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