Pharmaceutics 13 00963 v2

pharmaceutics
Article
Investigation on the Composition of Agarose–Collagen I
Blended Hydrogels as Matrices for the Growth of Spheroids
from Breast Cancer Cell Lines
Alessandra Quarta 1, * , Nunzia Gallo 2 , Daniele Vergara 3 , Luca Salvatore 2 , Concetta Nobile 1 ,
Andrea Ragusa 1,3 and Antonio Gaballo 1, *
1 CNR Nanotec, Institute of Nanotechnology, via Monteroni, 73100 Lecce, Italy;

[email protected] (C.N.); [email protected] (A.R.)
2 Department of Engineering for Innovation, University of Salento, via Monteroni, 73100 Lecce, Italy;
[email protected] (N.G.); [email protected] (L.S.)
3 Department of Biological and Environmental Sciences and Technologies, University of Salento, via Monteroni,
73100 Lecce, Italy; [email protected]
* Correspondence: [email protected] (A.Q.); [email protected] (A.G.)
Abstract: Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue
microenvironment and are gaining increasing importance as they resemble the extracellular matrix
(ECM)–cell and cell–cell physical interactions occurring in vivo. Several scaffold-based culture
systems have been already proposed as valuable tools for large-scale production of spheroids, but

they often suffer of poor reproducibility or high costs of production. In this work, we present a
Citation: Quarta, A.; Gallo, N.;
reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation
Vergara, D.; Salvatore, L.; Nobile, C.; in the agarose percentage affects the physical and mechanical properties of the resulting hydrogel.
Ragusa, A.; Gaballo, A. Investigation The influence of the different physical and mechanical properties of the blended hydrogels on the
on the Composition of growth, size, morphology, and cell motility of the spheroids obtained by culturing three different
Agarose–Collagen I Blended breast cancer cell lines (MCF-7, MDA-MB-361, and MDA-MB-231) was also evaluated. As proof of
Hydrogels as Matrices for the Growth concept, the cisplatin penetration and its cytotoxic effect on the tumor spheroids as function of the
of Spheroids from Breast Cancer Cell hydrogel stiffness were also investigated. Noteworthily, the possibility to recover the spheroids from
Lines. Pharmaceutics 2021, 13, 963. the hydrogels for further processing and other biological studies has been considered. This feature,
https://doi.org/10.3390/
in addition to the ease of preparation, the lack of cross-linking chemistry and the high reproducibility,
pharmaceutics13070963
makes this hydrogel a reliable biomimetic matrix for the growth of 3D cell structures.
Academic Editor: Hassan Bousbaa

Keywords: spheroids; tumoroids; hydrogel; collagen; agarose; mammary spheroids; tissue engineer-
ing; breast cancer; cisplatin; cancer therapy
Received: 19 May 2021
Accepted: 23 June 2021
Published: 26 June 2021
Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in Three-dimensional (3D) cell culture models, such as multicellular spheroids and
published maps and institutional affil- organoids, have been demonstrated to mimic several biological processes in vitro much
iations. better than monolayer cell cultures [1,2]. There are essentially two methods to produce
uniform-size multicellular spheroids: scaffold free, in which cells are prevented from
adhering to the substrate but not to each other, thus forming spheroids, and scaffold- or
matrix-based, in which cells are embedded into a three-dimensional biomaterial, such as a
Copyright: © 2021 by the authors. hydrogel [3]. The latter has the advantage of providing a physical structure comparable,
Licensee MDPI, Basel, Switzerland. in terms of stiffness and viscoelasticity, to the in vivo extracellular matrix (ECM), thus
This article is an open access article being able to reproduce not only cell–cell interactions, but also cell–matrix interplays
distributed under the terms and directly related to phenomena associated with cell growth (cell fate), such as cytoskeletal
conditions of the Creative Commons organization, gene expression, nutrients diffusion, pH, and hypoxia [4]. This makes matrix-
Attribution (CC BY) license (https:// based spheroids extremely important for studying solid tumors microenvironment and
creativecommons.org/licenses/by/ their response to drug treatments [5].
4.0/).
Pharmaceutics 2021, 13, 963. https://doi.org/10.3390/pharmaceutics13070963 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 963 2 of 22
Among 3D biomaterials, hydrogels of complex biological origin have been used in

many studies [6–8]. The most common are Matrigel™, derived from Engelbreth-Holm-
Swarm mouse tumor sarcoma, and other basement membrane-rich matrices. These hy-
drogels contain matrix membrane proteins, hormones, and soluble growth factors whose
composition may vary among different batches. This aspect limits severely their use
because, although they allow for the growth of spheroids, the batch-to-batch variability
can alter cell culture systems and experimental results. Thus, simpler 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 for growing
spheroids. Hydrogels with these features are made of natural, synthetic, or hybrid materi-
als, such as alginate, agarose, collagen, hyaluronic acid, and polyethylene glycol [1,9].
Among them, agarose is an inert, inexpensive, and easily available linear polysaccha-
ride derived from red marine algae and consisting of alternating units of D-galactose and
3,6-anhydro-α-L-galactopyranose joined by α-1,3- and β-1,4-linkages. It possesses excellent
biocompatibility, optimal gelling features, and tuneable mechanical properties that boosted
its use as biomaterial for the manufacturing of tissue engineering scaffolds [10,11]. 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 [12,13]. Nevertheless, due to its poor bioadhesivity, 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 mam-
mals. The presence of cellular binding sites (e.g., the “RGD” and “GxOGER” sequence,
where “R” is arginine, “G” is glycine, “D” is aspartate, “O” is hydroxyproline, “E” is
glutamate, and “x” is a hydrophobic amino acid) that promote cell adhesion, proliferation,
and signalling makes collagen highly bioactive and suitable for the development of biocom-
patible hydrogels [14–17]. In addition, collagen plays a crucial role in tumor progression
and invasion [18]. Nevertheless, it suffers of poor mechanical properties, limited stability
over time, and high costs [19]. In this sense, blended hydrogels composed of agarose and
collagen combine the mechanical properties of the former and the biomimetic nature of
the latter.
As far as we know, only one study described the use of hydrogels containing agarose
and collagen, as well as alginate, as matrices for producing tumor spheroids and their
viability was monitored up to 14 days [20]. In addition, two studies described the use of
agarose–collagen blended hydrogels to unravel the effects of biophysical cues on cellular
mechanobiology of 2D intervertebral disc cells [21] and previously prepared glioblastoma
spheroids [22]. Both works highlighted 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. In living tissues, the stiffness ranges
from few tens of Pa in intestinal mucus to GPa in bones, and variations of the mechanical
properties are typically associated with cancer development and other diseases [23]. In the
case of breast cancer, the stiffness has been shown to increase from hundreds of Pa to a few
kPa when the normal tissue undergoes tumor transformation, due to the remodeling of the
ECM [24].
Therefore, artificial matrices mimicking the physical properties of the naïve tumor
environment can facilitate the study of the behaviour of 3D tumors in vitro and predict
their response to modifications of the mechanical cues or to drug treatments, as it occurs
during cancer progression and metastasis [4].
In this work, we developed collagen hydrogels blended with variable amounts of
agarose (from 0.5% to 0.125%) and we investigated the possibility to grow within them
tumor spheroids of three different breast cancer cell lines, i.e., the luminal estrogen receptor
positive cells, MCF-7 and MDA-MB-361, and the triple negative model, MDA-MB-231.
The physical properties of the matrices were analyzed in detail and the morphological
characteristics of the spheroids were correlated over time to the hydrogel features. Prelim-
inary drug testing studies with cisplatin and the possibility to recover the spheroids for
additional studies were also evaluated.
2. Materials and Methods

Soluble type I collagen from calf skin was purchased from Symatese (Chaponost,
France). Agarose, β-agarase from Pseudomonas atlantica, FITC-labeled hyaluronic acid,
transferrin-TRITC, cisplatin, Dulbecco’s Modified Eagle Medium high glucose (DMEM),
fetal bovine serum (FBS), penicillin and streptomycin were purchased from Sigma–Aldrich
(Milan, Italy). Live/dead assay and MitoTracker Red CMXRos were purchased from
Thermo Fisher (Rodano, Italy). Ultrapure grade water with a conductivity of 18.2 MΩ cm
was used in all experiments. All chemicals were used as received.
2.1. Preparation of the Hydrogels

Agarose was dissolved in sterile phosphate-buffered saline (PBS, pH 7.4) to obtain a
1% (w/v) stock solution by heating on a hot plate. An aqueous suspension of 0.1% (w/v)
type I collagen was obtained by slowly hydrating collagen flakes in distilled water for
3 h, under magnetic stirring at 4 ◦ C. The blended hydrogels were prepared inside the
wells of a 24 well plate, and each well was loaded to a final volume of 1 mL. In detail,
the warm agarose solution (at around 45 ◦ C, the gel point is at 36 ◦ C) was added to the
collagen suspension to obtain 3 different final concentrations, 0.5%, 0.25%, and 0.125%.
The starting collagen solution was always diluted to 0.02% in the final blend. The mixture
in each well was gently stirred with a glass rod and allowed to gel at room temperature.
The gelation time varied from 30 s, in the case of hydrogels containing 0.5% agarose, up to
10 min, for those with 0.125% agarose. The gelation time was longer (up to 20 min) when
the cells were embedded into the hydrogel, as all the preparation steps were carried out
at 37 ◦ C. All the glassware was sterilized in an autoclave before use, and the preparation
of the hydrogels was carried out under a laminar flow hood. The preparation scheme is
summarized in Figure S1.
2.2. Characterization of the Hydrogels

2.2.1. Scanning Electron Microscopy (SEM)
Surface morphology and porosity of the hydrogels were investigated by using SEM
imaging techniques. SEM images were recorded with a Carl Zeiss–Merlin field emis-
sion scanning electron microscope (Carl Zeiss, Oberkochen, Germany) equipped with a
Gemini column and integrated with high efficiency in-lens and SE2 detectors, for high
spatial/depth-of-field resolution secondary electrons (SE) imaging of surface structure and
topography. The microscope was used in high vacuum and high-resolution acquisition
mode and the images were recorded at an accelerating voltage of 5 kV and a few seconds
frame-integration time, in order to minimize charging effects and sample damage. The
hydrogels were cut, frozen, and lyophilized before imaging. The pore size was determined
by measuring 50 random pores followed by statistical analysis using ImageJ software (NIH,
Bethesda, MD, USA).
2.2.2. Stability Test

The analysis was carried out keeping the hydrogels at 37 ◦ C either in PBS or DMEM,
and the samples were weighted at determined time points (up to 14 days). The stability of
the hydrogels was then calculated as residual weight percentage:
w − w0
Rw% = × 100%
w0
where w0 is the starting weight of the hydrogel at t = 0, and w is the weight at each
time point.
2.2.3. Swelling Behavior

The swelling property of the hydrogels was determined by using a conventional
gravimetric method [25]. Briefly, the dry weight of hydrogels was recorded before soaking
them in PBS at 37 ◦ C. The swelled weight of hydrogels was then measured at various time
points, up to 48 h. The swelling behaviour was estimated as the percentage of the swelling
ratio using the following equation:
ws − wd
Sr% = × 100%
wd
where wd is the dry weight of the hydrogel and ws is the wet weight after hydration in PBS.
2.2.4. Collagen Release

The agarose–collagen (A-C) hydrogels were kept in PBS at 37 ◦ C and the volume of
buffer (equal to 1 mL) was collected at determined time intervals to estimate the protein
content via bicinchoninic acid (BCA) assay [26]. A calibration curve was set using collagen
solutions at known concentrations. For each time interval, a blank sample (i.e., the PBS
collected from agarose hydrogels prepared without collagen) was also measured.
2.2.5. Fourier-Transform Infrared (FTIR) Spectroscopy

FT-IR spectra were recorded in transmittance mode on a Jasco 6300 spectrometer
(Jasco Corp., Tokyo, Japan) between 4000 and 500 cm−1 with 40 scans and a resolution of
4 cm−1 and analyzed with the Spectra Manager software (Jasco). The measurements were
performed by directly depositing the hydrogels onto the ATR crystal. The spectrum of each
sample was acquired against a background obtained with the crystal without any sample.
All analyses were carried out at room temperature.
2.2.6. Mechanical Compression Test

The effect of the agarose concentration on the Young’s modulus of the A-C hydrogels
was evaluated by uniaxial compression test. The correlation with time and temperature
was also investigated. Briefly, the samples were incubated in PBS at 37 ◦ C in a humidified
atmosphere with 5% CO2 . Cylinders of 8 mm were punched out and loaded into the
testing chamber at fixed time points (0, 1, 4, and 8 days). All tests were performed with a
universal ZwickiLine (Zwick Roell, Ulm, Germany) testing machine fitted with 10 N load
cell. Loaded samples were hydrated in PBS at 37 ◦ C and subjected to compression with a
displacement rate of 0.01 mm/s, until 80% strain [27,28]. The compressive modulus was
calculated by linear fitting between 2% and 10% of strain of the stress-strain curve. The test
was performed in triplicate for each sample type and time point.
2.2.7. Diffusion Test

PBS solutions containing either FITC-labeled hyaluronic acid (10 kDa molecular
weight) or transferrin-TRITC (80 kDa molecular weight) at known concentration were
prepared and deposited over the A-C hydrogels. The photoluminescence signal of the
loaded solutions was recorded over time and the resulting concentration was extrapolated
from the calibration curve of the corresponding standard staining solution. The diffusivity
of the two molecules within the hydrogels was assessed determining the ratio between
the concentration of the fluorescent molecule (either hyaluronic acid or transferrin) at the
analyzed time point and that measured in free PBS.
2.2.8. Drug Diffusion Test

1 mL of cisplatin solution (100 µM in PBS) was loaded above 1 mL of either 0.25–0.02%
or 0.125–0.025% A-C hydrogels. Five time points (1, 2, 4, 8, and 24 h) were set and three
duplicates for each time point and type of hydrogel were defined. The hydrogels were
incubated at 37 ◦ C. The cisplatin solution was withdrawn at the determined time points and
the hydrogel was frozen before being lyophilized. Soon after, the samples were digested
overnight in 65% nitric acid and the amount of Pt was estimated via elemental analysis
using an Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES) Varian
720-ES (Santa Clara, CA, USA).
2.3. Tumor Spheroid Preparation and Characterization

Human tumor cells were purchased from the American Type Culture Collection
(ATCC). The human breast cancer cell lines, i.e., MCF-7, MDA-MB-361, and MDA-MB-231,
and the human neuroblastoma SH-SY5Y cells were cultured as 2D monolayers in DMEM
medium (4500 mg/L glucose) supplemented with 10% FBS, 100 U/mL penicillin, and
100 µg/mL streptomycin at 37 ◦ C in an atmosphere of 5% CO2 .
The cells were trypsinized, counted and added to the hydrogels at a density of
2.5 × 104 cells/mL in complete growth medium. The cell suspension containing agarose
and collagen was let to gel into the well plate. As an example, to prepare 1 mL of 0.25–0.02%
A-C hydrogel, 250 µL of 1% agarose solution in PBS was added to 750 µL of a cellular
suspension containing 200 µL of 0.1% collagen solution. After gelation, 1 mL of DMEM was
added over the cell-embedded hydrogels before transferring the plate into the incubator.
The medium was changed every 4 days.
Morphological Analysis of the Tumor Spheroids

The morphological characteristics of spheroids, including their diameter and shape,
were determined by optical analysis using a EVOS XL Cell Imaging System microscope
(Thermo Fisher, Waltham, MA, USA). The progressively developing spheroids were ob-
served at 24 h intervals. The mean diameter of the 3D structures was calculated by using
ImageJ Software (1.48 v).
For the SEM imaging, the tumor spheroids embedded within the hydrogels were
fixed overnight with glutaraldehyde (2.5%) in cacodylate buffer (0.1 M) at 4 ◦ C. The
fixed specimens were washed three times with PBS and then 1% osmium tetroxide in a
cacodylate buffer was added. After 6 h, the samples were washed three times with PBS, cut
and lyophilized. Finally, the as-prepared samples were transferred to the SEM microscope
for imaging. The operating conditions of the microscope were the same as those used for
imaging the hydrogels.
2.4. Cellular Assays

2.4.1. Live/Dead Assay
Viability of spheroids was investigated by using a live/dead assay kit (Thermo Fisher
Scientific Inc., Waltham, MA, USA). Briefly, the activity of intracellular esterase induces
non-fluorescent, cell-permeant calcein acetoxymethyl ester to become fluorescent after
hydrolysis, giving a green fluorescence to the viable spheroids. On the other hand, ethidium
homodimer enters and binds to nucleic acids only into damaged cells, producing a red
fluorescence that indicates dead cells. The assay was performed at three time points to
monitor the viability of the spheroids during their growth. In detail, the medium was
removed from the plates containing the spheroids-embedded hydrogels and the samples
were washed twice with PBS. A phosphate buffer solution containing calcein and ethidium
homodimer was then added, and the plate was kept in incubation at 37 ◦ C for 1 h. Finally,
the solution was replaced with fresh PBS before imaging the samples under a Fluorescence
Microscope (EVOS FLoid Cell Imaging Station, ThermoFisher, Waltham, MA, USA).
2.4.2. Mitochondria Toxicity Assay

MitoTracker Red from Life Technologies was dissolved in PBS and added to the hy-
drogel containing the spheroids at the 5th day of growth (working concentration: 250 nM).
The samples were kept in incubation for 1 h at 37 ◦ C and then imaged under a Fluorescence
Microscope (EVOS FLoid Cell Imaging Station, ThermoFisher, Waltham, MA, USA).
2.4.3. Cisplatin Treatment

MCF-7 spheroids were grown in 0.25–0.02% and 0.125–0.02% A-C hydrogels up
to 12 days. A DMSO solution of cisplatin was then added to the spheroids-embedded
hydrogels to reach a final concentration of 100 µM. After 24 h incubation, the medium was
removed and the hydrogels were carefully rinsed with fresh medium before performing
the live/dead assay (ThermoFisher), as already reported.
2.5. Immunofluorescence Microscopy Analysis

After 5 days of growth within the hydrogels, the spheroids were fixed with ice-
cold 4% paraformaldehyde for 30 min. Then, the spheroids were washed twice with
PBS and incubated overnight with a mouse anti-E-cadherin (E-cad; Santa Cruz, sc-8426)
antibody, according to the manufacturer’s protocol (1:1000 dilution in PBS). Subsequently,
the samples were incubated with an anti-mouse Alexa Fluor 488 (AF488) conjugated
secondary antibody (Cell Signaling). The images of fluorescently labeled proteins were
captured using a fluorescence microscope (Leica LMD7000, Mannheim, Germany).
2.6. Enzymatic Digestion of Agarose for Spheroids Recovery

0.25–0.02% and 0.125–0.02% A-C hydrogels containing MCF-7 spheroids at different
days of growth were incubated with 40 U of β-agarase from Pseudomonas atlantica and
incubated overnight at 37 ◦ C. The supernatant was then collected and observed under an
optical microscope to visualize the presence of floating spheroids. Soon after, the spheroids
were fixed with 4% paraformaldehyde and stained with 40 ,6-diamidino-2-phenylindole
(DAPI). The labeled structures were imaged under a fluorescence microscope (EVOS FLoid
Cell Imaging Station, ThermoFisher, Waltham, MA, USA).
2.7. Ultrastructural Analysis of the Recovered Spheroids

The spheroids recovered from the hydrogels were fixed with glutaraldehyde (2.5%)
in cacodylate buffer (0.1 M) at 4 ◦ C for 1 h. The fixed specimens were washed three times
with the same buffer, and 1% osmium tetroxide in a cacodylate buffer was added for 1 h.
The cells were then washed again and dehydrated with 25%, 50%, 75%, and 100% acetone.
Two steps of infiltration in a mixture of resin/acetone (1/1 and 2/1 ratios) were performed,
and then the specimens were embedded in 100% resin at 60 ◦ C for 48 h. Ultrathin sections
(70 nm thick) were cut with an Ultramicrotome and then stained with lead citrate. TEM
images were recorded on a JEOL Jem1011 microscope operating at an accelerating voltage
of 100 kV (Tokyo, Japan).
2.8. Statistical Analysis

All data represent the average value of at least three independent experiments, un-
less otherwise specified. Normally distributed data was compared with a two-tailed
Student’s t-test.
3. Results
3.1. Agarose–Collagen Hydrogels: Preparation and Characterization
Three agarose–collagen (A-C) hydrogels with different weight ratios were tested in
this study. The collagen amount was kept constant to 0.02% in all the hydrogel formulations
because it was already proven to be sufficient to provide enough anchoring sites to the
embedded cells [22]. On the other hand, the amount of agarose, that confers the structural
support, was varied from 0.5% to 0.125% to evaluate its impact on the biophysical properties
of the hydrogel and, consequently, its effect on the cellular spheroids development. To
prepare the hydrogels without denaturing the collagen and causing cell death, the agarose
solution was first heated until boiling (100 ◦ C) to dissolve the polysaccharide, and then
cooled down to 45 ◦ C. At this point, since gelation of pure agarose occurs at around 36 ◦ C,
it was rapidly mixed with collagen and the cell suspension (Figure S1).
pact (Figure 1).
In fact, the higher percentage of water in the 0.125–0.02% A-C hydrogel makes the
dry structure very brittle, displaying pores with a mean size of 71 ± 14 µ m that are quite
uniform and interconnected (Figure 1e,f). Although the measure of the pore size of a dry
Pharmaceutics 2021, 13, 963 structure cannot be considered realistic, it still provides a good estimation of the 3D
7 of or-
22
ganization of the hydrogels. The number of pores is smaller in the 0.25–0.02% and 0.5–
0.02% A-C hydrogels, while the average pore size appears to be slightly larger (81 ± 21
and 87 ± 25 µ m, respectively, Figure 1a–d). The higher turgidity and compactness of the
The SEM images of the resulting hydrogels showed that, by decreasing the agarose per-
hydrogels with higher percentage of agarose is also evident at a macroscopic level, where
centage, the porosity of the structure increases significantly, also appearing less
the 0.25–0.02% and 0.5–0.02% A-C hydrogels appeared self-standing, while that with
compact (Figure 1).
0.125% agarose did not (bottom panel of Figure S1).
Figure1.1.SEM
Figure SEMimages
imagesatatlower
lower(a,c,e)
(a,c,e)and
andhigher
higher(b,d,f)
(b,d,f)magnification
magnificationofofthe
theA-C
A-Chydrogels
hydrogelswith
with
different agarose concentration: (a,b) 0.5%; (c,d) 0.25%; (e,f) 0.125%, respectively. Type I collagen is
different agarose concentration: (a,b) 0.5%; (c,d) 0.25%; (e,f) 0.125%, respectively. Type I collagen is
always 0.02%. Scale bar is 100 µ m.
always 0.02%. Scale bar is 100 µm.
FTIR
In fact,spectroscopy was performed
the higher percentage on the
of water hydrogels
in the to investigate
0.125–0.02% whether
A-C hydrogel collagen,
makes the
despite the low percentage amount used, was detectable on the outer
dry structure very brittle, displaying pores with a mean size of 71 ± 14 µm thatsurface of theare
hy-
drogels. To this aim, the analysis was performed directly on the synthetized
quite uniform and interconnected (Figure 1e,f). Although the measure of the pore size hydrogels
deposited
of on the ATR
a dry structure crystal,
cannot without any
be considered further itmanipulation
realistic, still provides(Figure
a good2a).
estimation of
the 3D organization of the hydrogels. The number of pores is smaller in the 0.25–0.02%
and 0.5–0.02% A-C hydrogels, while the average pore size appears to be slightly larger
(81 ± 21 and 87 ± 25 µm, respectively, Figure 1a–d). The higher turgidity and compactness
of the hydrogels with higher percentage of agarose is also evident at a macroscopic level,
where the 0.25–0.02% and 0.5–0.02% A-C hydrogels appeared self-standing, while that with
0.125% agarose did not (bottom panel of Figure S1).
FTIR spectroscopy was performed on the hydrogels to investigate whether collagen,
despite the low percentage amount used, was detectable on the outer surface of the
hydrogels. To this aim, the analysis was performed directly on the synthetized hydrogels
deposited on the ATR crystal, without any further manipulation (Figure 2a).
Figure
Figure 2. 2.
(a)(a)FTIR
FTIRspectra
spectraof ofagarose
agarose (green
(green curve),
curve), collagen
collagen(pink
(pinkcurve),
curve),and 0.25–0.02%
and A-C
0.25–0.02% (red
A-C curve)
(red curve)hydrogels. (b) (b)
hydrogels.
Swelling
Swelling behaviorofofthe
behavior theA-C
A-Chydrogels
hydrogelskept
keptfor
for 22 days
days in
in PBS
PBS at
at 37
37 °C.
◦ C. (c,d)
(c,d)Degradation
Degradationcurves
curvesupuptoto2 weeks ofof
2 weeks thethe
A-A-C
C hydrogels either in PBS (c) or in DMEM (d). Diffusion tests performed up to one week with hyaluronic acid-FITC (e)
hydrogels either in PBS (c) or in DMEM (d). Diffusion tests performed up to one week with hyaluronic acid-FITC (e) and
and transferrin-TRITC (f).
transferrin-TRITC (f).
Pure agarose hydrogel (0.25% w/v) was recorded as reference (light green line in Fig-
Pure agarose hydrogel (0.25% w/v) was recorded as reference (light green line in
ure 2a), showing the typical signals at 988 and 1076 cm−1, relative to the C–H bending and
Figure 2a), showing the typical signals at 988 and 1076 cm−1 , relative to the C–H bending
to the C–O stretching of the glycosidic bonds, and two broad peaks at 1656 and 3421 cm−1,
and to the C–O stretching of the glycosidic bonds, and two broad peaks at 1656 and
3421 cm−1 , characteristic of the stretching of the H–O–H bound water and of the O–H
hydrogen bonded carbohydrate hydroxylic groups, respectively [29]. Two broad peaks
were also observed in the pure collagen hydrogel at about 3328 and 1645 cm−1 for the amide
C=O and N–H stretching, respectively (pink line in Figure 2a). Additional smaller signals
were detected at 1051 cm−1 for the C-OH stretching vibrations of carbohydrate moieties
attached to the protein [30]. The FTIR spectrum of the blend 0.25–0.02% A-C hydrogel (red
curve of Figure 2a) showed all the peaks characteristic of the pure compounds, i.e., smaller
signals at 989 and 1079 cm−1 , with a small side-bump at 1045 cm−1 , and much broader
peaks at 1649 and 3464 cm−1 . Interestingly, while the last two peaks had similar intensity
in pure collagen, a much higher intensity of the signal at lower frequencies was observed
in pure agarose as well as in the blend hydrogel. As expected, no significant differences
were observed in the spectra of the other two blend hydrogels (data not shown).
A critical feature of polymeric hydrogel is their capability to absorb and retain water,
i.e., their swelling behavior. This property depends on many factors, such as network
density, solvent used, and non-covalent interactions among all the components. In this case,
the hydrogels containing 0.5% and 0.25% agarose showed a similar trend with a swelling
ratio of about 20 and 25 times at t0 and a maximum swelling of about 27 and 30 times after
24 h in PBS, respectively (Figure 2b). These data are in accordance with those reported
in the literature [31]. On the other hand, the ability to absorb water of the hydrogel with
0.125% agarose was considerably lower, with a swelling ratio of about 5 times at t0 and 7
after 24 h. Thus, there appears to be a critical threshold of agarose percentage below which
the physical properties of the hydrogel are dramatically altered.
The stability over time of the three formulations was also investigated by measuring
the percentage residual weight at 37 ◦ C up to two weeks. The 0.5–0.02% and 0.25–0.02%
A-C hydrogels were shown to be quite stable and lost around 10% and 15% of their weight
after 14 days of incubation in PBS (Figure 2c) and DMEM (Figure 2d), respectively. On the
other hand, the 0.125–0.02% A-C hydrogel showed a maximum of degradation close to
40% after 2 weeks in both incubation media. In parallel, the amount of collagen potentially
released was estimated through the BCA assay (Figure S2). To this aim, the A-C hydrogels
were kept in PBS at 37 ◦ C and the volume of solvent collected and renewed every 24 h up
to 14 days. Some collagen release was detected in all hydrogels, although to a higher extent
and with a quicker trend in those with a lower agarose content. The overall percentage
amount of collagen leaked after 2 weeks was equal to 50%, 27%, and 8% of the whole
collagen present in the hydrogels containing 0.125%, 0.25%, and 0.5% agarose, respectively.
This loss, together with the less compact texture of the 0.125–0.02% A-C hydrogel, would
explain the higher degradation of this matrix.
A further parameter that deserves investigation is the capability of a hydrogel to allow
the diffusion throughout the matrix of biomolecules and nutrients, such as growth factors
and serum proteins having molecular weight of several tens of kDa. To this aim, a diffusion
test was performed by using two fluorescent probes, FITC-conjugated hyaluronic acid
and transferrin-TRITC, with a MW of around 10 and 80 kDa, respectively. Once loaded
onto the three hydrogel formulations, the variation of concentration of the feeding solution
was monitored over time and reported versus the diffusion in pure PBS. Hyaluronic acid
diffused rapidly into the softest hydrogel, reaching 100% diffusion after 72 h (Figure 2e).
The other two hydrogels performed similarly but with a more gradual diffusion that
decreased by increasing the percentage of agarose, reaching a maximum of 82% and 75%
after one week, respectively. On the other hand, transferrin-TRITC spread much more
slowly, reaching after 24 h a diffusion value of almost 48% in the case of the 0.125–0.02%
A-C hydrogel, while it was close to 10% in the case of the other two blends (Figure 2f).
Noteworthily, after one week the diffusivity did not exceed 50% even in the softest matrix.
The structural properties of the A-C samples were also evaluated by mean of com-
pression test under physiological-like conditions showing how the agarose concentration
deeply affects the hydrogels’ mechanical properties (Figure 3a).
were evaluated after 0, 1, 4, and 8 days of incubation in physiological-like conditions (PBS
at pH 7.4, 37 °C, humified atmosphere with 5% CO2). While the 0.125–0.02% hydrogel
could not be tested over time due to its low consistence, the 0.5–0.02% and the 0.25–0.02%
blends retained their structural integrity until the 8th day of measure. As shown in Figure
Pharmaceutics 2021, 13, 963 3b, no significant changes in the E modulus were registered in the case of the 0.5–0.02%
10 of 22
and the 0.25–0.02% hydrogels. These data are in accordance with those obtained by the
degradation tests, in which a minimum weight loss was recorded.
Figure
Figure 3.
3. (a)
(a) Representative
Representative stress-strain
stress-strain curves
curves of
of the
the A-C
A-C hydrogels
hydrogels subjected
subjected to
to unconfined
unconfined compression
compression withwith aa dis-
dis-
placement
placement rate of 0.01 mm/s, until 80% strain. (b) Compressive moduli of the A-C hydrogels after 0, 1, 4, and 88 days
rate of 0.01 mm/s, until 80% strain. (b) Compressive moduli of the A-C hydrogels after 0, 1, 4, and days ofof
incubation in PBS at 37 °C,
◦ in humified atmosphere with 5% CO2.
incubation in PBS at 37 C, in humified atmosphere with 5% CO2 .
3.2. Growth
In fact,ofan
Mammary
increaseSpheroids in A-Cconcentration
in the agarose Hydrogels corresponded to an increase in the
One of the major
compressibility modulus.advantages
As expected,of hydrogels is theirratio
the 0.5–0.02% ability to provide
showed more realistic
the highest E modulus 3D
(5.7 ± 0.5
models forkPa),
in vitro studies.
followed In this
by the study, we
0.25–0.02% ± 0.4 kPa)mammary
(1.6generated (p = 0.0004) spheroids from three
and the 0.125–0.02%
(0.7 ± 0.2breast
different kPa) (pcancer
= 0.0001)
cell ones.
lines, A minor
i.e., two but still significant
luminal difference
estrogen receptor was found
positive cells between
(MCF-7
the 0.25–0.02%
and MDA-MB-361) andandthe a0.125–0.02%
triple negative ratios (p =(MDA-MB-231)
model 0.03). In order for to correlate
comparative degradation
analysis.
resistance
The cells wereto structural
seeded atstability
the density overoftime,2.5 ×the
104 mechanical
per mL inside performances
the three typesof the
of hydrogels
weretime
and evaluated
courseafter 0, 1, (up
studies 4, and 8 days
to 14 days) of were
incubation in physiological-like
performed to monitor the conditions
process of (PBS
multi-at
pH 7.4, 37 ◦ C, humified atmosphere with 5% CO ). While the 0.125–0.02% hydrogel could
cellular spheroid formation. MCF-7 and MDA-MB-361 2 successfully formed spheroids in
notthe
all be three
testedtypesover time due to itsOn
of hydrogels. lowthe consistence,
other hand, theMDA-MB-231
0.5–0.02% andcells the 0.25–0.02% blends
replicated during
retained their structural integrity until the 8th day of measure.
the first days, but they were not able to reach a defined 3D organization, thus not formingAs shown in Figure 3b, no
significantinchanges
spheroids in the
any of the E modulus
hydrogel were registered
conditions (Figure S3). in It
theis case
worth of to
thereport
0.5–0.02%
that noand the
mul-
0.25–0.02%
ticellular hydrogels.
spheroid These data
formation was are in accordance
observed with those
by culturing obtained
the three by the
cell lines indegradation
0.02% pure
tests, in which
collagen, whileathe minimum
spheroids weight
startedlosstowas growrecorded.
in the case of pure agarose hydrogel, but
they underwent senescence after few days (data not shown).
3.2. Growth of Mammary Spheroids in A-C Hydrogels
As reported, MCF-7 and MDA-MB-361 cells formed spheroids in the three blends,
One
but the sizeof and
the major advantagesofofthe
the morphology hydrogels is theirwere
3D structures ability to provide
different more realistic
depending on the
3D models for in vitro studies. In this study, we generated
experimental conditions. In particular, the spheroids grown in the stiffest hydrogel were mammary spheroids from
three different
spherical breast (Figure
but smaller cancer cell lines,those
S4) than i.e., two
grown luminal estrogen
in softer receptor
conditions positive
(Figure cells
4), espe-
(MCF-7 and MDA-MB-361)
cially in the case of MDA-MB-361 cells. and a triple negative model (MDA-MB-231) for comparative
analysis. The cells were seeded at the density of 2.5 × 104 per mL inside the three types of
hydrogels and time course studies (up to 14 days) were performed to monitor the process of
multicellular spheroid formation. MCF-7 and MDA-MB-361 successfully formed spheroids
in all the three types of hydrogels. On the other hand, MDA-MB-231 cells replicated during
the first days, but they were not able to reach a defined 3D organization, thus not forming
spheroids in any of the hydrogel conditions (Figure S3). It is worth to report that no
multicellular spheroid formation was observed by culturing the three cell lines in 0.02%
pure collagen, while the spheroids started to grow in the case of pure agarose hydrogel,
but they underwent senescence after few days (data not shown).
As reported, MCF-7 and MDA-MB-361 cells formed spheroids in the three blends,
but the size and the morphology of the 3D structures were different depending on the
experimental conditions. In particular, the spheroids grown in the stiffest hydrogel were
spherical but smaller (Figure S4) than those grown in softer conditions (Figure 4), especially
in the case of MDA-MB-361 cells.
Pharmaceutics 2021, 13,
Pharmaceutics 2021, 13, 963
963 11
11 of
of 22
22
Figure 4. Optical images (a,b) and average size (c,d) of the spheroids obtained with MCF-7 and MDA-MB-361 cells grown
0.25–0.02% (a,c)
up to 14 days either in 0.25–0.02% (a,c) or
or 0.125–0.02%
0.125–0.02% (b,d)
(b,d) A-C
A-C hydrogels.
hydrogels. Scale
Scale bar
bar is
is 100
100 µm.
µ m.
This effect is more evident as the 3D 3D structure

structure progressively
progressively grows
grows over
over time:
time: the
average size of MDA-MB-361 derived spheroids after 12 days grown in hydrogels with
0.5% agarose
0.5% agarose was
was around
around63.163.1±± 7.8 µ m, while
7.8 µm, while itit reached
reached81.381.3±± 6.3 µ m and
6.3 µm 94.7±
and94.7 9.5 µm
± 9.5 µm
in 0.25–0.02%
in 0.25–0.02%and and0.125–0.02%
0.125–0.02% A-CA-C hydrogels,
hydrogels, respectively
respectively (Table
(Table S1). S1).
In theIncase
theofcase
MCF-of
MCF-7 cells, the size gap was of about 7 and 27 µm respectively, being
7 cells, the size gap was of about 7 and 27 µ m respectively, being the spheroids in the the spheroids in
the hydrogels with 0.5% agarose about 64 µm large after 12 days, while
hydrogels with 0.5% agarose about 64 µ m large after 12 days, while they reached a diam- they reached a
diameter of about 70 µm in the 0.25–0.02% A-C hydrogel and about
eter of about 70 µ m in the 0.25–0.02% A-C hydrogel and about 91 µ m in the softest one 91 µm in the softest
one (Table
(Table S1). S1). The images
The images in Figure
in Figure 4 clearly
4 clearly evidence
evidence how howthe 3Dthestructures
3D structures
evolvedevolved
over
over time from a single cell to more complex aggregates, but the
time from a single cell to more complex aggregates, but the growth curves showed growth curves showed
that
that while the spheroids in the hydrogel with 0.25% agarose reached
while the spheroids in the hydrogel with 0.25% agarose reached a growth plateau after 14 a growth plateau
after 14
days, days,
they stillthey still displayed
displayed a positivea positive growth
growth trend in trend in theenvironment.
the softest softest environment. In
In fact, the
Pharmaceutics2021,
Pharmaceutics 13,963
2021,13, 963 12of
12 of22
22
spheroids in the softest

fact, the spheroids matrix
in the softestcontinued to grow exceeding
matrix continued the 100 µthe
to grow exceeding m diameter in both
100 µm diameter
cell lines after 28 days. On the other hand, the spheroids in the 0.25–0.02%
in both cell lines after 28 days. On the other hand, the spheroids in the 0.25–0.02% A-C A-C hydrogel
stopped
hydrogel their growth
stopped and,
their after 2and,
growth weeks, they
after startedthey
2 weeks, to shrink
startedandto exhibited
shrink and a dark intra-a
exhibited
cellular substance. substance.
dark intracellular In this sense, it appears
In this sense, itthat the softest
appears hydrogel
that the is capableisofcapable
softest hydrogel sustain-of
ing the spheroids’
sustaining growth growth
the spheroids’ for longerfor time
longer although, especially
time although, in the case
especially in theofcase
MCF-7, they
of MCF-7,
displayed irregular
they displayed contours
irregular and looser
contours structure
and looser [32,33].
structure As a As
[32,33]. general consideration,
a general we
consideration,
observed that MCF-7 cells could tolerate stiffer environments as
we observed that MCF-7 cells could tolerate stiffer environments as compared to MDA- compared to MDA-MB-
361, as confirmed
MB-361, as confirmedby thebyfact
thethat
factthey
that also
theygenerated small small
also generated spheroids in hydrogels
spheroids with
in hydrogels
1%
withagarose (Figure
1% agarose S5), while
(Figure MDA-MB-361
S5), while MDA-MB-361 did not.
did not.
In
Inconclusion,
conclusion, the the hydrogel stiffness
stiffnessand
andthethematrix
matrixcomposition
composition regulated
regulated thethe sphe-
spheroids
roids
growthgrowth and morphology
and morphology and, more
and, more interestingly,
interestingly, they affected
they affected the migration
the local local migration
of the
ofouter cells. cells.
the outer In fact, onlyonly
In fact, the softest matrix
the softest waswas
matrix ableable
to induce protrusion
to induce protrusionof cells from
of cells the
from
outer
the layer
outer layerandand
their local
their dissemination
local dissemination (Figure 5). 5).
(Figure
Figure
Figure5.5.Optical
Opticalimages
imagesofofthe
thetumor
tumorspheroids
spheroidsobtained
obtainedby
byMCF-7
MCF-7and
andMDA-MB-361
MDA-MB-361cells cellsafter
after
growing for 8 days in either 0.125–0.02% (top) or 0.25–0.02% A-C hydrogels (bottom). The red ar-
growing for 8 days in either 0.125–0.02% (top) or 0.25–0.02% A-C hydrogels (bottom). The red arrows
rows point to the cells disseminated by the spheroids grown in the soft matrix. Scale bar is 100 µ m.
point to the cells disseminated by the spheroids grown in the soft matrix. Scale bar is 100 µm.
AsAsalready
alreadystated,
stated,the motility
the motilityof of
these cells
these depends
cells depends on the
on interaction
the interaction withwith
the mi-
the
croenvironment, mainly with collagen [21,34]. It is likely that the less
microenvironment, mainly with collagen [21,34]. It is likely that the less dense hydrogel dense hydrogel fa-
cilitates thethe
facilitates protrusive
protrusive behavior,
behavior,alsoalsofacilitating
facilitatingcontact
contactwithwiththethecollagen
collagen anchoring
anchoring
points for the spatial dissemination of the cells. On the other hand,
points for the spatial dissemination of the cells. On the other hand, the reduced the reducedmigration
migration of
ofthe
the outer
outer cells
cells inin
thethe hydrogels
hydrogels with
with 0.25%
0.25% agarose
agarose should
should be be related
related to the
to the tighter
tighter pres-
pressure
sure
that that the matrix
the matrix exertsexerts
on theon theleading,
cells, cells, leading,
as already as described,
already described, to morespheroids.
to more compact compact
spheroids.
Based on these observations and with the aim to study the effects of the mechanical
Based
features of on
thethese observations
environment on theand with thefeatures,
tumoroids aim to study the effects
the following of the were
analyses mechanical
carried
features of the environment
out comparing on the (0.25%
the two conditions tumoroidsand features, the followinghydrogels)
0.125% agarose-based analyses were car-
in which
ried out comparing the two conditions (0.25% and 0.125% agarose-based
both cell lines were able to form healthy and stable spheroids up to two weeks. hydrogels) in
which both cell lines were able to form healthy and stable spheroids up to two weeks.
3.3. Mammary Spheroids Viability and Epithelial Markers Expression

3.3. Mammary Spheroids
The viability of theViability
spheroidsandobtained
Epithelialfrom
Markers Expression
the MCF-7 and MDA-MB-361 cell lines
Thetype
in both viability of the spheroids
of hydrogels obtainedthrough
was investigated from theaMCF-7
live/deadandfluorescence
MDA-MB-361 cell(Figure
assay lines in
both
S6). type of hydrogels was
The homogeneous investigated
green through
fluorescence a live/dead
evidenced fluorescence
that all the cells inassay
the (Figure
spheroids S6).
The
werehomogeneous
viable after 8 green fluorescence
days; while few red evidenced
spots werethat all the
already cellsafter
visible in the
14 spheroids
days in thewere 3D
viable aftergrown
structure 8 days;inwhile few red
the matrix withspots were
0.25% already
agarose, visible after
showing their 14 daysaging.
initial in theOn3Dthe
structure
other
grown in the matrix with 0.25% agarose, showing their initial aging.
hand, the spheroids embedded in the 0.125–0.02% A-C hydrogel resulted absolutely via-On the other hand, the
spheroids embedded in the 0.125–0.02% A-C hydrogel resulted absolutely
ble. These data are in accordance with the previous analysis of the growth curves of the viable. These
data are in accordance
3D structures in the twowith the previous
systems (Figureanalysis of the growth
4). To further confirmcurves of thethe
this trend, 3Dassay
structures
was
in theperformed
also two systems (Figure
after 28 days.4). Notably,
To furtherwhile
confirm this trend,grown
the spheroids the assay was0.125%
in the also performed
agarose-
after
based28hydrogels
days. Notably, while
were still the spheroids
viable, grownininthe
those prepared thestiffer
0.125% agarose-based
matrix were dead.hydrogels
Further-
were
more,still viable, those
MitoTracker red,prepared in theofstiffer
an indicator matrix were
mitochondrial dead. Furthermore,
membrane potential able MitoTracker
to selec-
red, anstain
tively indicator
active of mitochondrial
mitochondria, wasmembrane
used to gainpotential
informationableon tothe
selectively stain func-
mitochondrial active
mitochondria,
tion (Figure 6). was used to gain information on the mitochondrial function (Figure 6).
Figure
Figure6.6. Mitochondrial
Mitochondrial labeling with
with MitoTracker
MitoTracker redred of
ofliving
livingspheroids
spheroidsofofMCF-7
MCF-7and
andMBA-MB-
MBA-MB-
361cells
361 cellsafter
after 55 days
days of
of growth.
growth. Scale bar is 50 µµm.
m.
MitoTracker
MitoTrackerstaining
stainingshowed
showedthe
thepresence
presenceofofactive
activemitochondria
mitochondriaininthe
theperiphery
peripheryas
well as in the center of the spheroids.
as well as in the center of the spheroids.
The
The expression
expression of
of E-cadherin,
E-cadherin, a typical epithelial
epithelial marker
markerinin3D
3Dspheroids,
spheroids,waswasthen
then
determined
determined by by immunofluorescence, clearly showing
immunofluorescence, clearly showingthatthatthe
themammary
mammaryspheroids
spheroidsmain-
main-
tained
tained the
the expression
expression ofofthe
thetransmembrane
transmembraneglycoprotein
glycoprotein and
and evidencing
evidencing thethe presence
presence of
of tight cell–cell interactions, both typical features of an epithelial phenotype (Figure 7).
Similar results were obtained with the 0.125–0.02% A-C hydrogel for both MitoTracker and
E-cadherin staining (data not shown).
Pharmaceutics 2021, 13, 963 tight cell–cell interactions, both typical features of an epithelial phenotype (Figure 7). Sim-
14 of 22
ilar results were obtained with the 0.125–0.02% A-C hydrogel for both MitoTracker and
E-cadherin staining (data not shown).
Figure 7.7. Staining

Figure Staining of E-cadherin
E-cadherin inincellular
cellularspheroids
spheroidsofofMCF-7
MCF-7and
and MBA-MB-361
MBA-MB-361 after
after 5 days
5 days of of
growthin
growth in A-C
A-C hydrogels.
hydrogels. Scale
Scale bars
barscorrespond
correspondtoto5858µµm.
m.
The
Themorphology
morphologyof ofthe
theMCF-7
MCF-7spheroids
spheroidsgrown
grownforfor1212days
daysandandtheir arrangement
their arrangement into
the
into0.25–0.02% A-C A-C
the 0.25–0.02% hydrogel waswas
hydrogel alsoalso
investigated by by
investigated ultrastructural
ultrastructuralanalysis,
analysis,showing
show- a
3D
ingstructure wrapped
a 3D structure into theinto
wrapped hydrogel matrix (Figure
the hydrogel S7). TheS7).
matrix (Figure protrusion of the spheroids
The protrusion of the
spheroids
from from the
the hydrogel hydrogel
can canappreciated
be clearly be clearly appreciated
and they canand they can betocompared
be compared to co-
cocoons anchored
coons anchored
to a branch. to a branch.
Overall, these data
Overall, data suggest
suggestthat
thatthis
thistype
typeofofhydrogel
hydrogelisisa asuitable
suitableapproach
approach forfor
thethe
generation and
generation and growth of mammary
mammaryspheroids.
spheroids.
3.4.
3.4. Cisplatin
Cisplatin Delivery to
to the
the Embedded
EmbeddedSpheroids
Spheroids
To
To evaluate
evaluate the
the exploitation
exploitation of of these
these3D3Dsystems
systemsasasa adrug-screening
drug-screening platform,
platform, thethe
MCF-7 spheroids embedded either in the 0.25–0.02% or 0.125–0.02%
MCF-7 spheroids embedded either in the 0.25–0.02% or 0.125–0.02% A-C hydrogels were A-C hydrogels were
treatedwith
treated with 100
100 µM
µ M cisplatin.
cisplatin. This
Thisconcentration
concentrationwaswaschosen
chosen inin
accordance
accordance with recently
with recently
published studies
published studies in which
which thethedrug
drugresponse
responseofofhydrogel-embedded
hydrogel-embedded spheroids
spheroids was as- as-
was
sayed[35–37].
sayed [35–37]. In
In aapreliminary
preliminary experiment
experiment thethe diffusion
diffusion time
time of
ofcisplatin
cisplatin into
into the
thehydrogels
hydro-
gels (without
(without the spheroids)
the spheroids) waswas determined
determined viavia elemental
elemental analysis.As
analysis. Asshown
shownininFigure
FigureS8,
S8, drug
the the drug diffusion
diffusion waswas faster
faster in the
in the softer
softer hydrogel,
hydrogel, reachinga a100%
reaching 100%rate
rate(concentration
(concentra-
tion
at theatequilibrium)
the equilibrium) already
already after
after 2 h2 h incubationatat37
incubation 37 ◦°C.
C. In
In the
thecase
caseofofthe 0.25–0.02%
the 0.25–0.02%
A-C hydrogel,
A-C hydrogel, thethemaximum
maximum diffusion
diffusion waswas
detected afterafter
detected 8 h of8incubation. Based on
h of incubation. theseon
Based
findings the incubation time of the spheroids with the drug was
these findings the incubation time of the spheroids with the drug was set at 24 h.set at 24 h. After drug
After
drug treatment, the cell mortality was estimated by the live/dead assay: dead cells were
detected in the hydrogels administered with cisplatin, while control samples were brightly
green fluorescent (Figure 8).
Pharmaceutics 2021, 13, 963

treatment, the cell mortality was estimated by the live/dead assay: dead cells were de-
15 of 22
tected in the hydrogels administered with cisplatin, while control samples were brightly
green fluorescent (Figure 8).
Figure 8. Live/dead
Live/dead assay
assayperformed
performedwith
withthe
the MCF-7
MCF-7 spheroids
spheroids embedded
embedded intointo the
the 0.25–0.02%
0.25–0.02% (a) and 0.125–0.02% A-C
hydrogels (b)
(b) after
after24
24hhincubation
incubationwith
with100
100µM
µM cisplatin.
cisplatin. (c)(c) Mean
Mean fluorescent
fluorescent intensity
intensity detected
detected in the
in the 0.25–0.02%
0.25–0.02% (left)(left)
and
0.125–0.02% (right) A-C hydrogels. Green bars correspond to the fluorescence signal of calcein, while red bars to ethidium
homodimer, respectively. (* indicates p < 0.01 when comparing CTRL spheroids with cisplatin-treated spheroids in both
types of hydrogel. Statistical significance was assessed by t-test).
The analysis of the distribution of the fluorescent pixels performed on 25 spheroids

for each type of sample evidenced that the difference between the number of dead cells
of the control and those of the drug-treated samples is statistically significant (p < 0.01)
(Figure 8, lower panel). This assay confirmed that the drug diffusion depends on the
agarose percentage amount, as the number of dead cells was higher in the hydrogel
containing 0.125% agarose. Interestingly, the structure of the spheroids was dramatically
altered if the samples, after 24 h incubation with the drug, were kept in fresh medium for
additional 5 days. A large number of dead cells detached from the spheroids and many
cellular debris were scattered in the matrix, while small residues of the 3D structures were
still visible (Figure S9). This effect was observed in both types of hydrogels after 5 days
post drug treatment.
3.5. Enzymatic Digestion of Agarose for Spheroids Recovery

To evaluate the possibility to recover the spheroids from the hydrogels for additional
processing and/or other biological studies, the blends with 0.25% and 0.125% agarose
containing MCF-7 spheroids at different days of growth were incubated with β-agarase
from Pseudomonas atlantica [38,39]. However, only the spheroids located in the outer layer
of the 0.25–0.02% A-C hydrogel were recovered after overnight incubation at 37 ◦ C with
the enzyme (upper panels in Figure S10). Most of the hydrogel remained intact and the
spheroids continued to grow inside it as in control hydrogels not treated with agarase.
On the other hand, the overnight enzymatic treatment completely dissolved the hydrogel
containing 0.125% agarose, and all the spheroids could be recovered (lower panels of
Figure S10). Noteworthily, the stability test (Figure 2) already showed a higher degree of
spontaneous degradation of the hydrogel with 0.125% agarose compared to that higher
amount. Thus, the addition of agarase boosted the degradation process, leading to the
complete dissolution of the softer matrix. The morphology of the spheroids collected from
both types of hydrogels after agarase treatment was preserved even at different times
of growth, up to 12 days (Figure S10). The vitality of the recovered spheroids was also
confirmed by the live/dead assay (Figure S11) as well as by DAPI staining (Figure 9),
showing their suitability for further processing and study. Only a few free individual
Pharmaceutics 2021, 13, 963 cells could be observed, probably detached from the surface of the spheroids during 17 of the
22
centrifugation steps required by the staining protocol.
Figure 9. Cont.
Figure9.9.Optical
Figure Opticalimages
imagesofofMCF-7
MCF-7spheroids
spheroidsgrown
grownininthe
the0.25–0.02%
0.25–0.02%(upper
(upperpanels)
panels)and
and0.125–0.02%
0.125–0.02%(lower
(lowerpanels)
panels)A-C
A-
C hydrogels, recovered after agarase treatment, fixed and stained with DAPI. Scale bars correspond to
hydrogels, recovered after agarase treatment, fixed and stained with DAPI. Scale bars correspond to 100 µm.100 µ m
Nevertheless, the spheroids preserved their shape, morphology, and vitality character-
4. Discussion
istics,In
confirming
this work their suitability
blended for subsequent
hydrogels composed ofbiological studies (Figure
agarose (variable weight S11).
amount from
As proof of concept of the applicative potential of the recovered
0.125% to 0.5%) and collagen (fixed weight amount equal to 0.02%) were spheroids, theyaswere
prepared en-
processed for ultrastructural imaging. The TEM images of Figure S12 show neighbour-
abling matrices for the growth of 3D cellular structures. These hydrogels combine the bio-
ing cells tightly
mechanical connected
properties through
of agarose andcellular junctions and
the bioadhesivity cellular The
of collagen. organelles
amounttypical of
of colla-
metabolically active cells.
gen is 6.25-, 12.5-, and 25-times lower than that of agarose, and the formation of the hy-
drogel is probably to be ascribed mainly to agarose. In fact, although the self-assembling
4. Discussion
capability of collagen molecules in vitro under physiological conditions is well known,
In this work
reconstituted blended
collagen hydrogels
fibrils, that arecomposed of agarose
held together (variable weight
by non-covalent amount
interactions from
(hydro-
0.125% to 0.5%) and collagen (fixed weight amount equal to 0.02%)
gen bonding, hydrophobic and electrostatic interactions) are free to slide and do not formwere prepared as
enabling
a stable 3D matrices
networkfor [40–42].
the growth of 3D cellular
In addition, structures.
in preliminary These hydrogels
experiments we have combine the
observed
biomechanical properties of agarose and the bioadhesivity of collagen.
that collagen itself does not go into the gel state under the same experimental conditions The amount of
collagen is 6.25-, 12.5-, and 25-times lower than that of agarose, and
we used to obtain the A-C hydrogels (data not shown). Thus, the gelation likely depends the formation of the
hydrogel
upon theisformation
probablyof tointra-
be ascribed mainly to agarose.
and inter-molecular In fact,
hydrogen although
bonds in thethe self-assembling
agarose backbone
capability of collagen molecules in vitro under physiological
at a temperature lower than <40 °C, as elsewhere reported [43]. conditions is well known,
reconstituted
Although collagen
agarose fibrils, that are held
and collagen havetogether
already by non-covalent
been interactions
used to prepare (hydrogen
hydrogels, they
bonding, hydrophobic and electrostatic interactions) are free to slide
still represent valuable candidates for creating and exploiting viable hydrogels because and do not formofa
stable 3D network [40–42].
their biocompatibility and Inlow addition, in preliminary
cost. More experiments
complex hydrogel composedwe have observedpol-
of expensive that
collagen itself does not go into the gel state under the same experimental
ymers have been also developed for growing spheroids, but they suffer of limited availa- conditions we
used to obtain the A-C hydrogels (data not shown). Thus, the gelation likely depends upon
the formation of intra- and inter-molecular hydrogen bonds in the agarose backbone at a
temperature lower than <40 ◦ C, as elsewhere reported [43].
Although agarose and collagen have already been used to prepare hydrogels, they still
represent valuable candidates for creating and exploiting viable hydrogels because of their
biocompatibility and low cost. More complex hydrogel composed of expensive polymers
have been also developed for growing spheroids, but they suffer of limited availability and
high costs of the products. Furthermore, it is worth reminding that agarose gelation does
not require chemical crosslinking (whose residues would have effects on cell viability) and
occurs at temperatures compatible with cell growing conditions.
Here we suppose that the A-C hydrogel formulations combine the mechanical support
for 3D cell growth on the one hand, and the biomimetic component on the other. In
this sense, agarose acts as the structural backbone of the matrix while collagen provides
biological fingerprints for the growing 3D structures [20,32].
As expected, the characterization of the hydrogels evidenced that the agarose per-
centage governs the morphological, structural, and mechanical features of the matrix.
Reasonably, the greater the amount of agarose, the stiffer the hydrogel resulted. On the
other hand, the lowest agarose amount corresponded to the fastest hydrogel degradation
and diffusion of the molecules through the matrix. The higher degree of degradation of
the softest hydrogel was also associated to a higher release of collagen. Thus, it can be
expected that a less compact hydrogel facilitates the release of collagen, as determined by
the protein quantification assay.
Similarly, the entry and movement of biomolecules through the blended hydrogels
appears to be associated to the agarose percentage. The bigger the biomolecule and
the stronger the type of non-covalent interactions it can establish with the matrix, the
slower they are and the lesser the total amount of molecules that may reach the cells
embedded into the matrix. In this sense, the hydrogel could act as a physical barrier to the
diffusive transport of specific nutrients and drugs to the spheroid, similarly to what occurs
in vivo [44].
The hydrogels were exploited for growing tumoroids from three breast cancer cell
lines, namely MCF-7, MDA-MB-361, and MDA-MB-231 cells. However, while MCF-7 and
MDA-MB-361 cells formed nice 3D structures, MDA-MB-231 cells, a triple negative breast
cell line, did not organize into any three-dimensional arrangement, in accordance with
previous findings [45,46], probably because of their lack of adherens junctions.
The spheroids generated by MCF-7 and MDA-MB-361 cells presented size and com-
pactness strictly related to the stiffness of the surrounding matrix, and thus to the percent-
age of agarose. Apparently, both breast cancer cells prefer hydrogels with a stiffness from
about 1.5 to 0.7 kPa, i.e., with a percentage of agarose equal or below 0.25%, while stiffer
matrices, such as that with 0.5% agarose, did not result suitable to support the growth of the
spheroids. A different tissue-specific tropism of the cellular models probably contributes to
this result. In fact, MCF-7 cells have a low metastatic potential and are not tissue-specific,
while MDA-MB-361 cells were derived from a brain metastasis and their growth on a
softer hydrogel could match their in vivo metastatic microenvironment. With the intent
to partially confirm this hypothesis, the 3D growth of a neural cell model was examined.
SH-SY5Y neuroblastoma cells generated spheroids in the 0.125–0.02% A-C hydrogel, while
in the stiffer matrix after a slow cellular duplication, the structures did not grow further
over time (Figure S13). As a general remark, SH-SY5Y spheroids reached larger diameters
(121.7 ± 14.2 µm) than those obtained with MCF-7 and MDA-MB-361 cells grown for
14 days in the same matrix (i.e., 98.3 ± 10.0 and 101.3 ± 11.4 µm, respectively), and this
heterogeneity could be probably related to the different origin of the cell line.
The live/dead assay and the mitochondrial staining showed that the spheroids are
viable up to 14 days in both types of hydrogels. Notably, the 3D cultures were monitored for
up to one-month, evidencing a continued growth of the spheroids in the softer hydrogel,
reaching an average size larger than 100 µm. On the other hand, the spheroids in the
0.25–0.02% A-C hydrogel stopped their growth after 2 weeks, resulting completely dead
after 4 weeks. This different behavior might be related to the tighter interactions between
the cells and the surrounding environment as the 3D structures grow over time, i.e., the
limited degradation and capability of the stiffer hydrogel to accommodate the spheroids
induced their slow aging and death.
These results confirm that soft agarose–collagen hydrogels allow for long-term spheroid
growth although cells derived from different tissues sense the change in stiffness of the
substrate and significantly modify their behavior. In this respect, it has been shown that
cells respond to ECM environment by regulating a plethora of transcription factors and
other signals that affect cytoskeleton, cellular uptake, and cell cycle, that in turn determine
their morphology, proliferation, differentiation, tumor invasion and metastasis [47–50].
Among them, it is worth mentioning the transmembrane glycoprotein E-cadherin that is
expressed in epithelial cells and connect them through lateral adherent junctions. It has
been demonstrated that the level of expression of E-cadherin represents a crucial feature in
cancer progression as it is involved in the epithelial-mesenchymal transition [51]. Loss of
E-cadherin expression is generally associated to a lack of intercellular contacts and to an
increased tumor cell invasiveness through the activation of signaling pathways that regu-
late metastatic progression [52]. E-cadherin expression is also associated to the formation
of multicellular tumor spheroids, as already demonstrated [1]. Expression of E-cadherin

was also observed in the spheroids derived from MCF-7 and MDA-MB-361 cells, as shown
in Figure 7, confirming its importance in tumor development and progression, and the
suitability of these spheroids for mimicking natural tissues. This was also demonstrated by
successfully delivering a common chemotherapy drug, i.e., cisplatin, through the matrix.
In conclusion, as a general consideration from all the experiments performed, softer
hydrogels allowed for the establishment of long-term culture of large and irregular breast
tumor spheroids, while stiffer environmental conditions favored the growth of small and
compact 3D cell strictures viable for shorter periods (Figure S14). Finally, the possibility
to recover the tumoroids was demonstrated by treating the hydrogels with agarase. The
yield of the recovery process was quantitative and highly reproducible in the softest gel,
that already underwent partial spontaneous degradation, while in the matrix with 0.25%
agarose only a small percentage of 3D structures could be successfully recovered for
additional biological and biomolecular studies.
5. Conclusions
A simple (no crosslinking steps are required), cost-effective, and highly reproducible
method for the generation of tumor spheroids has been reported. The optical transparency
of the matrix facilitates the daily monitoring of the cellular growth and the morphological
variations due to drug testing. The procedure consists in wrapping individual tumor cells
in agarose hydrogels (with either 0.25% or 0.125% weight percentage) blended with 0.02%
collagen, where agarose reproduces the biomechanical features of the ECM, while collagen
provides the anchoring sites for the membrane proteins. The growth of tumoroids deriving
from three breast cancer cells lines was investigated in detail and compared. Interestingly,
one cell line, MDA-MB-231 did not form spheroids in any of the conditions employed,
while the other two lines, MCF-7 and MDA-MD-361, displayed quite similar behavior.
The variation in the agarose amount affected the physical and mechanical features of
the resulting hydrogel and the growth of the spheroids. In fact, the stiffer the hydrogel,
the more compact and slightly smaller the tumoroids resulted. Therefore, depending on
the type of tumoroids to be prepared and studied, the composition of the hydrogel can
be easily tuned. The growth of the spheroids was monitored for up to 2 weeks and the
qualitative analysis of their viability evidenced few dead cells only after 14 days.
Preliminary studies of drug testing with cisplatin showed that the blended hydrogels
allow for tracking the response of the spheroids to the drug administration. This aspect
makes the system potentially useful for routine drug screening.
Finally, the degradability of the hydrogels upon enzymatic treatment was demon-
strated, leading to complete recovery of the tumoroids in the case of the softest hydrogels,
while to a partial recovery in the hydrogel with 0.25% agarose. The possibility to recover
the spheroids is of a paramount importance as it enables further biomolecular studies
on the collected samples, showing the full potential of this cheap and easily scalable
biomimetic hydrogel.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/pharmaceutics13070963/s1, Figure S1: schematic drawing of hydrogels’ preparation and
pictures of the obtained hydrogels, Figure S2: BCA assay, Figure S3: optical images of MDA-MB-231
spheroids grown in the 0.25–0.02% A-C hydrogel after 2, 4, 6, and 8 days, Figure S4: optical images of
MCF-7 and MDA-MB-361 spheroids grown in the 0.5–0.02% A-C hydrogel after 6, 8, and 12 days,
Table S1: average size of the tumor spheroids after 12 days, Figure S5: optical images of MCF-7
spheroids grown in the 1–0.02% A-C hydrogel after 6, 8, and 12 days, Figure S6: live/dead assay
performed with the cellular spheroids embedded into the A-C hydrogels at 8, 14, and 28 days,
Figure S7: SEM images of MCF-7 spheroids embedded in the 0.25–0.02% A-C hydrogel after 12 days
of growth, Figure S8: cisplatin diffusion in 0.25–0.02% and 0.125–0.02% A-C hydrogels estimated via
elemental analysis, Figure S9: optical images of MCF-7 spheroids grown in 0.125–0.02% A-C hydrogel
after 24 h cisplatin treatment and additional 5 days of incubation with fresh medium, Figure S10:
optical images of MCF-7 spheroids grown in 0.25–0.02% and 0.125–0.02% A-C hydrogel and recovered
after incubation of the hydrogels with agarose, Figure S11: live/dead assay performed with the
MCF-7 spheroids grown for 8 days in the 0.125–0.02% A-C hydrogel and recovered after agarase
treatment, Figure S12: a–d) TEM images of MCF-7 spheroids grown for 8 days in the 0.125–0.02% A-C
hydrogel, Figure S13: optical images of SH-SY5Y spheroids grown in the 0.125–0.02% and 0.25–0.02%
A-C hydrogels up to 14 and 6 days, respectively, Figure S14: sketch depicting the effects over time of
the hydrogel stiffness on the size and viability of breast tumor spheroids derived from MCF-7 and
MDA-MB-361 cells.
Author Contributions: Conceptualization, A.Q. and A.G.; methodology, A.G., A.Q., A.R., L.S., N.G.
and C.N.; validation, A.Q. and A.G.; formal analysis, A.R.; investigation, A.Q., A.G. and D.V.;
resources, A.G., A.Q. and L.S.; data curation, A.Q., A.G., L.S. and N.G.; writing—original draft
preparation, A.G. and A.Q.; writing—review and editing, A.G., A.Q., A.R. and D.V.; visualization,
A.Q., A.G. and L.S.; supervision, A.Q., A.G., L.S., A.R. and D.V.; project administration, A.Q. and
A.G.; funding acquisition, A.Q. and A.G. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by the Italian project “Lab on a Swab” (cod. OTHZY54).
Acknowledgments: The authors would like to acknowledge the support of the following Italian
projects: “Tecnopolo per la medicina di precisione” (TecnoMed Puglia)—Regione Puglia: DGR n.
2117 del 21/11/2018 (CUP: B84I18000540002), and “Tecnopolo di Nanotecnologia e Fotonica per
la medicina di precisione” (TECNOMED)—FISR/MIUR-CNR: delibera CIPE n. 3449 del 7-08-2017
(CUP: B83B17000010001).
Conflicts of Interest: The authors declare no conflict of interest.
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pharmaceutics

1 CNR Nanotec, Institute of Nanotechnology, via Monteroni, 73100 Lecce, Italy;

Academic Editor: Hassan Bousbaa

Publisher’s Note: MDPI stays neutral

Pharmaceutics 2021, 13, 963. https://doi.org/10.3390/pharmaceutics13070963 https://www.mdpi.com/journal/pharmaceutics

Among 3D biomaterials, hydrogels of complex biological origin have been used in

2. Materials and Methods

2.1. Preparation of the Hydrogels

2.2. Characterization of the Hydrogels

2.2.2. Stability Test

2.2.3. Swelling Behavior

2.2.4. Collagen Release

2.2.5. Fourier-Transform Infrared (FTIR) Spectroscopy

2.2.6. Mechanical Compression Test

2.2.7. Diffusion Test

2.2.8. Drug Diffusion Test

2.3. Tumor Spheroid Preparation and Characterization

Morphological Analysis of the Tumor Spheroids

2.4. Cellular Assays

2.4.2. Mitochondria Toxicity Assay

2.4.3. Cisplatin Treatment

2.5. Immunofluorescence Microscopy Analysis

2.6. Enzymatic Digestion of Agarose for Spheroids Recovery

2.7. Ultrastructural Analysis of the Recovered Spheroids

2.8. Statistical Analysis

This effect is more evident as the 3D 3D structure

spheroids in the softest

3.3. Mammary Spheroids Viability and Epithelial Markers Expression

Figure 7.7. Staining

Pharmaceutics 2021, 13, 963

The analysis of the distribution of the fluorescent pixels performed on 25 spheroids

3.5. Enzymatic Digestion of Agarose for Spheroids Recovery

of multicellular tumor spheroids, as already demonstrated [1]. Expression of E-cadherin

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

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