if two cell types which are equally
adhesive are mixed in equal proportions the effective collision rate and thus the
aggregation rate, when there is complete specificity of adhesion, will be half that
which would occur if there was no specificity of adhesion, or would be found for
either cell type in isolation. If the two cell types which show specificity of adhesion
are of differing adhesiveness the combined aggregation rate for the mixture will
be proportional to cc0nl + OLPY\%. It is assumed in this treatment that the individual
cells of each type are of identical average radius. If they are not a more complex
treatment must be used.
Thus the technique for the detection of specific adhesion is to aggregate
mixtures of two cell types in a Couette viscometer using the technique described
by Curtis (1969 a) to measure the collision efficiency. Measurements of the collision efficiency are obtained for both cell types alone and for mixtures in various
proportions. The values of collision efficiency plotted against the proportion of
one cell type in the mixture are then compared with the plots which would be
expected if there is either no specificity of adhesion or complete specificity. The
experimental plots are examined by regression analysis to test whether they are
compatible with either complete specificity or its lack or an intermediate situation.
There is one further problem which required examination. If the cells show no
specificity of adhesion but the two types are of very dissimilar adhesiveness then
as aggregation proceeds relatively more and more of unaggregated cells are of the
less adhesive cell type. It might be expected that this effect would result in the
measured values of collision efficiency obtained on such a mixture falling as
aggregation proceeds. This expectation can be shown to be incorrect from the
following argument.
The aggregation of cell types of equal adhesiveness mixed in equal proportions
will give rise to a fraction of aggregates containing only one cell type (assuming
no specificity). This fraction is given by \fin where n is the number of cells in each
aggregate of the aggregate class considered. In general, for two cell types o and/?
mixed in proportions m0 to mp, where o is the more adhesive type, (x times as
256
A. S. G. CURTIS
adhesive as type p) the proportion of aggregates of one size class containing only
the more adhesive type is given by the relationship
•/(*£T-
(4)
Thus unless the adhesiveness of the two cell types or/and their relative proportions are very different, aggregation leads rapidly to the production of a preponderance of mixed aggregates. As aggregation proceeds the collision rate for
an arbitrarily chosen small portion of the aggregate population is at any moment
proportional to its population density and to the cube of the root mean square
aggregate radius, see equation (1). The aggregate radius is a direct function of the
population density in the period of aggregation immediately preceding, and if
the population density be written as n then the aggregate radius can be written
as {f)^/n. The population density in any aggregate class is also a function of the
population density in the immediately preceding period. Thus if two sections of
the population are compared, one composed mainly of cells of the more adhesive
type, and the other of cells of the less adhesive type, the relative collision rates
for types o and p are given by the relationship
/*^Vj = x
\np x nj
where x is the relative difference in adhesiveness. Therefore the collision rate for
an aggregate class falls linearly as a function of« because of the decrease in total
particle population density but rises as a linear function of n as aggregates
increase in radius. As a consequence the relative contributions of the more
adhesive and less adhesive cells to the collision rate do not change during
aggregation, so that the measured value for the collision efficiency will remain
constant.
(b) Materials
Cell suspensions were prepared from the neural retinae and livers of 7-day
chick embryos (De Kalb stock). These tissues were dissected from the embryos
in Hanks's saline, washed twice with modified calcium and magnesium free
saline (see Curtis 19696) and then dispersed by one or other of the two following
techniques. Method 1. This method is designed to be the same as that employed
by Roth & Weston (1967). The tissues were transferred to a solution of trypsin
(Difco) 0-5 % (w/v) in the CMF saline. This trypsin solution assayed at between
2000 and 2500 BAEE units of tryptic activity per ml. Since it showed collagenolytic activity no additions of collagenase were made to the trypsin solution.
Roth & Weston added collagenase to their trypsin. The tissues were incubated in
this solution for 7 min at 37 °C in the case of the neural retina and 2 min at 25 °C
for the liver. At the end of this period the trypsin solution was removed and the
tissue was washed with two changes of the CMF saline at 2 °C, the first contained
25 % serum to stop trypsin activity. A third wash with CMF saline was made and
>
Specific adhesion between cells
257
the tissues were broken up by the use of a pipette. Immediately this had been
done enough horse serum (Burroughs-Wellcome No. 2) was added to bring
serum concentration to approximately 20 %. The suspension was then centrifuged at ca. 300 g for 5 min to sediment the cells. The supernatant was removed
and the cells resuspended in CMF. Any small aggregates were then removed by
passing the suspension through a wire mesh sieve (Endecotts No. 400 Stainless
steel mesh). All manipulations of the suspension were carried out at 2 °C. Method
2. The tissues were transferred to a 0001 M-EDTA solution in CMF (pH 80) at
2 °C. They were incubated in this solution for 7 min for neural retina and 2 min
for the liver tissue. The EDTA solution was then replaced by three washes of
CMF and the tissues were dispersed by pipetting. The suspension was centrifuged at 25 g to remove any substantial pieces of undispersed tissue and then at
300 g, after this second centrifugation the supernatant was discarded and the
cells resuspended in CMF saline. Small aggregates were removed from the
suspension by passing it through a wire mesh sieve. Throughout this procedure
the cells were cooled at ca. 2 °C.
These suspensions were made up in CMF saline to contain approximately
6 x 106 cells/ml. One volume of the suspension was diluted with three volumes of
culture medium to obtain a cell suspension in a medium suitable for re-aggregation. This operation was carried out at 2 °C. The culture medium used contained
43 % Hanks's saline, 43 % 199 medium, and 14 % Horse serum (BurroughsWellcome No. 2). These media were filtered through a Millipore HA filter
shortly before use to remove any particulate contamination. When the reaggregation of mixed cell types was being investigated appropriate proportions
of two types of cell suspension were mixed while cool. The suspensions were then
rapidly warmed to 37 °C and either re-aggregated immediately in a Couette
viscometer or incubated in a reciprocating shaker before measurement of their
adhesiveness. The purpose of the incubation was to prevent adhesion of the
cells so that any changes in adhesiveness which may take place in the period
shortly after cell dispersion can be investigated. Re-aggregation of the cells
during the period of their incubation was prevented by shakingthecell suspensions
at 140 impulses per min in a reciprocating shaker. Viability tests were carried
out on the cell suspensions by the methods described in my paper (Curtis,
19696).
Measurement of adhesiveness
These measurements were carried out by the method described in an earlier
paper (Curtis 19696). The suspensions were aggregated under a shear rate of
between 8 and 9 sec -1 at 37 °C in a Couette viscometer, similar to that described
by Curtis (1969a). The collision efficiency a is obtained by use of equation (2).
Samples of the cell suspensions were removed at the start of aggregation to give
measurements of Nœ0 and at intervals thereafter to provide values of Nmt.
Counts of NœQ and N„t were made by use of haemocytometers. Since it has been
shown that the collision efficiency is affected by the shear rate under which
258
A. S. G. CURTIS
aggregation has been produced (Curtis, 1969 a) all measurements were carried out
within a very small range of shear rates. This technique for measuring adhesiveness produces extensive aggregation: examples of such aggregates have been
figured by Curtis (19696).
Results. Preliminary experiment
The initial experiment was to use this technique to examine the adhesiveness
of 7-day neural retina and liver cells which had been separated by trypsinization,
aggregating in the serum medium. Thirty measurements of the collision efficiencies of suspensions containing various proportions of neural retina and liver
I
I
I
0
10
20
I
I
i
i
l
30 40 50 60 70
Neural retina cells(%)
i
80
I
l
90 100
Fig. 1. Relation between collision efficiency and the percentage of neural retinal
cells in the re-aggregation of mixtures of neural retina and liver cells. Cells prepared by trypsinization and re-aggregated at 37 °C immediately thereafter. Plotted
points + are the means of a minimum of five measurements. The continuous line is
the plot of the linear regression equation fitted to the individual original measurements. The pecked line shows the relationship between collision efficiency and the
proportion of each cell type that would be expected if specific adhesion took place.
cells were made. The relationship between adhesiveness and the proportion of
each cell type in the suspension is shown in Fig. 1. When this plot was examined
by linear regression analysis a very good fit was found for a straight line relationship y = -0134.x:+14-6, where x is the percentage of the less adhesive
cell type in the mixture and y the collision efficiency as a percentage,
sb = 0019,
t = b\sh = 705 (P < 0001)
(where sb is the standard deviation of the regression coefficient). This plot is not
significantly different from that which would be expected on the theory that
Specific adhesion between cells
259
there is no specificity of adhesion. The plot which would be expected if there was
complete specificity of adhesion is shown in Fig. 1 also. It can be seen that the
greatest difference in collision efficiency according to these alternative hypotheses
occurs for a mixture containing 50 % neural retina. Using the test given by
Snedecor & Cochran (1967, p. 154) for the significance of a difference between a
given point on the regression curve and a chosen value, it is found that there is a
probability (P < 0-025) of 16 % of the cells showing specificity of adhesion.
Discussion. Preliminary experiment
This result suggests either that Roth & Weston's findings are (i) typical of
only a very small proportion of the cells in the system, or that (ii) their findings
were the result of especial features of their technique which were not reproduced
in the experiment just described. Although there is some evidence to support the
first of these hypotheses, it is necessary to explore the group of differences
covered by the second.
One of the main differences between the experimental situation in Roth &
Weston's system and that described above lies in the fact that in their system
aggregation of the suspension is allowed to proceed for between 4 and 6 h,
whereas in my experiments aggregation was followed for only approximately
90 min. Thus it is possible that the specificity of adhesion develops towards the
end of the period used by Roth & Weston (1967) and Roth (1968) has actually
advanced this suggestion. This was tested by allowing the cells to incubate in the
re-aggregation medium for 5 h under conditions in which aggregation is
suppressed; at the end of this period their aggregation was tested. It should be
appreciated that the experimental situation in this experiment is similar but
not identical to that used by Roth & Weston.
Another difference between Roth & Weston's experiments and those described above is that Roth & Weston's experimental situation required the
presence of aggregates of one cell type formed from cell suspensions whose
aggregation had begun 24 h previously. Conceivably aggregates have developed
a specificity of adhesion not found amongst the single cells. This was tested by
measuring the adhesion of cells onto aggregates. Finally, it should be pointed
out that when freshly prepared cell suspensions made by trypsinizing tissues are
added to a suspension of aggregates a small amount of trypsin is added to the
aggregates. Does this transfer of trypsin affect the adhesiveness of the aggregates ?
This possibility was investigated by adding a small amount of trypsin to a
suspension of aggregates which had reached an equilibrium between aggregation and break up under shear forces and measuring whether there was any
diminution in the extent of aggregation of the system. Edwards & Campbell
(unpublished) have made measurements of the amount of trypsin which is
likely to be carried over into the reaggregation system from the dispersion
system. The amount of trypsin added to the reaggregation systems was based on
their work.
17
E MB 23
260
A. S. G. C U R T I S
RESULTS
(i) Is the development of specificity of adhesion delayed?
In order to investigate this question the cell suspensions were incubated in the
re-aggregation medium at 37 °C for 5 h. They were placed in a reciprocating
shaker and agitated at 140 impulses per min which prevents aggregation during
this incubation. At the end of this period the cell suspensions were transferred
0
i
i
10
20
i
i
i
i
i
30 40 50 60 70
Neural retina cells (%)
i
i
i
80
90
100
Fig. 2. Relation between collision efficiency and the percentage of neural retinal cells
in the re-aggregation of mixtures of neural retina and liver cells. Cells prepared by
trypsinization, incubated for 5 hr in a medium suitable for re-aggregation and then
re-aggregated. Otherwise as Fig. 1.
to a Couette viscometer for measurement of their collision efficiency using the
technique described for the preliminary experiment to test for specificity of
adhesion. The collision efficiencies of mixtures of neural retina and liver cells in
various proportions were measured: in such cases the cell types were incubated
separately and only mixed just before transfer to the Couette viscometer. As in the
preliminary experiment the relationship between adhesiveness and the proportion of each cell type is shown in Fig. 2. Use of linear regression analysis shows
that the data are fitted very well by the regression equation y = 0T62x + 5-61,
where x is the percentage of the more adhesive cell type in the mixture,
and y the collision efficiency as a percentage, sb = 0013, t = 12-4 (P < 0-001).
The plot which would be expected if there was complete specificity of adhesion
is also shown in Fig. 2. It can be seen that the greatest difference between the
Specific adhesion between cells
261
plot of actual measured collision efficiencies and those expected on the hypothesis of specificity of adhesion occurs for a mixture containing 50 % of neural
retina cells. Use of the test for calculation of the significance of a difference
between a given point of a regression curve and a chosen value showed that
there is a probability of < 2-5 % of 20 % of the cells showing specificity of
adhesion.
Preincubation period (h)
Fig. 3. Collision efficiency of neural retinal cells (continuous line and plotted
points O) and of liver cells (pecked line and plotted points x ) in relation to the
period of preincubation of the cells in re-aggregation medium before re-aggregation.
Standard deviations shown for each point, a minimum offivemeasurements per point.
Curves fitted by eye.
It is remarkable that although there is no sign of a development of specificity
of adhesion over the first 5 h of aggregation the relative adhesiveness of the two
cell types has completely reversed. Neural retina cells have increased their
collision efficiency very considerably while liver cells have decreased in adhesiveness. As a result of this finding I decided to investigate the time course of these
changes in adhesiveness of the two cell types. This was carried out by incubating
either cell type in re-aggregation medium, under conditions of shaking which
suppress re-aggregation for various periods. The collision efficiency of the cell
type was then measured. The results are given in Fig. 3.
(ii) Do aggregates show specificity of adhesion?
Aggregates were prepared from trypsinized neural retina or liver cells as
described in the section on Methods. They were re-aggregated in a gyratory
shaker at 148 gyrations per min. The shear rate corresponding to such a speed of
17-2
262
A. S. G. CURTIS
agitation (Curtis to be published) ensures that the aggregates are of a fairly
small size but that very few unaggregated cells or minute aggregates remain.
After 24 h the suspension of aggregates was transferred to a Couette viscometer
run at a lower shear rate (C = 8-6 sec-1) than that in the gyratory shaker. The
Table I . A . Collision efficiency of cell suspensions in the
presence of aggregates
Cell
suspension
type
Aggregate
type
(a)
(b)
(c)
(d)
(e)
(ƒ)
(g)
(//)
Neural
Neural
Liver
Liver
Neural
Neural
Liver
Liver
retina
retina
(/)
0')
(k)
(/)
Neural retina
Neural retina
Liver
Liver
retina
retina
Dispersal
method
Mean
collision
efficiency
n
/o
Trypsin
Neural retina
1-2
5
Neural retina
EDTA
18-8
5
Trypsin
Neural retina
8-7
17
Neural retina
EDTA
8-6
9
Trypsin
Liver
4-3
17
EDTA
Liver
17-4
9
Trypsin
Liver
18-2
5
Liver
EDTA
6-8
5
For comparison in the absence of aggregates
—
Trypsin
2-8
5
—
EDTA
15-3
12
—
Trypsin
5
15-9
EDTA
3-7
5
—
S.D.
2k 2
0-07
1-48
400
110
110
2-76
2-38
0-92
002
8-8
256-1
9-7
19-7
61-4
22-7
3-4
0-56
1-97
0-21
0-45
1-3
42-8
018
0-8
B. Tests of the significance of differences in mean collision efficiency for cell
suspensions in the presence and in the absence of aggregates
Suspension
Aggregate
type
type
/
D.F.
Comparison
(i) Cell suspensions prepared by trypsinization
Neural retina
Neural retina
Liver
Liver
Neural retina
Neural retina
Liver
Liver
Neural retina
Liver
6-28**
2-87**
8
20
Neural retina
3-95**
20
2-15
8
Liver
(ii) Cell suspensions prepared with EDTA
3-54**
15
Neural retina
Liver
2-03*
19
Neural retina
9-39**
12
Liver
6-33**
8
(a) with (/')
(e) with (/)
(c) With (Â:)
(g) with (k)
(b) with
(ƒ) with
(d) with
(h) with
0)
(ƒ)
(/)
(/)
**Significant below 1 % level; * significant below 5 % level.
effect of the lower shear rate means (Curtis to be published) that the maximal size of aggregate for a given cellular adhesiveness is greater. Thus if single
cells are now added to the aggregate suspension they will aggregate mainly
onto the existing aggregates, provided that there is no specificity of adhesion to
prevent their aggregation. If there is complete specificity of adhesion the measured
collision efficiency for the cell suspension will be the same as it would be without
Specific adhesion between cells
263
the presence of aggregates. The finding of a greater collision efficiency will
indicate that there is either no or an incomplete specificity of adhesion. Unfortunately no precise analysis of the collision efficiency which would be expected
for a given population density and size of aggregates and a chosen value of
adhesion specificity is yet possible, but it is clear that the greater the measured
collision efficiency the smaller the extent of specificity to be expected. The experiment was carried out in two ways. In the first the cell suspension added to the
aggregates was prepared by trypsinization. But since (see (iii)) there is evidence
that traces of trypsin transferred with the suspension may diminish the adhesiveness of the aggregates, a variation of this experiment was carried out using cell
suspension separated by EDTA treatment. The following measurements of
collision efficiency were obtained; see Table 1. It can be seen from this table that
the collision efficiency of neural retina cells (separated with EDTA) is considerably increased by the presence of either neural retina or liver aggregates.
Moreover, the extent of the increase is of similar value for either aggregate type.
An exactly similar situation obtains when either aggregate type is aggregated in
the presence of a suspension of liver cells separated with EDTA. Although the
increase in collision efficiency in the presence of the aggregates is evidence
that there is no complete specificity of adhesion shown by the aggregates, it
might be arguable that the increase is compatible with a very small lack of specificity. However the fact that either aggregate type increases the collision efficiency of either type of cell suspension to a similar extent argues that there is no
factor causing the cell suspensions to adhere preferentially to aggregates of
their own type. Somewhat different results were obtained when cell suspensions
prepared by trypsinization were used, see Table 1. The collision efficiency of
neural retina cells is appreciably increased by the presence of liver aggregates,
whereas that of liver cells is lowered in the presence of neural retina aggregates. However, the collision efficiency of neural retina cells is lowered in the
presence of neural retina aggregates, which argues that this lowering may be not
specific in nature. It should be borne in mind that the experimental situation
used in this experiment is identical with that reported by Roth & Weston.
(iii) The problem of the effect of the transfer of trypsin
In both Roth & Weston's experiments and those described in section (ii) the
adhesiveness of the aggregates may have been affected by the addition of the
fresh cell suspension to the system. Since it is inevitable that a certain amount of
trypsin would be carried over with the freshly dispersed cells, and trypsin is
known to decrease the adhesiveness of cells, it can be asked whether the adhesiveness of the aggregates is affected by this transfer of trypsin. This question was
resolved by the following experimental design. Aggregates of one cell type were
prepared by aggregation in a gyratory shaker for 24 h at 37 °C. They were then
transferred in their original aggregation medium to a Couette viscometer. Two
units of trypsin (BAEE units) in 001 ml volume of Hanks's saline were added to
264
A. S. G. CURTIS
20 ml of the suspension containing aggregates: this addition provides a level of
tryptic activity in the medium comparable with that which is present in freshly
dispersed cell suspensions after the cells have been washed free from the trypsin
medium. The adhesiveness (collision efficiency) was then measured on both those
suspensions to which an addition of trypsin had been made and a control series.
12
O
11
° O
0
o
10
o °
8
o
I
°o
"5 8
+ +
+
+
6 -
•2
Î +
4
13
10
20
30
40
50
60
Time from start of re-aggregation (min)
70
Fig. 4. The effect of the addition of small amounts of trypsin on the further re-aggregation of mixtures of neural retina cells and small aggregates in a Couette viscometer
after 24 h of re-aggregation in a gyrated flask. The results of three separate experiments and controls are shown. All runs were started at a particle concentration of
9-5 x 105 per ml. Points plotted as + refer to controls in the absence of trypsin,
points plotted as O refer to suspensions in the presence of trypsin. Release of cells
from the aggregates can be seen during the first 10 min of the aggregation of suspensions in the presence of trypsin, thereafter the value of N«, drops more slowly
than in the controls indicating that the adhesiveness of these cells and aggregates is
lower in the presence of trypsin than in its absence. At the start of the original reaggregation the population density of single cells was ca. 2-4 x 106/ml.
Specific adhesion between cells
265
The Couette viscometer was set at a shear rate slightly below the shear rate
obtaining in the gyratory shaker while the preliminary aggregation was carried
out. The reason for adjusting the shear rate to a slightly lower value was to
ensure that there would be no break up of the aggregates into small bodies or
single cells due merely to an increase in shear rate. The shear rate determines the
equilibrium size of the aggregates (Curtis, in preparation). If the presence of
trypsin lowers the adhesiveness of the aggregates, there will be an increase in the
proportion of single cells and small aggregates after a period of exposure to
shear in the viscometer, whereas if there is no change in the adhesiveness a very
slight further aggregation occurs (because of the reduction in shear rate). The
results of the counts of total particle concentration N^ for this experiment are
shown in Fig. 4. It is clear that those neural retina aggregates which have been
exposed to a low concentration of trypsin show a reduced adhesiveness relative to
their subsequent adhesiveness for a period of about 1 h after addition of the
enzyme. By comparison the aggregates which have not been exposed to low
concentrations of the enzyme show no sign of a decrease in adhesiveness. After
1 h those suspensions which were treated with the enzyme begin to show an
increasing adhesiveness.
DISCUSSION
The experiments which have been described above, show that if there is any
specificity of adhesion between neural retina and liver cells it is of very small
extent and must be greatly overshadowed by the relatively large amount of nonspecific adhesion which takes place. This statement applies not only to the cells
at the start of aggregation, but also to those which have been cultured for some
hours after their separation. Does a specificity reside in the surface of the
aggregates rather than in the cells? There is good evidence that the aggregates
themselves even after 24 h of aggregation are still capable of forming many nonspecific adhesions with cell suspensions. Thus both aggregates and individual
cells are without specificity in the majority of their adhesions. At this point
consideration should be given to the reason for the apparent discrepancy
between the interpretation which Roth & Weston drew from their results and the
results of the present work which provide no evidence for the occurrence of
specific adhesion. It should be remembered that the experimental situation
described in Results (iii) when trypsin was used to separate the cells, exactly
reproduces the situation used by Roth & Weston (1967) and by Roth (1968).
Perhaps the most remarkable result obtained in this work has been the discovery that the relative adhesiveness of neural retina and liver cells completely
reverses over the first 6 h of aggregation. This suggests a completely alternative
explanation of the results which were obtained by Roth & Weston. First,
consider a situation which obtains in the first set of experiments described in
this paper. At the start of the aggregation the liver cells are very adhesive, while
the neural retina cells are of low adhesiveness. During this phase the majority of
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A. S. G. CURTIS
aggregates will be composed of liver cells because they are some six times as
adhesive as the neural retina cells. The situation reverses itself after about 3 h
and thereafter the neural retina cells will tend to aggregate preferentially with
themselves. For a short period the two cell types are of comparable adhesiveness
and presumably during this period adhesion takes place between the two types
to a considerable extent. In the situation described by Roth & Weston aggregates were mixed with cell suspensions. If it is presumed that the adhesiveness of
the aggregates is altered by traces of trypsin added with the cell suspension then
their system can be interpreted in the same manner. Evidence has been given
that aggregates are affected in this way by the presence of trypsinized cell suspensions. Initially the cells in liver cell suspensions will be of considerable
adhesiveness and thus they will adhere appreciably to liver aggregates but
inappreciably to neural retina aggregates. Suspensions of neural retina cells will
show inappreciable aggregation at this stage. Later in aggregation the system
will have reversed the relative adhesiveness of the two cell types and then the
neural retina cells will adhere to the neural retina aggregates but not to liver
aggregates. At this stage the liver cells will be relatively non-adhesive. Thus when
aggregates are mixed with cells from another tissue, one cell type is adhesive
while the other is not. The consequence of this is that the majority of cells will
stick only to aggregates of their own type even if there is no specificity of adhesion.
A small amount of adhesion will take place between unlike types, particularly in
the cross-over period.
In this work I have shown that (i) the adhesiveness of cells in suspension,
prepared by trypsinization, changes over the first 6 h of culture; liver and
neural retina reversing their relative adhesiveness, (ii) the cell suspensions show
no specificity of adhesion either at the start of aggregation or after a culture
period of 6 h and (iii) aggregates show no evidence of specificity of adhesion
when mixed with cell suspensions prepared with a chelating agent, though it is
impossible to exclude totally the occurrence of some specificity. However, the
behaviour of the cell suspensions, prepared by the use of trypsin, in the presence
of aggregates, might be interpreted in part as evidence for the occurrence of a
specific form of cell adhesion. Liver cell suspensions (prepared by trypsinization)
show an apparent lowering of their adhesiveness in the presence of neural
retina aggregates by comparison with such suspensions in the absence of
aggregates. Indeed if there was no increase in the adhesiveness of such a suspension when in the presence of aggregates there would be reason to conclude
that adhesion might be specific. However, this apparent lowering of adhesiveness
is non-specific in nature, for neural retina aggregates lower the collision efficiency of cells of the same type. Small additions of trypsin to the aggregates
decreases their adhesiveness and cells detach themselves from the aggregates. It
seems probable that the small transfer of trypsin which is made when trypsinized
cell suspensions are added to aggregates results in the release of cells from the
aggregates with the consequence that the total particle density rises at first
Specific adhesion between cells
267
and then falls less rapidly than would otherwise be expected. This interpretation
(of non-specificity) is supported by the fact that liver aggregates increase the
apparent adhesiveness of neural retina cells, implying of course that adhesions
between the two cell types are often formed. Since trypsin in small amounts
increases the adhesiveness of liver cells it can be appreciated that this interpretation is consistent with the experimental results. The finding that liver aggregates apparently increase the adhesiveness of neural retina cells argues very
strongly that the aggregates themselves do not show specificity of adhesion.
Thus there appears to be no evidence for the occurrence of specific adhesion in
this system, although it is, as yet, impossible to discount completely the possibility of there being a small amount of specific adhesion.
It may be felt that there is some contradiction between Roth & Weston's
experiments and these, in that I have found situations in which the mixture of
unlike aggregates and cell suspensions gives rise to higher values of collision
efficiency for the suspension type than when it is measured for a suspension in the
absence of aggregates. Roth & Weston of course found little adhesion between
unlike cell types. It should be remembered that neural retina cells are of very low
collision efficiency immediately after dispersion, and thus in the presence of liver
aggregates, of fairly high adhesiveness, will show an increased collision efficiency
even though it is still of so low a value that there will only be a slight amount of
adhesion between the two types. At a later time, presumably, the relative
adhesiveness of the two types will reverse and then the neural retina will aggregate on itself predominantly.
A very approximate estimate of the possible extent of specific adhesion
which might occur in the experiments with mixtures of aggregates and cell
suspensions can be obtained as follows: using equation (3) the instantaneous
collision rate of a neural retina suspension with itself for conditions of shear
used in these experiments would be 2-66 x 103/s/ml compared with a value of
ca. 2-7 x 103/s/ml between single cells and aggregates. Therefore the collision rate
should approximately double in the presence of the liver aggregates and if the
aggregates have a collision efficiency (a value suggested by experiments on liver
cell suspensions) six times that of the neural retina suspension the apparent
collision efficiency for the suspension should increase 75 %. The actual increase
found was 65 % and although this is an imprecise treatment it would argue that
there can be little if any specificity of adhesion due to the aggregates themselves.
These results show that there is no evidence for the occurrence of specific
adhesion in mixtures of embryonic chick liver and neural retina cells and the
quantitative measurements suggest that if there is any specificity of adhesion it
is so small in extent as to be completely overshadowed by non-specific adhesion.
The sequence of changes in cell adhesiveness which have been revealed by these
experiments provide an alternative explanation for the results of Roth & Weston.
These findings also suggest an alternative explanation for the sorting out
phenomena which are supposed to occur in aggregates. If one cell type becomes
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A. S. G. CURTIS
adhesive before the second it would be expected that the first cell type would
aggregate first, later the second cell type would aggregate onto the core formed
by the first cell type as an outer coating. During most of the phase of aggregation
of the second cell type the first cell type would be insufficiently adhesive for
appreciable involvement in aggregation of any of its cells which failed to aggregate earlier. Thus the fact that the first cell type is of diminished adhesiveness
while the second cell type is adhesive and vice versa helps to ensure that the
various regions of the aggregate formed during the period of high adhesiveness
of a given cell type are substantially free from other cell types. In other words,
the apparent sorting out represents nothing more than the sequence of changes
in the adhesiveness of the various cell types during the building of the aggregates. This hypothesis suggests that the aggregates are built up within a sorted
out structure, and that our impression that extensive cell movement takes place
inside the aggregate, in which the cell types are initially randomly arranged, is
erroneous. Since no one as far as I know has yet examined the distribution of cell
types in an aggregate during its formation, the rather disquieting hypothesis
outlined above, cannot be regarded as disproven. Nor do we yet know whether
the apparent sorting out hides a proportion of cells which are wrongly placed.
Consideration of both Roth & Weston's results and my own suggests that a
proportion of cells make adhesions with those of another type and thus it
might be expected that a proportion of cells will be wrongly placed.
Roth (1968) and Roth & Weston (1967) have claimed that the use of kinetic
studies of re-aggregation are not adequate for the investigation of the occurrence
of specific adhesion. Their main grounds of criticism are that the technique does
not take account of differences in cell size or concentration. Though this criticism does apply to earlier methods of following the kinetics of re-aggregation,
such as those used by Curtis & Greaves (1965), it is not relevant to the technique
used in the present work. Indeed the technique used here provides more precise
control of these factors than is available in the system used by Roth & Weston.
This has been discussed in detail by Curtis (1969«), but it is apposite to point
out that unless aggregate diameter and the viscosity of the suspending medium
are kept constant when using Roth & Weston's technique no accurate comparison can be made of the ability of aggregates to collect cells of different types
or under different conditions of the composition of the medium. Nor does their
technique take into account the effect of differences in diameter between
different cell types on the collision probability. Another ground on which Roth
has criticised the use of aggregation kinetics is that the methods used for
dispersing cells into suspensions may alter cellular adhesiveness from the normal
state. Thus studies of cell adhesion on dispersed cells may not be relevant to the
behaviour of cells in vivo. This is undoubtedly a criticism which is potentially
valid in all such studies, when the behaviour of the cells is referred to the in vivo
situation. It is also a criticism which applies to Roth & Weston's work. However,
it is not a criticism which is applicable to re-investigations of their work provided
Specific adhesion between cells
269
that no conclusions are drawn about in vivo behaviour. Interestingly my results
suggest that the results obtained could be due to just such effects of the dispersal
technique altering the adhesiveness of cells for a period. The fact that Roth
found that fragments of tissue which had never been dispersed behaved in the
same way as aggregates may represent nothing more than the fact that traces of
trypsin carried into the re-aggregation system affect the adhesiveness of aggregates and fragments in the same way. The differences observed in this work
between the adhesiveness of cells separated with trypsin and those dispersed
with EDTA raise the question of whether either or neither technique has changed
the adhesiveness of the cells from that found in the embryo. Alternatively, if it
is claimed that trypsin does not alter the adhesiveness of the cells the changes
seen during re-aggregation must either represent a response to the conditions of
culture or are a repetition of processes which take place in the embryo at this
period. The interesting effects reported by Roth of such factors as various chemicals, conditioned medium or the addition of other cell types on the capture of
cells by aggregates can be interpreted as effects on the sequences of changes in
adhesiveness which aggregates and cells undergo after exposure to trypsin.
It should, however, be appreciated that these findings are not inconsistent
with various of the alternative hypotheses which have been put forward to
account for sorting out in aggregates. In particular the changes in cellular
adhesiveness revealed in this work are very similar to those required in the
' timing ' hypothesis (Curtis, 1967) for the explanation of sorting out in aggregates.
SUMMARY
1. The theoretical basis and experimental details of a new test for the occurrence of specific adhesion are described. The test does not require the identification of the species or tissue type of individual cells in aggregates. It depends upon
measurement of the adhesiveness (collision efficiency) of re-aggregating cells.
Measurements of collision efficiency are made on two cell types separately and
on mixtures of the two types in varying proportions. In the absence of specific
adhesion the collision efficiency for a mixture of two cell types is a mean value
ofthat for the two cell types alone weighted according to the proportion of each
cell type in the mixture. If adhesion is specific, however, all collisions between
unlike cells will be ineffective, so that the measured collision efficiency will be
depressed below the value found in the absence of specific adhesion. The full
theory and method of calculating the expected collision efficiency for mixtures
of two cell types in the absence or presence of specific adhesion is given. This
method can also be used to detect partial specificity of adhesion, and statistical
tests are given to test the probability of occurrence of various degrees of specificity.
2. This method was applied to the investigation of the adhesion of 7-day
embryonic chicken neural retina and liver cells. Previous work by Roth &
Weston led to the claim that these cell types showed specific adhesion.
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A. S. G. CURTIS
3. No evidence could be found for the occurrence of specific adhesion of
these cell types using the collision efficiency test, when two suspensions were
mixed. Nor could evidence be found that aggregates of one cell type could only
or preferentially adhere to single cells of the same type. However, it was observed that the adhesiveness of liver and neural retina cells prepared by trypsinization, altered over a 5 h period so that the liver cells which were initially much
more adhesive than the neural retina cells became less adhesive than them, while
the retinal cells showed a considerable increase in adhesiveness.
4. These findings provide an alternative explanation of those experimental
results which have led to the conclusion that specific adhesion of cells occurs in
this system. It is suggested that in Roth & Weston's experiment transfer of
small amounts of trypsin with the cell suspensions affected the adhesiveness of
the aggregates. When liver cell suspensions are added to liver aggregates they are
initially very adhesive and the cells adhere to the aggregates. If, however,
neural retinal cells are added to liver aggregates the retinal cells are initially of
very low adhesiveness so that they make very few effective collisions with the
aggregates. By the time the neural retinal cells have become very adhesive the
aggregates are of much lower adhesiveness so that the retinal cells aggregate
amongst themselves and do not adhere appreciably to the liver aggregates.
Conversely when neural retina cells are added to neural retina aggregates they
both become adhesive at the same time and the cell suspension adheres to the
aggregates, but when liver cells are added to retinal aggregates they are each
adhesive at different times so that little adhesion takes place between them.
5. Furtherexperimental evidence in favour of this interpretation was obtained.
It was shown that freshly trypsinized cell suspensions transfer sufficient trypsin
to affect the adhesiveness of aggregates in the manner postulated.
6. Evidence was obtained which suggests that the behaviour of the cells was
due to their trypsinization since cells dispersed with a chelating agent immediately
showed behaviour comparable with that of cells which had been cultured for
5 h to recover from trypsinization.
7. It is concluded that there is good evidence against the occurrence of
specific adhesion in the neural retina—liver system.
RÉSUMÉ
Sur Vexistence d'une adhésion spécifique entre cellules
1. On décrit la base théorique et les détails expérimentaux d'un nouveau test
pour établir la présence d'une adhésion cellulaire spécifique. Le test n'exige pas
l'identification de l'espèce ou du type tissulaire des cellules individuelles dans
l'agrégat. Il dépend de la mesure de l'adhésivité (efficacité des collisions) des
cellules en cours de réagrégation. Les mesures de l'efficacité des collisions sont
faites séparément sur deux types de cellules et sur des mélanges des deux types en
proportions variées. En l'absence d'adhésion spécifique, l'efficacité des collisions
Specific adhesion between cells
271
pour un mélange de deux types cellulaires est une valeur moyenne de celle des
deux types cellulaires seuls, établie selon la proportion de chaque type cellulaire
dans le mélange. Si l'adhésion est spécifique, toutes les collisions entre cellules
différentes seront néanmoins inefficaces, de sorte que l'efficacité des collisions
mesurée sera abaissée au-dessous de la valeur trouvée en l'absence d'adhésion
spécifique. On expose la théorie complète et la méthode de calcul de l'efficacité
prévue des collisions pour des mélanges de deux types cellulaires en présence ou
non d'adhésion spécifique. Cette méthode peut aussi être utilisée pour déceler
la spécificité partielle d'adhésion, et on donne des tests statistiques pour
éprouver la probabilité de réalisation de divers degrés de spécificité.
2. On a appliqué cette méthode à l'étude de l'adhésion de cellules rétiniennes
neurales et hépatiques d'embryon de poulet de 7 jours. Un travail antérieur de
Roth & Weston a conduit à l'affirmation que ces types cellulaires présentaient
une adhésion spécifique.
3. On n'a pu mettre en évidence l'existence d'une telle adhésion spécifique
pour ces types cellulaires en utilisant le test d'efficacité des collisions, quand deux
suspensions étaient mélangées. On n'a pas non plus pu trouver que des agrégats
d'un seul type cellulaire pouvaient seulement ou préférentiellement adhérer à
des cellules isolées du même type. Néanmoins, on a observé que l'adhésivité de
cellules isolées hépatiques et rétiniennes neurales préparées par trypsinisation,
changeait au cours d'une période de 5 h, de sorte que les cellules hépatiques qui
étaient initialement beaucoup plus adhésives que les cellules neurales devenaient
beaucoup moins adhésives qu'elles, tandis que les cellules rétiniennes présentaient un accroissement considérable d'adhésivité.
4. Ces résultats fournissent une autre possibilité d'explication des résultats
expérimentaux qui ont conduit à la conclusion que l'adhésion spécifique des
cellules survient dans ce système. On suggère que dans l'expérience de Roth &
Weston, le transfert de petites quantités de trypsine avec les suspensions cellulaires affectait l'adhésivité des agrégats. Quand des suspensions de cellules
hépatiques sont ajoutées à des agrégats hépatiques, elles sont initialement très
adhésives et les cellules adhèrent aux agrégats. Si néanmoins des cellules neurales
rétiniennes sont ajoutées à des agrégats de foie, les cellules rétiniennes ont
initialement une adhésivité très basse, de telle sorte qu'elles réalisent très peu de
collisions efficaces avec les agrégats. Au moment où les cellules neurales rétiniennes sont devenues très adhésives, les agrégats ont une adhésivité beaucoup plus
faible, de sorte que les cellules rétiniennes s'agrègent entre elles et n'adhèrent pas
de façon appréciable aux agrégats hépatiques. Inversement, quand des cellules
neurales rétiniennes sont ajoutées à des agrégats des mêmes cellules rétiniennes,
ils deviennent tous adhésifs en même temps et la suspension cellulaire adhère aux
agrégats, mais quand on ajoute des cellules hépatiques à des agrégats rétiniens,
les deux composants sont adhésifs chacun à des moments différents, de sorte
qu'il y a peu d'adhésion entre eux.
5. On a obtenu des preuves expérimentales supplémentaires en faveur de
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A. S. G. CURTIS
cette interprétation. On a montré que des suspensions cellulaires fraîchement
trypsinisées renferment assez de trypsine pour affecter l'adhésivité des agrégats
de la manière postulée.
6. On a obtenu des résultats qui suggèrent que le comportement des cellules
était dû à leur trypsinisation, étant donné que des cellules dispersées par un
agent chélateur montrent immédiatement un comportement comparable à
celui de cellules qui avaient été cultivées pendant 5 heures pour se remettre de la
trypsinisation.
7. On conclut que les faits observés fournissent de bons arguments contre
l'existence d'une adhésion spécifique dans le système rétine neurale—foie.
I am very grateful to Miss E. Davies who has given invaluable technical assistance in these
experiments. I would also like to thank Mr P. Hannah who assayed tryptic activities and to
Miss A. Baker and Miss F. MacFarlane who prepared histological material. The work was
carried out with the support of a grant from S. R. C.
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CURTIS,
CURTIS,
{Manuscript received 21 April 1969)