KR101908056B1 - Electrical impedance monitoring system and method of c2c12 myoblast differentiation on an indium tin oxide electrode - Google Patents

Abstract

The present invention discloses an electrical impedance monitoring system of muscular cells. The present invention includes a cell chip, an extracellular matrix, an impedance measuring unit, and a control unit. The cell chip has a cell culture chamber attached over a patterned working electrode, counter electrode and electrodes on a transparent substrate. The extracellular matrix forms in the cell culture chamber. The impedance measuring unit applies an alternating current to the cell chip and measures the response potential of the culture liquid, culture cells and measuring electrode in the culture chamber. The control unit receives the measured value of the impedance measuring unit and determines whether the cultured cells are differentiated. According to the present invention, it is possible to quantitatively evaluate the differentiation of myofibroblasts in real time without labeling, and can be utilized in the development of a therapeutic agent for muscle diseases or a drug candidate.

Description

TECHNICAL FIELD [0001] The present invention relates to an electrical impedance monitoring system and a method for monitoring an electrical impedance of a muscle myoblast,

TECHNICAL FIELD The present invention relates to a system for monitoring electrical impedance of muscle myoblasts, and more particularly, to a method for monitoring myocyte differentiation by quantitatively evaluating myoblast differentiation in real time without a label using a transparent electrode and an extracellular matrix, To an electrical impedance monitoring system for myofibers that can be utilized.

The skeletal muscle, the largest organ of the body, accounts for 40% to 50% of the total body mass, and plays an important role in exercise, insulaion and metabolism of internal organs. Damage to muscle function can cause many pathological changes such as diabetes, obesity, muscle wasting, and aging. Thus, in vitro studies using muscle cells have been performed as a means of predicting in vivo effects.

However, for these studies it is necessary to reproduce well-defined differentiated cells, yet it is difficult to reproduce them in sufficient quantities. In order to achieve consistent results, new techniques are needed to quantitatively evaluate cell functions.

The C2C12 cell line is a myoblast cell line that expresses various proteins during myogenesis that differentiate into myotubes. C2C12 cells differentiated from C2C12 myofibroblasts are used in a variety of in vitro studies, including epidemiological studies on diseases such as diabetes, chronic heart failure and chronic kidney disease, as well as studies on muscle cell regeneration, metabolism, insulin action and muscle atrophy .

Accurate study of cell responses or actions to external stimuli should use real-time and nondestructive measurements that characterize physiological and morphological changes in the cell. Electrical cell-substrate impedance sensing (ECIS), developed by Giaver and Keese, is a method for detecting real-time and unlabeled cell behavior.

The frequency-dependent electrical impedance of the cell covering the electrode is obtained from the in-phase and reverse-phase potentials measured while applying a weak alternating electric field. The value of the cell parameter can be extracted using an equivalent circuit model designed to describe the current path and the potential distribution at the interface between the electrode and the cell.

ECIS allows high throughput and non-destructive screening of cellular responses to drug candidates or culture conditions. ECIS can be used to quantify cell adhesion, proliferation, metastasis, necrosis, apoptosis, wound healing, and differentiation.

Up to now, electrical impedance characteristics of human mesenchymal stem cell (hMSC) growth, hMSC differentiation into adipocytes, bone differentiation of hMSC, neural differentiation of hMSCs, growth of adipose tissue stem cells (ADSC) and aging of ADSCs have been reported.

In addition, the effects of electrode material and structure patterned by nanoparticles, graphene or nanoparticles and a graphene mixture on stem cell differentiation were investigated. Experiments measuring electrochemical signals of differentiated or undifferentiated stem cells have shown that electrochemical markers can be used to quantify the pluripotency of stem cells.

However, the ECIS monitoring system and protocol for myofibroblast differentiation has not yet been proposed.

Accordingly, an object of the present invention is to provide an electrical impedance monitoring system capable of quantitatively evaluating myofacial differentiation in real time without a label.

It is another object of the present invention to provide a cell chip structure which can be optically monitored by using a transparent electrode for monitoring electrical impedance of myofibroblast differentiation.

It is still another object of the present invention to provide an optimum measurement condition and method of an electrical impedance monitoring system using a transparent electrode.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

According to one aspect of the present invention, there is provided an electrical impedance monitoring system for muscular cells, comprising: a cell chip having a working electrode patterned on a substrate, a counter electrode, and a cell culture chamber attached to the working electrode; An extracellular matrix formed in the cell culture chamber; An impedance measuring unit for applying an alternating current to the cell chip and measuring the response potential of the culture solution, the cultured cells and the measuring electrode in the culture chamber; And a controller for receiving the measured value of the impedance measuring unit and determining whether the cultured cells are differentiated.

According to another aspect of the present invention, there is provided a method of monitoring electrical impedance of muscle myoblasts, comprising: patterning a working electrode and a counter electrode on a substrate and attaching a cell culture chamber; Forming an extracellular matrix in the cell culture chamber; Seeding myofibroblasts into the extracellular matrix, and adding a culture medium; Adding a differentiation medium to the myoblast; Applying an alternating current to the transparent electrode and measuring an impedance using a potential generated in response; And outputting the differentiation characteristics of the myoblasts at the impedance.

According to another aspect of the present invention, the frequency of the alternating current may be between 1 kHz and 100 kHz.

According to another aspect of the present invention, the muscle fiber may be C2C12.

According to another aspect of the present invention, the extracellular matrix may include laminin, collagen, and entatin.

According to another aspect of the present invention, the extracellular matrix may be formed to a thickness of 200 nm to 600 nm.

According to another aspect of the present invention, the working electrode and the counter electrode are formed of indium tin oxide (ITO), indium zinc oxide (IZO), aluminum zinc zinc oxide (AZO), zinc tin oxide (ZTO), gallium zinc oxide (GZO), antimony tin oxide (ATO), iron tin oxide (FTO) A-IGZO (In2O3: Ga2O3: ZnO) TiO2, In2O3, ZnO, SnO2, polyaniline, polypyrrole, (PEDOT), graphene, carbon nanotube (CNT), metal nano wire, or a mixture of these materials. Can be used.

According to another aspect of the present invention, the step of outputting the differentiation characteristic comprises: obtaining a resistance value of muscle myoblasts at an impedance obtained at the step of measuring the impedance; Dividing the initial resistance value by the resistance value to obtain a resistance change; Integrating the resistance change; And outputting the result of the integration.

Since the electrical impedance monitoring system of myofibroblast according to the present invention can quantitatively evaluate the differentiation of myofibroblasts in real time without labeling, it can be utilized in the development of a therapeutic agent for muscle diseases or a candidate drug.

The electrical impedance monitoring system of muscular atrophy according to the present invention can also optically monitor during electrical impedance monitoring using a transparent electrode. In addition, by coating the transparent electrode with an extracellular matrix, the myoblast can adhere well to the transparent electrode and proliferate and differentiate.

According to the method for monitoring electrical impedance of myofibroblast according to the present invention, the electrical impedance characteristics of myofibroblasts on a transparent electrode coated with an extracellular matrix can be explained by equivalent circuit modeling, It can be judged.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic block diagram of a system for monitoring electrical impedance of muscular atrophy according to an embodiment of the present invention; FIG.
2 is a perspective view of a cell chip according to an embodiment of the present invention.
3 is a graph showing an AFM measurement image and thickness of an extracellular matrix layer.
4 is a graph showing impedance measurement results according to the frequency of 0.9% aqueous NaCl solution in the cell culture chamber 180. FIG.
FIG. 5 shows microscopic photographs and cell counts of C2C12 cells cultured on ITO electrodes with and without differentiation media.
6 is a graph showing the relative levels of mRNAs differentiated from the cell culture dish and the ITO electrode during the differentiation process of myoblasts.
FIG. 7 shows the results of immunofluorescence analysis of changes in MHC and appearance expressed in canalicular cells differentiated from C2C12 myofibroblasts.
8 is a graph showing resistance and resistance change measured by the ECIS method during the growth of C2C12 myofibrobium on the ITO electrode.
Figure 9 is a graph showing the integration of resistance and resistance change at 21.5 kHz with and without differentiation media.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings and the following description. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the embodiments disclosed herein are being provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. Like reference numerals designate like elements throughout the specification. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. &Quot; comprises "and / or" comprising ", as used herein, unless the recited element, step, operation, and / Or additions.

Hereinafter, a detailed description will be given of an electrical impedance monitoring system for muscular cells according to an embodiment of the present invention, with reference to FIGS. 1 to 9. FIG.

FIG. 1 is a schematic block diagram of a system for monitoring an electrical impedance of muscle myoblasts according to an embodiment of the present invention, and FIG. 2 is a perspective view of a cell chip according to an embodiment of the present invention.

Referring to FIGS. 1 and 2, an apparatus for monitoring an electrical impedance of muscular cells according to an embodiment of the present invention includes a cell chip 100, an impedance measuring unit 300, and a controller 400.

The electrodes 142 and 146 of the cell chip are connected to the impedance measuring unit 300 through the electric circuit 350. The impedance measuring unit 300 includes a voltage generator, a lock-in amplifier, and a multiplexer. The control unit 400 is connected to the impedance measuring unit.

The cell chip 100 includes a working electrode 146 and a counter electrode 142 formed by coating an electrode layer 140 on a substrate 120 and then patterning the electrode layer 140. The cell chip may have a chamber 180 capable of containing the culture medium so that cell culture on the working electrode is possible. The working electrode and the counter electrode may be connected to the terminal pad 144 via the transmission line 148. The transmission line 148 may be coated thereon with a photoresist 160 to insulate it.

In order to optically monitor cell growth during cell growth, transparent material such as glass, quartz, or transparent plastic can be used as the substrate 120.

The working electrode and the counter electrode may also be transparent electrodes. The transparent electrode can be made of a conductive oxide, a conductive polymer, a nano device, or the like.

Examples of the conductive oxide include indium tin oxide (ITO), indium zinc oxide (IZO), aluminum zinc oxide (AZO), zinc tin oxide (ZTO), gallium zinc Gallium zinc oxide (GZO), antimony tin oxide (ATO), iron tin oxide (FTO), A-IGZO (In2O3: Ga2O3: ZnO) titanium dioxide (TiO2) (In2O3), zinc oxide (ZnO), tin oxide (SnO2), and mixtures thereof.

Examples of the conductive polymer include polyaniline, polypyrrole, poly (3,4-ethylenedioxythiophene) (PEDOT), and the like.

As the nanomaterial, graphene, carbon nanotube (CNT), metal nano wire, or the like can be used.

The insulating layer 160 may also be a transparent insulating layer. The transparent insulating layer may be made of silicon dioxide (SiO ₂ ), silicon nitride (SiN x), parylene, poly (4-vinylphenol), polyvinylphenol (PVP), poly melamine-co-formaldehyde (PMF), polyethylene naphthalate (PEN), transparent epoxy, or the like.

The cell culture chamber 180 can be fabricated using polystyrene or the like, and can be attached to an electrode or an insulator with an adhesive silicone or the like. In Figure 2, eight reservoirs are shown to accommodate the cell culture medium, but the number can be increased or decreased as needed.

The extracellular matrix 200 may be formed on the working electrode 146 of the cell culture chamber 180. The extracellular matrix can be composed of materials such as laminin, collagen and entactin. Enstatin is a cross-linking molecule that interacts with laminin and collagen and contributes to the structural organization of these extracellular matrix molecules.

The extracellular matrix thickness, cell differentiation, and electrical impedance measurement sensitivity are mutually related. Thus, for an electrical impedance monitoring system of muscle myoblasts, it is necessary to select an optimal extracellular matrix thickness.

To do this, first coat the extracellular matrix and check its thickness. The cell culture / differentiation is checked whether it is normal, and the sensitivity is analyzed by measuring the impedance. This process can be repeated to select the optimal substrate thickness.

The extracellular matrix 200 preferably has a thickness of 200 nm to 600 nm. If the thickness is too thin, the thickness may become uneven or the adhesion between the cell and the electrode may be insufficient. If the thickness is too thick, the capacitive impedance may increase and the impedance error of the cell may increase.

Referring to FIGS. 1 and 2, a method of operating an electrical impedance monitoring system for muscular cells according to an embodiment of the present invention will be described.

The working electrode and the counter electrode are patterned on the substrate, the cell culture chamber is attached, and the extracellular matrix 200 is formed in the cell culture chamber 180. A predetermined amount of the cultured cells is seeded in a cell culture chamber in which the extracellular matrix is formed, and a culture medium is added.

As the cultured cells, myoblast, C2C12, C2, Sol8, L6 and the like can be used.

The cell chip 100 including the cell culture chamber containing the cultured cells is placed in an incubator (not shown). The incubator can use a device capable of controlling the temperature, humidity and CO ₂ concentration. The incubator is preferably maintained at a temperature of 32 to 42 ° C and a CO ₂ concentration of 2 to 8% so as to be suitable for culturing and differentiation of myoblast cells.

The cell chip entering the incubator is connected to the impedance measuring unit 300 through an electric circuit 350 connected to the terminal pad 144 of the cell chip by a connector or the like not shown in the figure.

An impedance measuring unit (300) flows an alternating current and measures the response potential generated in the cell chip. The response potential measured by the impedance measuring unit is transmitted to the control unit 400 through the GP-IB or the like.

The control unit can obtain the total impedance at the response potential. The total impedance can be determined by the sum of the constant phase element (CPE) of the electrode interface impedance, the parallel impedance of the _cell impedance (R _cell ) and the capacitor (C _cell ), and the resistance of the _solution (R _solution ) (See the equivalent circuit of Fig. 8A).

The control unit 400 not only controls the impedance measuring unit, but also calculates the total impedance, and can grasp and display the degree of differentiation of muscular cells based on the calculation results.

The resistance value (R _cell ) of myoblasts can be calculated by the equivalent circuit fitting analysis on the total measured impedance. The degree of differentiation was determined by dividing the resistance value (R ₀ ) of the myoblast initially measured in the resistance value (R _cell ) of the myoblast by the resistance change (ΔR = R _cell / R ₀ ). When the frequency is too small or too large, the resistance change (ΔR) due to differentiation of myoblasts may be observed to be relatively small, and the measurement sensitivity may be reduced. Therefore, it is preferable to use 1 kHz to 100 kHz as the alternating frequency in the myocardial electrical impedance monitoring system.

The degree of myoblast differentiation can be monitored by monitoring the resistance value (R _cell ) of myoblasts, but the result of integrating the resistance change (ΔR) can be used for faster and more accurate judgment.

Impedance measurement is advantageous in that measurement can be performed even when the cell chip 100 is in an incubator. When a transparent substrate, a transparent electrode, or the like is used, optical analysis can be performed simultaneously with the impedance measurement.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1: Fabrication of cell chip structure based on ITO electrode

Referring to FIG. 2, an ITO electrode 140 having a thickness of 480 nm is patterned on a slide glass (75 mm x 25 mm x 1.1 mm) to form eight working electrodes 146, a common counter electrode 142, (148) and the terminal pad (144).

An epoxy-based photoresist (SU-8 2002, Microchem, Newton, MA, USA) was coated (160) to a thickness of 2 袖 m using spin coating and photolithography to insulate the transmission line 148 of the ITO electrode chip . As a result, the exposed working electrode 146 was disk-shaped with a radius of 250 mu m and separated from the counter electrode 142 by a distance of 3 mm.

Eight polystyrene cell culture chambers 180 were then attached to the ITO electrode-based chips using adhesive silicone, providing a reservoir to receive the cell culture medium.

Example  2 : C2C12 Myoblast  culture Preparatory cell  Substrate gel coating

In order to achieve stable cell adhesion to the ITO electrode, the surface of the electrode substrate was immersed in an extracellular matrix (ECM) gel of 100 μL of Matrigel (0.9 to 1.2 mg · mL ^-1 , BD Matrigel ™ Matrix, BD Biosciences , San Jose, CA, USA).

Matrigel is derived from tumor cells rich in extracellular matrix proteins and consists of approximately 60% laminin, 30% collagen IV and 8% entactin. Enstatin is a cross-linking molecule that interacts with laminin and collagen and contributes to the structural organization of these extracellular matrix molecules.

Matrigel contains perlecan, TGF-beta, epithelial growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator and other growth factors. And residual substrate metalloproteinases derived from tumor cells.

When the thickness of the extracellular matrix gel is thin, the thickness becomes uneven or the adhesion of the cell becomes problematic. If the thickness of the extracellular matrix gel is large, there is a problem that the capacitive impedance increases. Therefore, the thickness of the extracellular matrix gel is preferably 200 nm to 600 nm.

The thickness of the Matrigel layer, an extracellular matrix gel coated on the ITO electrode, was measured using an atomic force microscope (AFM, XE-100, Parks Systems, Suwon, Korea). 3 is a graph showing an AFM measurement image and thickness of an extracellular matrix layer. FIG. 3A is an AFM image of the surface topology of the extracellular matrix layer, and FIG. 3B shows the thickness according to position.

From the measurement results, it can be seen that the extracellular matrix layer was uniformly coated on the ITO electrode surface to a thickness of about 410 nm.

The impedance of the ITO electrode coated with the extracellular matrix gel was compared with that of the ITO electrode coated with the extracellular matrix gel. 4 is a graph showing impedance measurement results according to the frequency of 0.9% aqueous NaCl solution in the cell culture chamber 180. FIG. 4A shows the magnitude of the impedance according to the frequency, and FIG. 4B shows the change in the phase according to the frequency.

The impedance characteristics of the electrodes are determined by the electrode interface impedance at low frequencies and by the solution resistance at high frequencies. When the ITO electrode was coated with an extracellular matrix gel, the reactance slightly increased at low frequencies, resulting in an increase in the impedance magnitude and a decrease in phase. These results can be derived from the additional capacitance characteristics of the extracellular matrix gel coat layer connected in series with the capacitive ITO electrode impedance.

Example  3: C2C12 Myoblast  differentiation

10 ⁵ cells / mL of C2C12 myoblast were seeded on Matrigel coated ITO electrodes. Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Welgene, Daegu, Korea), 100 IU · mL- ¹ penicillin, 0.1 mg · mL- ¹ streptomycin ; DMEM). The incubation temperature was maintained at 37 ° C and the CO ₂ concentration was maintained at 5%.

After 3 days of culture, the cells were cultured in DMEM, 2% horse serum (HS, Gibco, Carlsbad, CA, USA), 100 IU.multidot.s ^-1 penicillin, 0.1 mg.multidot.l.sup.- ¹ streptomycin Lt; RTI ID = 0.0 > C2C12 < / RTI >

In the control group, the culture medium was converted into maintenance medium (DMEM, 0.5% FBS, 1% penicillin) to inhibit cell proliferation and prevent cell layer detachment due to excessive cell growth.

The number of cells cultured in the differentiation and maintenance media on the ITO electrode was measured using ImageJ software (NIH, Bethesda, MD, USA).

FIG. 5 shows microscopic photographs and cell counts of C2C12 cells cultured on ITO electrodes with and without differentiation media. 5A is a micrograph of the ITO electrode (black scale at the bottom right of each photograph is 100 μm), and FIG. 5B is the number of cells on the ITO electrode.

Referring to FIG. 5A, C2C12 cells adhered, spread and proliferated on gel coated ITO electrodes during culture in growth medium (DMEM) containing 10% fetal bovine serum (FBS).

When cultured for 3 days in growth medium and replaced with differentiation medium containing 2% horse serum (HS), myotube morphology was observed between 7 and 11 days of culture. On the other hand, control cells did not show any morphological changes in relation to differentiation into myotubes.

Referring to FIG. 5B, it can be seen that source differentiation affects cell proliferation. The number of C2C12 cells in the ITO electrode with differentiation medium was lower than the number of C2C12 cells in the control group from 7 days to 11 days of culture.

Example  4 : C2C12 Myoblast  Isolation and quantification of RNA during culture and differentiation PCR  Perform

The total RNA (total RNA) was isolated from the cells using Trizol reagent according to the manufacturer's protocol (RNAiso Plus, TaKaRa, Dalian, China). The amount of RNA was measured using an ND-1000 spectrophotometer (Thermo Scientific, Waltham, Mass., USA).

CDNA was synthesized from 2 μg of RNA using PrimeScript 1st strand cDNA synthesis kit (TaKaRa). Then, PCR (polymerase chain reaction) was performed. Quantitative real-time PCR (qRT-PCR) was performed in a reaction mixture containing a fluorescent dye SYBR green master mix (TaKaRa). PCR amplification was performed 40 times using the CFX384 Touch ™ real-time PCR detection system (Bio-Rad, Munich, Germany) and discontinued. Relative gene expression levels were normalized according to the CT (cycle threshold) method. The sequences of the primer pairs used in the PCR are shown in Table 1 below.

Kinds direction Sequences (5 '-> 3') SEQ ID NO: Cyclophilin Forward TGGAGAGCACCAAGACAGACA SEQ ID NO: 1 Reverse TGCCGGAGTCGACAATGAT SEQ ID NO: 2 MyoD Forward CTTCTATCGCCGCCACTC SEQ ID NO: 3 Reverse AAGTCGTCTGCTGTCTCAA SEQ ID NO: 4 Miogenen Forward CCAACCCAGGAGATCATTTG SEQ ID NO: 5 Reverse ACGATGGACGTAAGGGAGTG SEQ ID NO: 6 MHC
(Myosin H chain) Forward ACAAGCTGCGGGTGAAGAGC SEQ ID NO: 7 Reverse CAGGACAGTGACAAAGAACG SEQ ID NO: 8

MyoD is expressed in myoblasts and decreases during differentiation. On the other hand, myogenin and MHC are expressed at later stages of myogenesis.

6 is a graph showing the relative levels of mRNAs differentiated from the cell culture dish and the ITO electrode during the differentiation process of myoblasts.

6A, by analyzing the expression of myogenic factors involved in muscle differentiation, it was found that the level of MyoD mRNA expressed in the differentiation process of myoblasts decreased during the differentiation process in both the cell culture dish and the ITO electrode did.

Referring to FIG. 6B, it can be seen that the level of myogenin mRNA is increased in both the cell culture dish and the ITO electrode during the differentiation process of myoblasts.

Likewise, referring to FIG. 6C, the mRNA level of HMC in the differentiation process of myoblasts also increased in both the cell culture dish and the ITO electrode.

Example  5: C2C12 Myoblast  Immunofluorescence analysis during culture and differentiation

Cells in the differentiation process of myoblasts were fixed with 4% paraformaldehyde for 20 minutes at room temperature and washed with phosphate buffered saline (PBS). Cells were then infiltrated with PBS containing 0.27% Triton X-100 at room temperature and incubated with protein inhibitory solution (DAKO).

Cells were exposed to anti-MHC antibody overnight at 4 ° C and then stained with DAPI and incubated with secondary antibody conjugated with fluorescein isothiocyanate (FITC), a fluorescent labeling reagent. And fluorescence images of the cells were taken using a confocal microscope.

FIG. 7 shows the results of immunofluorescence analysis of changes in MHC and appearance expressed in canalicular cells differentiated from C2C12 myofibroblasts. MHC expression is detected with anti-MHC antibody (green), and nuclei detected with DAPI staining (blue).

In the second column (DAPI) of FIG. 7, the same blue fluorescence reaction was observed in the control group (Control) at the top and differentiated C2C12 cells at the bottom. Both the control and differentiated cells show that the nuclei are normally present.

In the first column (MHC) of Fig. 7, no fluorescence reaction was observed in the control group at the uppermost level, but distinctly fluorescent fluorescence was observed in the differentiated C2C12 cells at the lower part thereof. It can be confirmed that MHC expressed in the root canal is present only in the differentiated C2C12 cells.

In other words, only the nuclei were observed in the control group of undifferentiated C2C12 cells, but in the differentiated C2C12 cells, multinucleated canaliculus cells and MHC expression were increased. This result shows that C2C12 cells were successfully differentiated from ITO electrodes into canalic cells using the previous differentiation protocol.

Example  6: C2C12 Myoblast  Measurement of electrical impedance during culture and differentiation

Using a digital lock-in amplifier and a multiplexer (34970A, Agilent Technologies, Inc., Santa Clara, Calif., USA) equipped with a voltage generator (SR830, Stanford Research Systems, Sunnyvale, CA, USA) for cell culture and differentiation, The electrical impedance spectra were measured in the frequency range of 100 Hz to 100 kHz.

1 is a photograph of an experimental apparatus for measuring the impedance of a cell.

In order to block the influence of the electric field on the C2C12 cell, the resistance was connected in series with the voltage generator, and the amplitude of the current flowing was limited to 1 μA or less.

Instruments used for electrical cell-to-board impedance measurements (ECIS) were controlled by a LabVIEW program (National Instruments, Austin, Tex., USA) via general purpose interface bus (GP-IB) communication. Impedance data were calculated from the measured potentials when applying frequency-dependent currents.

8 is a graph showing resistance and resistance change measured by the ECIS method during the growth of C2C12 myofibrobium on the ITO electrode.

Figure 8a shows the resistance (R) values of C2C12 myofibroblasts on ITO electrodes measured during growth of C2C12 myoblasts. The line is the result of calculation using the equivalent circuit model.

In the equivalent circuit model, the total impedance is the constant phase element (CPE) of the electrode interface impedance, the parallel impedance of the _cell impedance (R _cell ) and the capacitor (C _cell ), the resistance of the _solution . The electrical impedance characteristics of the cells on the ITO electrodes fit well with the equivalent circuit model in the frequency range of 100 Hz to 100 kHz.

In FIG. 8B, the change in resistance (R = R _cell / R ₀ ) is shown for each incubation time. This value was most severely changed at the frequency of 21.5 kHz during cell growth. Referring to FIG. 8B, the impedance measurement was clearly distinguishable from 1 kHz to 100 kHz.

Figure 9 is a graph showing the integration of resistance and resistance changes at 21.5 kHz with and without differentiation media, i.e., C2C12 myoblast growth or differentiation.

In Fig. 9A, the symbols are n = 4 resistance averages of the cell culture samples, and the bars indicate their standard errors. Arrows marked near 72 hours in the figure indicate the times when the culture medium was replaced with differentiation medium or maintenance medium.

The line at the uppermost line in FIG. 9A is the measurement result of the control group without the differentiation. The dashed line gradually decreasing from 72 hours is the measurement result of the differentiation medium, and the lower line parallel to the x axis is the measurement result when there is no cell .

All culture media were replaced every day with the measurement of the impedance of the cells. Before applying the differentiation medium, the resistance increases with variability, due to cell attachment and diffusion to the ITO electrode.

However, the resistance value gradually decreased during myoblast differentiation, and was differentiated from the resistance value of the control group after the time of differentiation of 116 hours (188 hours after the start of the experiment, indicated by a dotted line and * in FIG. 9A) Were performed by IBM SPSS statistics, Student's t-test p-value <0.05). The decrease in resistance during differentiation was due to a decrease in cell density at the electrode, and there was also a change in cell morphology.

Figure 9b shows the integration of the resistance change at 21.5 kHz in C2C12 cells of the ITO electrode during ITO electrode cell growth or differentiation. The slope of the uppermost slope is the result of measurement of the non-differentiated control group. The dotted line showing the slope change from the 100th hour is the measurement result of the differentiation medium, and the dotted line showing the lowest slope is the measurement result to be.

The integration of the resistance change values led to a rapid detection of cell differentiation as well as a reduction in the deviation between the measured values. Referring to Figure 9b, which shows the integration of the resistance change at 21.5 kHz, cell differentiation was distinguishable from the control group after 30 hours of differentiation (102 hours after the start of the experiment, marked with * in Figure 9b and dotted line) (Student's t-test p-value <0.05).

That is, by using the integration of the resistance change value, it is possible to judge whether or not the C2C12 myofibroblast differentiates within a much shorter time than whether the resistance value is differentiated.

In the above examples, C2C12 myofibroblasts were well adhered, proliferated and differentiated on an extracellular matrix-coated ITO electrode. Differentiation at the electrodes of C2C12 cells was confirmed by analysis of myogenic gene expression, changes in cell morphology, and immunofluorescence.

The electrical impedance characteristics of the C2C12 cell on the ITO electrode coated with the extracellular matrix could be explained by equivalent circuit modeling. During the differentiation of C2C12 myofibroblasts, which resulted in changes in cell morphology and density, the integration of the resistance change at 21.5 kHz was separated from the control values at 30 hours of differentiation time.

Therefore, it is possible to monitor the C2C12 myofibroblast differentiation state in real time without labeling by using the electrical impedance analysis using the ITO electrode coated with the extracellular matrix. Therefore, it is expected that the electric impedance analysis system using the ITO electrode coated with the extracellular matrix can be used for the development of a therapeutic agent for muscle diseases or a drug candidate.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, I will understand. Therefore, the scope of the present invention should not be limited to the above-described embodiments, but should be determined by all changes or modifications derived from the scope of the appended claims and the appended claims.

100 cell chip
200 extracellular matrix
300 Impedance measuring unit
400 controller

Claims (13)

delete delete delete delete delete delete In a method for monitoring electrical impedance of muscle myoblasts,
(a) patterning a working electrode and a counter electrode on a substrate and attaching a cell culture chamber;
(b) forming an extracellular matrix comprising enstatin in a cell culture chamber in which the electrode is patterned and having a thickness of 200 nm to 600 nm;
(c) seeding myofibroblasts to the extracellular matrix and adding a culture medium;
(d) adding a differentiation medium to the myoblasts;
(e) applying an alternating current of 1 μA or less having a frequency of 21.5 kHz to the working electrode and the counter electrode and measuring an impedance using a potential generated in response;
(f) outputting the differentiation characteristics of the myoblasts at the impedance,
(F) outputting the differentiation characteristic comprises:
(f-1) obtaining a resistance value of myofibroblast by analyzing the equivalent circuit fitting at the impedance obtained in the step of measuring the impedance;
(f-2) dividing the initial resistance value by the resistance value to obtain a resistance change;
(f-3) integrating the resistance change;
(f-4) obtaining a graph of a result of the integration over time and obtaining a slope of the graph; And
(f-5) determining that the slope of the graph changes according to myofacial differentiation, and outputting the determined result. delete 8. The method of claim 7,
Wherein the myofibroblast is any one of C2C12, C2, Sol8, and L6. delete delete 8. The method of claim 7,
The working electrode and the counter electrode may be formed of indium tin oxide (ITO), indium zinc oxide (IZO), aluminum zinc oxide (AZO), zinc tin oxide (ZTO) Gallium zinc oxide (GZO), antimony tin oxide (ATO), iron tin oxide (FTO), A-IGZO (In2O3: Ga2O3: ZnO), titanium dioxide (TiO2) , Indium oxide (In2O3), zinc oxide (ZnO), tin oxide (SnO2), polyaniline, polypyrrole, poly (3,4-ethylenedioxythiophene); A method of monitoring an electrical impedance of muscovite characterized by using PEDOT, graphene, carbon nano tube (CNT), metal nano wire, or a mixture thereof. delete

Images