Three-Dimensional Culture Model of Tumoral Cells-Juniper Publishers
Juniper Publishers- Journal of cell science
The mammospheres are a multicellular tumor spheroid
model. These are a widely used culture system to mimic the
three-dimensionality of tumors. In this manuscript, we narrate the
developmentof a model using a murine mammary adenocarcinoma, which we
used to study tumor physiology and therapeutic applications. We test
three techniques published in previous works. The best condition was
used V-shaped 96-well plates with an agarose monolayer, with this we
were obtained compact and robust spheroids. After that, we studied the
interaction between tumoral cells and mesenchymal stem cells (MSC)
derived to human umbilical cord in three dimensional culture.
Additionally, for evaluate spheroid's structure by optical microscopy we
performed histological staining of the mammospheres with
hematoxylin-eosin. Moreover, we made immunehis to chemistry staining to
evaluate cell proliferation and VEGF expression index, with Brdu and
VEGF, respectively. We found that the presence of MSC could modify the
mammospheres structure. We can conclude that mamospheres can be perform
with more than one cell type similarly to what happens in a in vivo
grafted tumour and this can be used to study the tumour physiology and
therapeutic agents on solid tumors without the use of laboratory
animals.
Keywords: Mammospheres; Murine mammary mdenocarcinoma; Mesenchymal stem cells
Abbreviations: Brdu: Bromodeoxyuridine, MSC: Mesenchymal Stem Cells, VEGF: Vascular Endothelial Growth Factor
in vitro Solid tumors are formed by a mass of
cancer cells in direct contact with stromal cells (e.g., fibroblasts,
myofibroblasts, endothelial cells, pericytes, adipocytes and immune
cells) surrounded by a complex extracellular matrix (CEM). The
interaction of these cell populations influence the tumor growth, the
resistance to death, the invasiveness and metastasis. In addition, the
tumor growth and development needs adequate tissue perfusion to maintain
a high metabolic rate and prevent hypoxic zones [1-3].
The study in solid tumors traditionally is developed in two ways:
I. Model in vitro, a monolayer culture, which
is maintained in a controlled environment to study the effect produced
by modify a variable on cultured cells. The problem with this method is
that does not allow us to understand the behaviour in vivo, because it does not have of the interaction with the cells of the CEM.
II. Model in vivo, working with laboratory's
animals, it has a complex microenvironmental and can be manipulated, but
we can't control all cellular conditions. Further, this model is more
expensive and time-consuming than the first, and can require a large
number of animals [4].
In our cell culture laboratory, we have a cell line
called TN60, a murine mammary adenocarcinoma. These tumor cells (TC) are
undifferentiated and have fast growth [2].
With these cells, we created the spheroids or three-dimensional
cultures. These were called mammospheres and allow us to maintain good
control of the system and, simultaneously, generate spheroid structures
that resemble a solid tumor grafted into the animal [5,6].
How do you generate the mammospheres? We need to
growth the TC on non-adherent or poor adherent surface to promote the
interaction between cells and obtain cellular aggregates, and avoid a
conventional monolayer culture. We work with liquid overlay technique
(LOT) because it is simple and economical. We used V-shaped 96-wells
plates and non-adherent culture plates, using agarose as the
non-adherent substrate. [4,6-9]. The TC were incubated in DMEM medium supplemented with 10% FBS at 37 °C whit 5% CO2 for a week.
Cell aggregates began to grow 24 hours after
incubation at 37 °C, under the conditions with agarose-coated, on
96-wells plates
and non-adherent culture plates. These spheroid structures form a
necrotic and hypoxic core, surrounded by a layer of actively
proliferating cells, similar to that found in tumors grafted into mice.
We observed compact structures, which are quite resistant to mechanical
dissociation, i.e. by replacement of the culture medium, and in the
processes of fixation and paraffin embedding. When we compare the
cellular morphology of TC from the mammospheres or from the tumors, we
can see that they are very similar.
In the last years, mesenchymal stem cells (MSCs) have
been demonstrated to play important roles in tumor pathogenesis and,
for this reason, are the subject of intense investigation. We developed
mammospheres with the co-culture of TC and hUC-MSC to analyse the
interaction and investigate specific mechanisms existing among both kind
of cells [10-12].
The hUC-MSC would be mimicking the extracellular matrix. We chose to
work with culturing in V-shaped 96-wells plates with a monolayer of 1%
agarose on each well. We co-cultured 10.000 TC and different
concentrations of hUC-MSC, 1.000 and 5.000 cells, with culture medium
appropriate. These were incubated under the same conditions mentioned
before. The co- cultured of TC and hUC-MSC was successful, because it
was obtained cell grow as three-dimensional culture. We saw by optical
microscopy that these spheroids had a structure more compact and rounder
than mammospheres only composed of TC.
Spheroids have been manipulated to obtain
histological samples, they have been fixed, processed and analysed by
optical microscopy high-quality samples. Then, we have performed
immunostaining with selected antibodies to determine the index of DNA
synthesis of TC (with bromodeoxyuridine, Brdu, a marker of S phase cell
cycle) and the VEGF expression (with VEGF clone VG1 monoclonal of mice, a
marker of neovascularization) [2,13,14].
Based on the results, we can conclude that the
mammospheres culture under the condition of V-shaped 96-well plates with
1% agarose are the most representative of tumoral model in vivo
because these spheroids form a necrotic and hypoxic core, surrounded by a
layer of actively proliferating cells, similar to that found in tumors
grafted into mice. The mammospheres can be perform with more than one
cell type. This point is very interesting because we can include
microenvironment tumoral cells, to analyse effects or interaction
between them and TC, mimicking to in vivo model and far away the monolayer culture.
Understanding the mechanisms through which TC, hUC-
MSC and the tumoral microenvironment are interacting, it could reveal a
new target for cancer treatment and control, such as antiangiogenic
therapy, using this three-dimensional culture technique, which is
cheaper, require less work-time and without the use of laboratory
animals. The development of three- dimensional culture will contribute
to study the tumor physiology and test effects of various therapeutic
agents on solid tumors.
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