Abstract
In this paper, a bioimpedance sensor used to measure the impedance
behavior of the biological cells cultured on a scaffold of collagen thin
films. The collagen thin films with different concentrations were
created on a 1-hexadecanethiol modified surface of the interdigitated
electrodes(IDEs). The mesenchymal stem cells(MSCs) and human umbilical
vein endothelial cells(HUVECs) were cultured on the collagen thin films.
The interaction and the proliferation rate of these cells were
investigated by the microscopic, and bioimpedance approaches. Results
showed that the MSCs, and HUVECs excellently attached to the collagen
thin films. Impedance measurements of the cells cultured on the collagen
thin film were performed by a Hioki IM3570 impedance analyzer at the
frequency band of 10 kHz to 1MHz at 10 mV. Bioimpedance measurements
showed that the proliferation rate of both MSCs, and HUVECs increased by
decreasing the collagen thin films concentration. After 48 h, the
differential impedances () of the MSCs cultured on the collagen thin
layers with concentrations of , , and measured , , and , respectively.
Also, of the HUVECs obtained , , and for the corresponding frequency
band and collagen thin films concentrations.
Keywords: Collagen Thin film; Alkanethiolate-treated
electrodes; Bioimpedance spectroscopy; Biological cells.
Introduction
The main challenge encountering the biotechnology is the capability of
quantitative evaluation of biomarker expression in living cells.
Toxicology and drug screening rely on cell-based approaches, hoping that
quantitative cell respond will demonstrate a more trusty foreteller of
clinical results than molecular-scale approaches[1]. The development of modified matrices and
quantitative techniques help obtain more reliable, reproducible, and
meaningful cell-based measurements. Thin films of extracellular matrix
(ECM) proteins have been used as helpful apparatuses in various
biological examinations [2]–[9], which give
controlled surfaces to cell-based tests. A controlled surface is vital
for the examination of cell proliferation, migration, and
differentiation [7].
Collagen is the primary fibrous protein in the ECM. In type I collagen
molecule, there are three amino acid chains that form rod-shaped triple
helices. The triple helices are assembled and form fibrils; then bundles
and fibers are formed by lateral aligning of the fibrils[10], [11]. Thin films of collagen are
beneficial to coordinate different biological issues, for example, cell
morphology [4], [6], [12], the impact of
surface attributes on intracellular flagging [4],
and the collaboration between the surface and integrin receptors[8]. These films elicit the same cell conduct as
thick collagen gels, but they are generally more reproducible and
simpler to depict [3]. The collagen thin films are
more straightforward than thick collagen gels so that singular fibrils
can be portrayed by optical microscopy, permitting more thorough
investigations of the connection among cells and fibrils[7]. Besides, a disadvantage of thick collagen
gels is that they are challenging to plan methodically from one trial to
another [3]. The collagen gels can easily detach
from their supporting surfaces, and visible variety in the gel can be
seen by the eye. Also, quantitative examination of cell attributes on
these matrices can be convoluted on account of light scattering from the
fibrillar gel and catching of fluorescent reagents[3].
Bioimpedance points out the objection of the biological specimens like
bacteria, biological cells, and living tissues to the electric current
stream. Bioimpedance measurements are progressively used in biomedical
utilizations and cell investigations[13]–[17]. Electrical cell-substrate
impedance sensing (ECIS) is the phrase given to a specific kind of
bioimpedance measurement explicitly pertinent to measuring the
electrical impedance of adherent living cells on planar microelectrodes[18]. ECIS has been utilized to show various
properties of cells, for example, attachment and spreading[19]–[23], motility[24], [25], and growth and proliferation[26], [27]. Bioimpedance analyses have been
used as a reliable means to distinguish healthy and cancerous cells and
to determine metastatic attributes [28]–[37].
Impedance measurements of adherent cells can discover variations in cell
morphology on the order of nanometers [38],
providing sensitivity more significant than that got by visual
inspections. Generally, impedance measurements can discover a small
variation in cell capacitance and resistance attached to the measuring
microelectrodes.
In our previous work, the collagen thin films were created on the
electrode surface modified by oxygen plasma; and the impact of the
deionized water on the measured impedance of the collagen thin films was
examined [39]. Results showed that the collagen
thin films impedance in the presence of deionized water decreased by
increasing the collagen thin films concentration. Due to the dependence
of the collagen thin films impedance on the collagen concentration, in
the cellular experiments, the proliferation rate of the cells has been
investigated using the differential impedances () during the time.
In this paper, we portray the preparation and characterization of
collagen thin films on alkanethiol-treated gold interdigitated
electrodes (IDEs) and the evaluation of their biomimetic nature by
microscopic approaches such as field-effect scanning electron microscopy
(FESEM). Then, the proliferation rate of mesenchymal stem cells derived
from rabbit adipose tissue (MSCs), malignant breast cancer cells
(MDA-MB-231), and human umbilical vein endothelial cells (HUVEC) on the
collagen thin films with different concentrations was investigated by
bioimpedance measurements. The use of fibrillar collagen thin films
offers excellent cellular collaboration with the collagen scaffold and
reproducibility of the scaffold preparation.
Materials and methods
Biosensor construction
Microscope glass slides, as a substrate for electrodes, were cleaned
through the standard RCA #1 method (a mixed solution of
NH4OH:H2O2:H2O
in volume ratio of 1:1:5, respectively). Subsequently, a thin layer of
Ti (50 nm) was deposited on the glass substrate by sputtering to improve
the gold adhesion to the substrate. A ~200 nm thin layer
of gold was deposited on Ti by a sputtering system (Veeco Co.). The gold
was planned using soft lithography and the design of the IDEs with
100μm width and a distance of 100μm between every
two branches that transferred on the glass substrate.
Collagen solutions
preparation
Dry Type 1 Collagen, gotten from calf skin, was bought from the Zist
Farayand Tajhiz Sahand (Tabriz). A sum of 36 mg of collagen was
dissolved in 12 ml of 0.4 M acetic acid (Sigma Aldrich, CAS No:
64-19-7)
using a magnetic stirrer at \(4\ \) (refrigerator); and a stock solution
of 3 mg/ml concentration was prepared. Before dissolving, dry collagen
was exposed to ultraviolet radiation for 5-10 minutes to be sterilized.
To prepare the neutralized collagen solution, the cold stock solution
was blended with the cold concentrated (10X) PBS, and cold 0.5M NaOH
(Sigma Aldrich, CAS No. 1310-73-2) in a 8:1:1 ratio to attain
physiologically PH and ionic strength conditions under which collagen
monomer effectively polymerizes into fibrils at \(37\ \). The obtained
neutralized solution was diluted with (1X) PBS, to obtain the desired
concentration of collagen solution. The collagen solution was
neutralized on ice to keep the collagen from polymerization during the
neutralization process.
Preparation of alkanethiol-coated interdigitated electrodes
Chambers of Plexiglas were created and attached to the substrates in a
way that embedded the fabricated electrodes. The amount of 600 \(\mu\)L
of 2mM 1-hexadecanethiol (Sigma Aldrich, CAS No: 2917-26-2 ) solution in
absolute ethanol (Sigma Aldrich, CAS No. 64-17-5 ) was poured in the
chambers and remained overnight under the biological flow hood. After
that, the electrodes were washed with ethanol and dried under biological
flow hood before incubation with collagen solution. Alkanethiol-coated
samples could be held under ethanol at least for seven days without any
damage in efficiency [3]. 1-hexadecanethiol is a
chemical material with formula
CH3(CH2)15SH; its head
group (SH) chemically connected to the gold electrodes, and its
hydrophobic tail group (CH3) creates a chemical bond
with the collagen molecules.
Preparation of type 1 collagen thin films
The amount of 400 \(\mu\)L of cold neutralized collagen solution was
poured in each of the alkanethiol-treated IDEs and then were incubated
overnight at \(37\ \) in a 5% CO2 incubator to permit
collagen polymerization. Collagen thin films were constructed by
permitting collagen molecules to absorb to a hydrophobic alkanethiol
surface. The collagen-coated electrodes were rinsed several times with
(1X) PBS, and then with sterile deionized water to remove all loosely
adhered collagen molecules or fibrils from the surface. Subsequently,
the samples were briefly dried under a steam of air under biological
flow hood and then placed into a (1X) PBS solution at \(4\ \) ready for
use with cells.
For characterization of the collagen thin films, the samples were
pictured using a field-effect scanning microscopy (FESEM). Images were
obtained from various regions on any sample to make certain the
homogeneity of surface specifications. The FESEM images of the created
collagen thin films of various concentrations on the
1-hexadecanethiol-treated IDEs is shown in Fig. 1