1. Introduction
Three-dimensional cell culture models
such as multicellular spheroids and organoids have been demonstrated to
mimic several biological processes in vitro much better than
monolayer cell cultures (Cui, Hartanto, &
Zhang, 2017). There are essentially two methods to produce uniform-size
multicellular spheroids: scaffold free, in which the cells are prevented
from adhering to substrates but not to each other thus forming
spheroids, and scaffold- or matrix-based, in which cells are embedded
into a three-dimensional (3D) biomaterial such as a hydrogel. The
latter, usually composed of polymers, has the advantage to provide a
physical structure comparable, in terms of stiffness and
viscoelasticity, to the extracellular matrix (ECM) in vivo and it
is thus able to reproduce not only cell-cell interactions, but also
cell-matrix interplays directly related to phenomena associated with
cell growth (cell fate) such as cytoskeletal organization, gene
expression, nutrients diffusion, pH and hypoxia
(Singh, Brito, & Lammerding, 2018). This
makes matrix-based spheroids increasingly important for studying solid
tumors microenvironment and the response to drug treatments
(Nath & Devi, 2016).
Among several 3D biomaterials, hydrogels of complex biological origin
have been used in many studies. The most common are
MatrigelTM, derived from Engelbreth-Holm-Swarm mouse
tumor sarcoma, and other basement membrane-rich matrices. These
hydrogels contain matrix membrane proteins, hormones and soluble growth
factors whose composition may vary among different batches. This aspect
limits their use because, although they allow for the growth in three
dimensions, the batch-to-batch variability could potentially alter cell
culture systems and experimental results. Thus, other simple hydrogels,
without hormones and growth factors, able to maximize reproducibility
and offering the possibility to tune biochemical as well as mechanical
properties, have been considered to grow spheroids. Hydrogels with these
features are made of natural, synthetic or hybrid materials, such as
alginate, agarose, collagen, hyaluronic acid and polyethylene glycol
(Cui et al., 2017;
Teruki, Kimiko, & Yasuhiko, 2020).
Among them, agarose is an inert, inexpensive, and easily available
polysaccharide derived from red marine algae and consisting of repeated
units of b-1,3-linked-D-galactose and a-1,4-linked
3,6-anhydro-L-galactose. It possesses excellent biocompatibility,
optimal gelling features and tunable mechanical properties that boosted
its use as biomaterial for the manufacturing of tissue engineering
scaffolds (Anderson & Johnstone, 2017;
López-Marcial et al., 2018). In addition,
thanks to the ease of preparation, it has been recently exploited as
non-adhesive and micromolded substrate for the growth of tumor spheroids
based on multicellular aggregation
(Napolitano et al., 2007;
Tang, Liu, & Chen, 2016). Nevertheless,
due to its poor bioadhesivity, as it lacks cell adhesion motifs, its use
as ECM-mimicking material is very limited.
On the other hand, type I collagen is the main protein component of the
ECM in mammals. The presence of cellular binding sites (i.e. the
“GxOGER” sequence, where “R” is arginine, “G” is glycine, “D” in
aspartate, “O” is hydroxyproline, “E” is glutamate and “x” is a
hydrophobic amino acid) that promote cell adhesion and proliferation,
and regulate cell signaling pathways, makes collagen highly bioactive
and suitable for the development of natural material-based hydrogels for
cell culture studies (Davidenko et al.,
2018; Davidenko et al., 2016;
Hamaia & Farndale, 2014;
Tibbitt & Anseth, 2009). In addition, it
plays a crucial role in tumor progression and invasion
(Li & Kumacheva, 2018). It can form
hydrogels through self-aggregation of the fibers or by physical and
chemical crosslinking, but is suffers of poor mechanical properties,
limited stability over time and high costs
(Caliari & Burdick, 2016).
In this sense, blended hydrogels composed of agarose and collagen
combine the mechanical properties of agarose and the biomimetic nature
of collagen (Ulrich, Jain, Tanner, MacKay,
& Kumar, 2010). As far as we know, only one study described the use of
hydrogels containing agarose and collagen as matrices for the formation
of tumor spheroids. In detail, composite hydrogels made of alginate,
marine collagen, and agarose were prepared and the growth and viability
of spheroids derived from ovarian cancer and lymphoma cell lines were
monitored up to 14 days (Shin et al.,
2016). Notably, twoother studies described the use of agarose-collagen
blend hydrogels that were not used to promote the growth of spheroids,
but to unravel the effects of biophysical cues on cellular
mechanobiology of 2D intervertebral disc
cells(Cambria et al., 2020) and
glioblastoma spheroids that had been previously prepared by the hanging
drop method and then loaded into the hydrogel
(Ulrich et al., 2010). Both works
evidence the crucial role of the hydrogel stiffness and adhesivity as
driving forces that modulate the cell plasticity and connect the
biological functionality to the surrounding physical stimuli. Thus, the
interplay between the hydrogel and the cells embedded within it depends
on what the cells sense and how they reply to the host environment. In
living tissues, the stiffness spans from few tens Pa in intestinal mucus
to GPa of bones, and variations of the mechanical properties are
typically associated to diseases and cancer development
(Guimarães, Gasperini, Marques, & Reis,
2020). In the case of breast, it has been shown that the stiffness
increases from hundreds Pa to few kPa when the normal tissue undergoes
to tumor transformation, due to the remodeling of the ECM
(Butcher, Alliston, & Weaver, 2009).
Therefore, artificial matrices resembling the physical properties of the
naïve tumor environment may facilitate the study of 3D tumors behavior
in vitro and predict their response to modifications of the mechanical
cues or to drug treatments, as it occurs during cancer progression and
metastatization (Singh et al., 2018).
In this work we have developed agarose-collagen blended hydrogels with
variable agarose amount (from 0.5 to 0.125%) and analyzed the growth of
breast tumor spheroids of three different breast cancer cell lines: the
luminal estrogen receptor positive cells, MCF-7 and MDA-MB-361, and the
triple negative model, MDA-MB-231, for comparative analysis.
The preparation method is simple, quick and highly reproducible. By
embedding individual cells into the liquid mixture of agarose and
collagen, prior to the gelation, allows to follow up the growth of the
3D tumoroids over time. The physical properties of the matrices have
been thoroughly investigated and the characteristics of the spheroids’
growth were related to the hydrogel features. Upon variation of the
agarose percentage, the physical and mechanical properties of the
matrices can be tuned, and in turn, the growth kinetic and the
morphology of the spheroids also vary, confirming their sensitivity to
the environment stiffness. Noteworthy, the optical transparency of
agarose facilitates the daily analysis of spheroids that result more
compact in stiffer hydrogels, while they look loosened with cellular
protrusions and disseminated cells in softest matrices. Remarkably,
tumor spheroids can be recovered upon enzymatic treatment of the
hydrogels with agarase, with a yield of 100% in the case of the
matrices containing the lower amount of agarose (equal to 0.125%).
Preliminary drug testing studies with cisplatin evidenced a cytotoxic
response that depends on the agarose percentage concentration. Overall,
the results show that agarose-collagen blended hydrogels are suitable
modular matrices for tumor spheroids culturing and for evaluating drug
protocols efficacy.