The noncontact cocultivation system was developed for the study of the paracrine interactions between MCF-7 (breast carcinoma cells) and MT-4 (a line of human T-cell leukemia). Viability and proliferation rates were determined in the adhesion and suspension fractions of MCF-7 cells sampled from two model systems: monolayer culture and multicellular tumor spheroids (MTS). Cocultivation with MT-4 reduced the number of MCF-7 cells in the adhesion fraction and had no effect upon the suspension fraction, despite an increase in the total population of MCF-7 cells. The two model systems displayed a substantial difference in cell viability, alone and in the presence of MT-4 cells – the fraction of viable cells in the monolayers was greater than in the spheroids. It is suggested that cocultivation with MT-4 stimulates proliferation of MCF-7 cells via a paracrine mechanism, reduces adhesion to the substrate, and leads to MTS formation.

1. ISSN 0095-4527, Cytology and Genetics, 2009, Vol. 43, No. 5, pp. 305–310. © Allerton Press, Inc., 2009.
Original Russian Text © L.V. Garmanchuk, E.M. Perepelitsina, M.V. Sidorenko, L.I. Ostapchenko, 2009, published in Tsitologiya i Genetika, 2009, Vol. 43, No. 5, pp. 14–21.
305
INTRODUCTION
Multicellular tumor spheroids (MTS) represent an
intermediate complexity level among in vitro model
systems, between monolayer and organ cultures [1].
Cell interactions inside a tumor population, cell junc-
tions, influence of the tumor microenvironment, as well
as cytology and morphology of the tumor nodules have
been studied based on this model [1–5]. It is known that
a tumor represents a heterogeneous micropopulation
containing, apart from tumor cells, a dynamic combina-
tion of endothelial cells, fibroblasts, perivascular, and
immunocompetent cells (macrophages, lymphocytes,
and dendritic cells [6]). The presence or predominance
of one or another cell type may allow us to predict
tumor behavior [7]. All cellular elements of the
micropopulation are involved in constant interaction
and mutual influence. One of the mechanisms of such
mutual influence is the paracrine pathway involving a
network of cyto/chemokines that can be produced by
the cells of the microenvironment [8]. For instance, it
has been demonstrated that tumor-associated macroph-
ages (TAM), evolving from peripheral blood mono-
cytes, stimulate invasive activity of the tumor cells via
systems of chemokines and matrix metalloproteinases
[9]. Simultaneously, TAMs inhibit lymphocyte activity
at the site of the tumor by producing immune-suppress-
ing cytokines, such as IL-10 [9, 10].
In the present work, multicellular tumor spheroids and
monolayer culture are used to compare reactivity
(response to external effects) of these model systems in the
context of cocultivation of tumor cells and immunocom-
petent cells, lymphocytes. Human T-leukemia cells were
used for the cocultivation, because they produce a whole
range of cytokines, including the CXCL12-CXCR4 sys-
tem, which strongly influences primary tumor growth and
metastasis of the breast carcinoma [11]. Thus, it was
shown that an interaction between a receptor CXCR4 and
its ligand CXCL12 plays a key role in the chemotaxis and
migration of tumor cells to specific metastatic sites
[12, 13]. In addition, the ligand CXCL12 has been
detected in peripheral blood T-lymphocytes, monocytes,
subpopulations of killer-cells, dendritic cells, and endot-
helial cells [14–18].
The first major characteristic we studied was the
proliferative activity of human breast carcinoma cells
of the MCF-7 line in spheroid and monolayer culture
models in the presence of MT-4 cells. Cell number in
adhesion and suspension fractions was chosen as the
second characteristic for investigation, based on the
fact that adhesion, or substrate dependence, of neoplas-
tic cells correlate with the rate of tumor progression
towards invasion and metastasis.
MATERIALS AND METHODS
Cell lines. The adhesive cell line MCF-7 of human
breast adenocarcinoma, kindly provided by Dr. Gut
(Ludwig National Cancer Institute, London) was used as
an experimental model. These cells have an epithelial-
like morphology, a karyotype of 2n = 46 with 0.6 percent
polyploidy. Oncogenicity has been confirmed in nude
mice [19]. In MCF-7 cells, receptors to estrogens are
present, estradiol is synthesized. For the cocultivation,
Growth Kinetics of 2- and 3-D Cell Models as Influenced
by the Microenvironment
L. V. Garmanchuka, E. M. Perepelitsinab, M. V. Sidorenkob, and L. I. Ostapchenkoa
aBiological Faculty, Taras Shevchenko National University of Kiev
bDepartment of Biotechnical Problems of Diagnostic, Institute for Problems of Cryobiology and Cryomedicine,
National Academy of Sciences, Ukraine
e-mail: olenaquail@rambler.ru
Received September 08, 2008
Abstract—The noncontact cocultivation system was developed for the study of the paracrine interactions
between MCF-7 (breast carcinoma cells) and MT-4 (a line of human T-cell leukemia). Viability and prolifera-
tion rates were determined in the adhesion and suspension fractions of MCF-7 cells sampled from two model
systems: monolayer culture and multicellular tumor spheroids (MTS). Cocultivation with MT-4 reduced the
number of MCF-7 cells in the adhesion fraction and had no effect upon the suspension fraction, despite an
increase in the total population of MCF-7 cells. The two model systems displayed a substantial difference in
cell viability, alone and in the presence of MT-4 cells – the fraction of viable cells in the monolayers was greater
than in the spheroids. It is suggested that cocultivation with MT-4 stimulates proliferation of MCF-7 cells via
a paracrine mechanism, reduces adhesion to the substrate, and leads to MTS formation.
DOI: 10.3103/S0095452709050028

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GARMANCHUK et al.
we used line MT4 (human T-leukemia cells), provided
by the National Bank of Cell Cultures, IEPOR (Kiev).
MCF-7 cells were cultured in a full medium DMEM
(Sigma, USA) containing 10% FBS (Fetal bovine serum)
(Sigma, USA), 2 mM L-glutamine (Sigma, USA), and 40
µg/ml of gentamicin sulphate (Biopharma, Ukraine)
under standard conditions at 37°C, 5% CO2, 100% humid-
ity. Culture density was 2.0–4.0 × 104 cells/cm2. The
medium was changed every other day, after the cells
reached confluency. MT-4 suspension culture was grown
at 1 mln/ml on an RPMI medium (Sigma, USA) and in the
presence of 10% FBS (Fetal bovine serum) under standard
conditions. The medium was changed at 2–3day intervals.
Generation of spheroids. For the generation of sphe-
roids, we used an addition of an anti-adhesion agent car-
boxymethylcellulose (Sigma, USA) to MCF-7 cells, as
described in [20, 21]. For this purpose, the cells, after
formation of a tight monolayer, were disengaged from
culture plates using Versene solution (0.025 M EDTA)
and 0. 25% trypsin solution (Sigma, USA). After that,
the cells were seeded on plates of two types—with low
and high adhesive properties (Nunc, Denmark)—at 2.5 ×
104 cells/ml, followed by addition of carboxymethylcel-
lulose solution to a final concentration of 0.24% [20].
The plates were incubated for 1 h with low-speed rota-
tion under standard conditions prior to a 6-hour adapta-
tion period. For the monolayer culture, cells were also
seeded on two types of surfaces and incubated for 1 h
with mild stirring (this time no CM-cellulose was added
to the medium), followed by a 6-hour adaptation period.
Cocultivation. For the cocultivation, 5.0–9.0 × 105
of MT-4 cells, that had been seeded into diffusion
chambers with nitrocellulose filters (pore diameter
0.22 µm), were added to the obtained spheroid and
monolayer MCF-7 cultures (12 ± 1 × 104 cells) after the
adaptation period (the addition was the starting point of
coculturing, or the zero point). Cocultivation periods
were 1 and 2 days. Upon termination of the cocultiva-
tion periods, the cells located on the substrate (the
adhesion fraction) and in suspension (the suspension
fraction) were sampled separately for counting.
Experimental design. The viability, proliferative
activity, and adhesive properties of MCF-7 cells were
estimated in two steps using spheroid and monolayer
culture models. In the first stage, cells were sampled
from the suspension fraction for the monolayer culture
analysis; and for the spheroid culture, a suspension
fraction was used, which had been thermostated for
2 min for spontaneous sedimentation. Then, two thirds
of the sample volume and the supernatant were centri-
fuged for 2 min at 200 g. This approach allowed us to
separate the cell suspension into three subfractions:
spheroids comprising more than 50 cells, those contain-
ing less than 50 cells, and single cells. The total cell
number and the numbers of dead and viable cells were
counted in each subfraction using trypan blue.
At the second stage the cells, attached to the plate
surface, (the adhesion fraction) were removed using a
trypsin solution, also followed by counting the dead
and living cell fractions. MCF-7 cells, grown as spher-
oid and monolayer cultures under standard conditions
with no MT-4 cells added, were used as a control. The
counts were done according to the same scheme as
described above. The specified experimental design
allowed us to estimate the influence of MT-4-produced
humoral factors upon the following MCF-7 parameters:
survival potential, proliferative activity, and substrate
dependence (adhesive properties), based on the models
of spheroid and monolayer growth. The procedures
were performed using plates with high and low adhe-
sion characteristics.
Statistical data analysis was performed using the
Student’s t-test; the graph was plotted with an applica-
tion package Origin 7.5; the diagrams were created
using Microsoft Excel.
RESULTS OF THE INVESTIGATION
AND DISCUSSION
The results of our investigation showed that the pro-
liferative activity, survival potential, and adhesive prop-
erties of MCF-7 in vitro, in spheriod and monolayer
growth systems, with and without the influence of
humoral factors, depend on the type of model system,
substrate characteristics, and the time of incubation.
An estimate of the MCF-7 proliferative activity in
different model systems, as influenced by paracrine
factors produced by MT-4, is given in Fig. 1 and 2. The
number of MCF-7 cells in a monolayer, in the presence
of T-lymphocytes of the line MT-4, increases by 13–
25% (p < 0.05), when cultured on both types of sub-
strate (plates with low and high adhesive properties).
By contrast, the cell number in a spheroid MCF-7 cul-
ture, when cocultured with MT-4, decreases by an aver-
age of 17 ± 4% (p < 0.05), as compared to the analo-
gous control. It is also worth mentioning that the overall
number of cells growing as spheroids and in the mono-
layer, in the control cultures (with no cocultivation),
changes exponentially with the two culture types differ-
ing only in proliferation rates (Fig. 1 and 2).
Another important observation was that on the first
day of cultivation on a low-adhesion substrate in the
presence of MT-4 no reliable differences were detected
in the proliferation rates between the monolayer and the
spheroids, whereas on the second day the cell number in
the monolayer exceeded that in the spheroids by 24%.
Meanwhile, on high-adhesion plates, the cell number in
the spheroid culture model was 27 percent lower than
that in the monolayer after 24 h of culturing, and 19 per-
cent lower by the second day of culturing (p < 0.05)
(Fig. 2). Thus, we showed that MCF-7 reactivity in
spheroid and monolayer cultures, under the influence of
humoral factors produced by MT-4 cells, exhibits an
inverse correlation, as compared to the corresponding
control: the cell number’s parameter increases in mono-
layer and decreases in MTSs.

3. CYTOLOGY AND GENETICS Vol. 43 No. 5 2009
GROWTH KINETICS OF 2- AND 3-D CELL MODELS 307
Results of the investigation of the adhesion ability of
cells in two model systems alone and under the influ-
ence of the external factors provided by MT-4, are pre-
sented in Fig. 3 and 4.
Substrate dependence, or adhesive properties of
cells, were the second key characteristic that we exam-
ined in spheroid and monolayer growth models cocul-
tured with MT-4. As is evident from the above data, the
cell number in the suspension and adhesion fractions of
the control and trial cultures (indirect cocultivation
with MT4 cells) depended upon three parameters.
The first parameter is the type of substrate (Figs. 3
and 4). For the monolayer culture grown on low-adhe-
sion substrate, the suspension fraction was virtually
equal to the adhesion fraction in control on the first day
of incubation (Fig. 3a) (8 ± 0.2 × 104 cells, as compared
to 6.7 ± 0.6 × 104 cells, respectively). Humoral factors
produced by MT-4 cells effected a 2.5-fold increase in
the suspension fraction as compared to the adhesion frac-
tion (p < 0.05) (Fig. 3a). By the second day of incubation,
the redistribution in the cell population between suspen-
sion and adhesion fractions was expressed in values of
the same magnitude both for control and trial cultures
(Fig. 3a); i.e., both in control and trial cultures, the suspen-
sion culture’s fraction was considerably (4.4-fold in the
control and 2.2-fold in the presence of MT-4, p < 0.01)
larger than the adhesion fraction (Fig. 3a). When plates
with high adhesive properties were employed for the
monolayer growth, no similar effect was observed either
in the control or in the presence of MT-4 (Fig. 4a). With
regard to MCF-7, in the case of the spheroid culture
model (Figs. 3b and 4b), cultivation on plates with low
adhesive properties resulted in virtually equal cell num-
bers in suspension and adhesion fractions after 1 day of
culturing (Fig. 3b), with a prevalence of the suspension
cell’s fraction, both in the control and in the presence of
MT-4. Notably, by day 2, the control culture exhibited a
significant increase in the adhesion cell’s fraction (18 ±
1.1 × 104 cells versus 7.8 ± 0.8 × 104 in the suspension
fraction). In the presence of the humoral factors, pro-
duced by MT-4 cells, the cell number in both fractions is
virtually equal (Fig. 3b). On the high-adhesion substrate,
for all MCF-7 cultures, the spheroid model retains a con-
stant cell distribution – more in the suspension than in the
adhesion fraction (Fig. 4b).
The second parameter that also influences the distri-
bution of MCF-7 cells between adhesion and suspension
fractions in spheroid and monolayer growth systems is
the time of cocultivation. In the presence of humoral fac-
tors, produced by MT-4 cells, a more pronounced redis-
tribution of the cell fractions was observed by day 2, as
compared to the control (Figs. 3 and 4).
The third parameter was the cell number in both
fractions that depended on the model type used. More
specifically, in the presence of MT-4 cells, the MCF-7
monolayer culture model exhibited an increase in the
total cell number, on both types of substrate (with the
exception of a cocultivation model after 1 day of
growth on a substrate with low adhesive properties). A
drop in the total cell number was observed in the
300
280
260
240
220
200
180
160
140
120
100
zero point 24 h 48 h
1
2
3
4
Fig. 2. Proliferation of MCF-7 cells in monolayer and
spheroid growth models on high- and low-adhesion sub-
strates without MT-4: vertically – 103 cells.
300
280
260
240
220
200
180
160
140
120
100
zero point 24 h 48 h
1
2
3
4
Fig. 1. MCF-7 proliferation in monolayer and spheroid
growth models on high- and low-adhesion substrates in the
presence of MT-4: vertically – 103 cells. Here and subse-
quently in Fig. 2: 1—low-adhesion monolayer; 2—high-
adhesion monolayer; 3—low-adhesion spheroids; 4—high-
adhesion spheroids.

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GARMANCHUK et al.
MCF-7 spheroid growth system in the presence of
MT-4 (Figs. 3b and 4b). It is worth mentioning that,
when the cultivation was performed on a low-adhesion
substrate, a prevalence of the suspension over the adhe-
sion fraction was observed in the monolayer culture,
both in the control and trial cultures (i.e., in the pres-
ence of MT-4 cells). Conversely, in a spheroid MCF-7
culture model, the adhesion fraction dominated over
the suspension fraction by day 2, both in the control and
trial cultures. As for the monolayer culture, the situa-
tion is clear: the cells diffuse into the suspension frac-
tion due to low affinity to the substrate. In the case of
the spheroid culture model, we assume that the
KM-cellulose used for the generation of spheroids may
play the role of a substrate modifier for the low-adhe-
sion substrate; otherwise, a different mechanism may
be involved.
The examination of the suspension fraction from
spheroids cultured on both types of substrate revealed
that the presence of MT-4 has a significant effect upon
the size of the spheroids (cells per spheroid) and their
number, as well as on the percent ratio of spheroids to
single cells (see the table).
As is evident from the data represented, for the cul-
ture grown on a low adhesion substrate, without MT-4,
the fraction of single cells in the suspension made up
18–25% of the culture population, while in the pres-
ence of MT-4 cells this parameter increased to 50–62%.
On the high-adhesion substrate, the same tendency was
observed—single cells comprised 15–20% of the total
cell number in the control and 30–40% in the cocultiva-
tion system.
Thus, we discovered that, in MCF-7 spheroid
growth systems, when cocultured with MT-4, the num-
ber of spheroids in the suspension declines with a pro-
portionate accumulation of single cells.
Probably, these cells possess a high metastatic
potential, and therefore, extravasate from spheroids. In
[12], Muller et al., showed that chemokines produced
by MT-4 cells increase the metastatic potential of the
cells of human breast carcinoma. In our case, the
extravasation of single MCF-7 cells from spheroids
may indicate a population shift in neoplastic cells and
an increase in the fraction of metastatically active cells.
Obviously, under in vivo conditions, the cytokines pro-
250
200
150
100
50
0
24 h 48 h 48 h24 h
control with MT4
24 h 48 h 48 h24 h
control with MT4
107
54
97
148
90
48
121
75
(a)
35
137
39
23
167
54
179
146
(b)
Fig. 4. Redistribution in the suspension ( ) and adhesion ( ) fractions of MCF-7 grown on high-adhesion plates: monolayer
growth (a); spheroid growth (b); vertically – 103 cells.
300
250
200
150
100
50
0
24 h 48 h 48 h24 h
control with MT4
24 h 48 h 48 h24 h
control with MT4
80
67
220
50
125
50
80
175
115
60
78
180
125
45
106
120
(b)(a)
Fig. 3. Redistribution in the suspension ( ) and adhesion ( ) fractions of MCF-7 grown on a low-adhesion substrate: monolayer
growth (a); spheroid growth (b); vertically – 103 cells.

5. CYTOLOGY AND GENETICS Vol. 43 No. 5 2009
GROWTH KINETICS OF 2- AND 3-D CELL MODELS 309
duced by T-lymphocytes may promote an expansion of
the pool of metastatic cells, as shown in [12]. Another
possible mechanism of the humoral influence of MT-4
on cells in spheroids when cultured on a substrate with
low adhesive properties may consist in enhancement of
their substrate dependence (development of cell popu-
lation towards prodifferentiation). It may be suggested
that the influence of MT-4 on MCF-7 microconglomer-
ates is mediated by a modulating action upon the sur-
face receptors responsible for the formation of adhe-
sion loci by metastatically active cells.
Viability measurements in spheroid and monolayer
culture models showed that dead cells comprised 8–
12% of the control culture’s population (with the
exception of the monolayer MCF-7 culture incubated
on plates with low adhesive properties, in which the
number of the dead cells reached 37% of the suspension
fraction and 21% of the adhesion fraction). The median
of MCF-7 viability in the suspension fraction, in the
presence of MT-4, is 94 ± 4%, whereas in the mono-
layer this parameter is estimated at 71 ± 3%. Accord-
ingly, the viability of cells in the monolayer culture was
significantly lower than that in spheroids. We demon-
strated that MT-4-secreted humoral factors stimulate
cell proliferation in the suspension fraction of a spher-
oid culture and in the adhesion fraction of a monolayer,
although the total cell number in the MCF-7–MT-4
spheroid coculture decreases as compared to the mono-
layer or the corresponding control. This pattern is
attributable to reduced proliferation of single cells in
the suspension due to changes in the phenotype of
actively proliferating cells (metastatically active cells
or cells with a marked substrate dependence).
In conclusion, the monolayer and spheroid cultures
display the following characteristic features in response
to humoral factors produced by MT-4 cells in noncon-
tact cocultivation models:
(1) in the monolayer MCF-7 culture, proliferative
activity in the adhesion fraction, cell migration into the
suspension, and the decline in proliferation are
enhanced;
(2) in the spheroid culture, an increase is observed
in the following parameters: the proliferative activity in
the suspension fraction, the number of slowly prolifer-
ating single cells, and the percentage of viable cells.
CONCLUSION
The mixed culturing of cells from a neoplastic line
MCF-7 and a human T-leukemia line MT-4 revealed
that reactivity of the cells in response to humoral agents
depended on the culture model (monolayer or sphe-
roids), the culture fraction (adhesion or suspension),
adhesive properties of the substrate, and the time of cul-
ture. The proliferative activity of the MCF-7 cells
grown as a monolayer without MT-4 is lower than in
spheroids, while cocultivation with MT-4 stimulates
cell proliferation in the monolayer and inhibits it in
MTSs. The cell distribution between suspension and
adhesion fractions of MCF-7 depends primarily upon
the type of substrate (plates with high and low adhesive
properties). The cytokines, secreted into the culture
medium by MT-4, stimulate proliferation of the adhe-
sion cell’s fraction of the monolayer and extravasation
of cells into the suspension (a prototype of the metasta-
sis). Notably, the most marked cell redistribution
between the suspension and the adhesion fractions was
detected on the second day of culturing. Cell viability
(adhesion fraction of the monolayer and suspension
fraction of the spheroids) is reliably increased in the
presence of MT-4. Thus, the comparison of two model
systems in vitro (spheroid and monolayer MCF-7 cul-
tures), in the presence of microenvironmental factors
produced by T-lymphocytes of the line MT-4 indicated
a stimulation of the neoplastic process on the key
stages.
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