KR20190041643A - Impedance sensor array for culturing cells and method of identifying the states of the cells using the same - Google Patents

Abstract

The present invention provides an impedance sensor array for culturing a cell and a method for checking the state of a cell using the same. The impedance sensor array for culturing a cell is installed on a cell culturing substrate for culturing a cell and monitors the culturing state of the cell. The impedance sensor array for culturing a cell includes multiple impedance sensors. The method for checking the state of a cell checks the state of the cell by using the impedance sensor array for culturing a cell, which monitors the culturing state of the cell by being installed on the cell culturing substrate to culture the cell. The cell culturing substrate includes N number of cell culturing areas from a first cell culturing area to an N^th cell culturing area. The impedance sensor array for culturing a cell includes N number of the impedance sensors from a first impedance sensor to an N^th impedance sensor. The first to N^th impedance sensor corresponds to the first to N^th cell culturing area one to one and has an impedance sensor in the single cell culturing area. Moreover, the method for checking the state of a cell includes a step of monitoring the culturing state of the cell by the cell culturing area in order from the first impedance sensor to the N^th impedance sensor.

Description

TECHNICAL FIELD [0001] The present invention relates to an impedance sensor array for cell culture and a method of identifying a cell state using the same. [0002]

TECHNICAL FIELD The present invention relates to an impedance sensor array for cell culture and a method for identifying a cell state using the same.

Mass cell cultures are increasing in the pharmaceutical industry for individual cell therapy and in the cosmetics industry for in vitro toxicity testing. However, conventional cell culture systems have limited versatility, including sensing and operation, smart feedback control and in situ culture medium exchange systems, and are unsuitable for low cost and factory scale mass cell culture.

In order to solve the above problems, the present invention provides an impedance sensor array for cell culture, which is excellent in cell culture.

The present invention provides an impedance sensor array for cell culture for culturing a large amount of cells.

The present invention provides a method for identifying a cell state using the impedance sensor array for cell culture.

Other objects of the present invention will become apparent from the following detailed description and the accompanying drawings.

The impedance sensor array for cell culture according to an embodiment of the present invention is an array of impedance sensors for cell culture that is disposed on a cell culture substrate for culturing cells and monitors the culture state of the cells, And includes a plurality of impedance sensors.

The cell culture substrate includes N cell culture regions from the first cell culture region to the Nth cell culture region, and the cell culture impedance sensor array includes N impedance sensors from the first impedance sensor to the Nth impedance sensor And the first to Nth impedance sensors correspond to the first to Nth cell culture regions at a ratio of 1: 1, and one impedance sensor may be disposed in one cell culture region.

The cell culture impedance sensor array may monitor the culturing state of the cells in each cell culture area sequentially from the first impedance sensor to the Nth impedance sensor.

The impedance sensor is electrically connected to a wireless communication system disposed adjacent to the cell culture substrate, and the wireless communication system can wirelessly communicate with an external device that controls the impedance sensor.

The method for confirming the cell state according to another embodiment of the present invention includes a method of confirming the state of the cell using an impedance sensor array for cell culture, which is disposed on a cell culture substrate for culturing the cells, Wherein the cell culture substrate includes N cell culture areas from the first cell culture area to the Nth cell culture area, and the cell culture impedance sensor array includes N impedance sensors from the first impedance sensor to the Nth impedance sensor, Wherein one of the first to Nth impedance sensors has one impedance sensor disposed in one cell culture region corresponding to the first to Nth cell culture regions at a ratio of 1: And monitoring the culture condition of the cells in the cell culture area sequentially to the Nth impedance sensor All.

According to embodiments of the present invention, it is possible to wirelessly control a plurality of cell culture platforms accommodated in a plurality of incubators with a small number of cells in a cell culture plant, thereby enabling a large amount of cells to be cultured at low cost.

The incubation state of the cells can be confirmed in real time without opening the incubator, and cell proliferation and differentiation can be promoted by stimulating the cells. In addition, it is possible to confirm the culturing state of the cells for each region of the cell culture substrate, and stimulate the cells only in the region where the culturing state is insufficient, thereby promoting cell proliferation and differentiation. As a result, cell culture can be uniformly performed on the whole area of the cell culture substrate.

The cell culture substrate can be easily laminated, which is suitable for mass culture of cells, and can maintain high cell viability even when laminated in multiple layers. The graphene oxide coated on the cell culture substrate can improve the adhesion of the cell and avoid the use of the cell adhesion aid.

1 and 2 show a cell culture platform according to an embodiment of the present invention.
Figure 3 schematically shows an array of sensors and heaters according to one embodiment of the present invention.
4 shows a schematic image of a cell culture substrate according to an embodiment of the present invention.
5 shows a manufacturing process of a cell culture platform according to an embodiment of the present invention.
FIG. 6 shows a stacked image of a cell culture platform according to an embodiment of the present invention.
7 is an image of a cell culture plant using a cell culture platform according to an embodiment of the present invention.
8 is a view for explaining a coating process of graphene oxide (GO) according to an embodiment of the present invention.
9 shows an AFM image of a bare PLA substrate and a PLA (GO-PLA) substrate coated with graphene oxide.
10 shows Raman spectra of the bare PLA substrate, GO-PLA substrate, and GO powder.
Figures 11 and 12 show SEM images of GO-PLA substrates.
13 shows SEM images of PLA and GO-PLA substrate cells according to the cell culture period.
14 is a graph showing the number of cells according to the type of cell culture substrate.
15 shows a cell image arranged on a cell culture substrate.
16 shows a cell culture platform comprising a laminated cell culture substrate immersed in a medium for testing cell viability.
17 to 19 are views for explaining the cell survival rate according to the number of laminates and the depth of the culture medium of the cell culture substrate.
20 shows an impedance sensor according to an embodiment of the present invention.
21 shows a calibration curve of the impedance sensor of Fig.
22 shows a heater and a temperature sensor according to an embodiment of the present invention.
23 shows a calibration curve of the heater and the temperature sensor of Fig.
24 shows a pH sensor according to an embodiment of the present invention.
25 shows a calibration curve of the pH sensor of Fig.
26 shows a potassium ion sensor according to an embodiment of the present invention.
27 shows a calibration curve of the potassium ion sensor of Fig.
28 to 33 show long term stability of the sensor and the heater according to an embodiment of the present invention.
Fig. 34 shows an impedance curve according to the cell type.
Fig. 35 shows fluorescence images of C2C12 myoblasts, which were subjected to thermal stimulation and electrical stimulation.
36 shows the impedance curve of the cell according to the type of the stimulus.
Figure 37 schematically shows a cell culture platform wirelessly connected to monitoring software.
38 to 41 schematically show the wireless control of the sensor and the heater.
Figure 42 shows an impedance monitoring calibration for C2C12 muscle cells of a cell culture platform comprising a laminated cell culture substrate according to an embodiment of the present invention.
Figure 43 shows the radio impedance monitoring of C2C12 myoblasts under normal growth conditions.
Figures 44 and 45 show the radio impedance monitoring of C2C12 myoblasts in growth using stimulation.

Hereinafter, the present invention will be described in detail with reference to examples. The objects, features and advantages of the present invention will be easily understood by the following embodiments. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments disclosed herein are provided so that the disclosure may be thorough and complete, and that those skilled in the art will be able to convey the spirit of the invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Although the terms first, second, etc. are used herein to describe various elements, the elements should not be limited by such terms. These terms are only used to distinguish the elements from each other. In addition, when an element is referred to as being on another element, it may be directly formed on the other element, or a third element may be interposed therebetween.

The sizes of the elements in the figures, or the relative sizes between the elements, may be exaggerated somewhat for a clearer understanding of the present invention. In addition, the shape of the elements shown in the drawings may be somewhat modified by variations in the manufacturing process or the like. Accordingly, the embodiments disclosed herein should not be construed as limited to the shapes shown in the drawings unless specifically stated, and should be understood to include some modifications.

The cell culture platform according to an embodiment of the present invention may include a cell culture substrate, a sensor for cell culture, a heat generator for cell culture, a wireless communication system, and a home placement exchange system.

The cell culture substrate may include a polymer substrate coated with graphene oxide. The polymer substrate may include a biocompatible polymer, for example, polylactic acid (PLA). The graphene oxide may be coated on the surface of the polymer substrate using a spray method. The cell culture substrate may have a plurality of perforations for easily penetrating the culture medium throughout the substrate. In addition, the cell culture substrate may be used by stacking two or more cells.

The cell culture sensor is disposed on a cell culture substrate. The cell culture sensor may include one or more of an impedance sensor, a pH sensor, a potassium ion (K +) sensor, and a temperature sensor.

The impedance sensor may monitor the condition of the cells cultured on the cell culture substrate. By connecting a bypass switch to the circuit of the impedance sensor, the impedance sensor can be used as an electric stimulator and can provide electrical stimulation to the cell.

The pH sensor and the potassium ion sensor can monitor the condition of the cell culture liquid by measuring pH and potassium ion concentration of the cell culture liquid. Thus, by monitoring the state of the cell culture fluid, information on the state change of the cells monitored by the impedance sensor can be grasped more accurately.

The temperature sensor may be disposed adjacent to the heat generator for cell culture to measure the heating temperature of the heat generator for cell culture, and the heat stimulus for the cell may be controlled by controlling the heat generator for cell culture.

The heat generator for cell culture stimulates cells cultured on the cell culture substrate to promote cell proliferation and differentiation. The heat generator for cell culture may provide heat stimulation to the cells.

The cell culture substrate may be divided into a plurality of cell culture regions, and the sensor and the heat generator for cell culture may be arranged to correspond to one or two or more cell culture regions. For example, the cell culture substrate may be divided into sixteen cell culture regions, and the impedance sensor may be arranged in each of sixteen cell culture regions to identify the cell state in each of sixteen regions. The heat generator for cell culture, the temperature sensor, and the pH sensor may be disposed one by one in four cell culture areas.

The wireless communication system can communicate wirelessly with an external device electrically connected to the sensor and the cell culture heat generator and disposed outside the incubator. The sensor and the heat generator for cell culture can be wirelessly controlled by the external device through the wireless communication system. That is, in-situ monitoring of cell culture by the sensor and cell stimulation by the heat generator for cell culture can be wirelessly controlled by the external device.

The plurality of impedance sensors disposed in each of the plurality of cell culture regions sequentially sense the state of the cells, and the impedance data for each cell culture region thus obtained is wirelessly transmitted to the external device sequentially through the wireless communication system, The cell culture condition for each culture area can be confirmed. In addition, the external device can wirelessly control the heat generator for cell culture to promote heat proliferation and differentiation by providing a thermal stimulus to a region where the cell culture condition is confirmed to be insufficient compared to other regions. Thereby, cell culture can be uniformly performed well in the entire region of the cell culture substrate.

The in-situ culture medium exchange system may supply and exchange the cell culture liquid through a well and / or a tube connected to an inlet and an outlet of the case where the cell culture substrate is disposed.

FIG. 1 and FIG. 2 show a Smart Cell Culture Platform (SCCP) according to an embodiment of the present invention. FIG. 3 schematically shows an array of sensors and heaters according to an embodiment of the present invention. 4 shows a schematic image of a cell culture substrate according to an embodiment of the present invention.

1 to 4, the cell culture platform includes a polylactic acid (PLA) substrate (GO-PLA substrate) coated with graphene oxide (GO), a sensor and stimulator array, an Arduino- , And in-situ media exchange system.

The GO-PLA substrate may be formed by forming a substrate with PLA using 3D printing, and then coating a graphene oxide on the surface of the substrate using a spraying method. The GO-PLA substrate has a plurality of perforations (1 x 1 mm) for easily penetrating the culture medium throughout the cell culture substrate, and can provide high cell viability even when five GO-PLA substrates are laminated. The GO-PLA substrate may have a concave hole and a convex pillar formed at the upper and lower portions of the edge, respectively. The two GO-PLA substrates can be stacked while being automatically aligned by inserting protrusions formed on the bottom of the GO-PLA substrate disposed on the top of the GO-PLA substrate disposed below. The positions of the concave portion and the convex portion can be changed with each other. The medium can flow into each substrate through a space (1 cm) between two GO-PLA substrates stacked.

The sensor and stimulator arrays are disposed on the GO-PLA substrate. The sensor may include an impedance sensor, a pH sensor, a potassium ion sensor, and a temperature sensor, and the stimulator may include a heat generator (thermal stimulator) and an electric stimulator. For example, a temperature sensor and a first insulating layer covering the temperature sensor may be disposed on the GO-PLA substrate. A heater and a second insulating layer covering the heater may be disposed on the first insulating layer. And a third insulating layer covering the impedance sensor and the impedance sensor may be disposed on the second insulating layer. The pH sensor and the potassium ion sensor may be disposed on the second insulating layer together with the impedance sensor.

The impedance sensor can monitor the state of the cells cultured on the GO-PLA substrate. By connecting a bypass switch to the circuit of the impedance sensor, the impedance sensor can be used as an electric stimulator and can provide electrical stimulation to the cell. The pH sensor and the potassium ion sensor can monitor the condition of the cell culture liquid by measuring pH and potassium ion concentration of the cell culture liquid. Thus, by monitoring the state of the cell culture fluid, information on the state change of the cells monitored by the impedance sensor can be grasped more accurately.

The temperature sensor may be disposed adjacent to the heat generator to measure the heating temperature of the heat generator, and may control the heat stimulus to the cells by controlling the heat generator.

The stimulator may stimulate cells cultured on the GO-PLA substrate to promote cell proliferation and differentiation. The heat radiator may provide thermal stimulation to the cell, and the electric stimulator may provide electrical stimulation to the cell.

The GO-PLA substrate may be divided into a plurality of cell culture regions, and the sensor and the stimulator may be arranged to correspond to one or more cell culture regions. For example, the GO-PLA substrate may be divided into 16 cell culturing regions, and the impedance sensor may be arranged in each of 16 cell culturing regions to confirm the cell state by 16 regions. The heat generator, the temperature sensor, and the pH sensor may be disposed one by one in four cell culture areas.

An Arduino-based wireless communication system is capable of wirelessly communicating with an external device that is electrically connected to the sensor and the stimulator and disposed outside the incubator. The sensor and the stimulator may be wirelessly controlled by the external device through the Arduino-based wireless communication system. That is, in-situ monitoring of cell culture by the sensor and cell stimulation by the stimulator can be wirelessly controlled by the external device.

The in-situ media exchange system supplies and exchanges the cell culture liquid through a silicon tube connected to the inlet and outlet of the acrylic case.

5 shows a manufacturing process of a cell culture platform according to an embodiment of the present invention.

Referring to FIG. 5, a parylene C layer is deposited on a silicon wafer on which Ni of 30 nm is deposited, followed by patterning. To form a temperature sensor, a 150 nm Au layer is formed and patterned, and an epoxy (SU8) is deposited thereon. The process is repeated to form a heater, an impedance sensor, a potassium ion sensor, and a pH sensor. An epoxy layer and a parylene layer are deposited to form an encapsulation layer. A graphene oxide (GO) is coated thereon using a spray method and then patterned to form a graphene oxide pattern. Whereby a device for cell culture is formed on the silicon wafer. The Ni deposited on the silicon wafer is etched, the cell culture apparatus formed thereon is subjected to SAM treatment, and then transferred onto a PLA substrate using a scooping method. After connecting ACF (anisotropic conductive film) to the PCB substrate, iridium oxide was deposited on the pH sensor and PEDOT was deposited on the potassium ion sensor to form the working electrodes of the ion sensors, and the reference electrode was formed using the Ag / AgCl ink Then, an ion selective membrane is coated. The PLA substrate on which the cell culture device is transferred is laminated and then connected to a wireless communication system. The laminated PLA substrate is placed in a PDMS well, and a cell culture liquid is supplied and exchanged through a silicon tube.

FIG. 6 shows a stacked image of a cell culture platform according to an embodiment of the present invention.

Referring to Figure 6, each cell culture platform may be housed in an acrylic case having an air passage and an inlet / outlet to increase the efficiency of deploying the maximum number of cell culture platforms in an incubator. The acrylic case can standardize the dimensions of each cell culture platform and facilitate lamination.

7 is an image of a cell culture plant using a cell culture platform according to an embodiment of the present invention.

Referring to FIG. 7, the cell culture plant is provided with incubators accommodating a plurality of cell culture platforms. The cell culture platform continuously monitors the current state of the adherent cells such as cell viability, proliferation and differentiation, and accordingly stimulates cell proliferation or differentiation by appropriately stimulating and communicating with the software wirelessly. Appropriate measures can be taken if abnormal conditions such as impedance, temperature, pH, and / or sudden increase or decrease in potassium ion concentration occur. Since the smart cell culture platform can be managed on a factory scale by a small number of persons by wireless communication, the management and operation cost of labor costs and the like can be greatly reduced.

8 is a view for explaining a coating process of graphene oxide (GO) according to an embodiment of the present invention. Referring to FIG. 8, graphene oxide may be coated on a 3D printed PLA substrate by a spray method.

9 shows an AFM image of a bare PLA substrate and a graphene oxide coated PLA (GO-PLA) substrate, FIG. 10 shows a Raman spectrum of a bare PLA substrate, a GO-PLA substrate, FIGS. 11 and 12 show SEM images of GO-PLA substrates, FIG. 13 shows SEM images of PLA substrates and GO-PLA substrate cells according to cell culture period, FIG. 14 shows cell numbers FIG.

9 to 14, the coated graphene oxide improves the cell adhesion force to have high viability, and the GO-PLA substrate has better cell adhesion than the bare PLA substrate, and the proliferation And the number of differentiated cells was significantly increased.

15 shows a cell image arranged on a cell culture substrate. Referring to FIG. 15, patterning of graphene oxide provides cell alignment to cells that need to mimic native tissue.

FIG. 16 shows a cell culture platform including a laminated cell culture substrate immersed in a culture medium in order to test cell viability. FIGS. 17 to 19 show the cell survival rate according to the number of the cell culture substrates and the culture medium depth FIG.

16 to 19, the designed substrate of the present invention maintains a cell survival rate of about 100% even when the number of stacked cell culture substrates is 5, Cell viability was significantly improved. In addition, the cell survival rate was maintained at almost 100% until the depth of the culture medium reached about 10 mm.

FIG. 20 shows an impedance sensor according to an embodiment of the present invention, and FIG. 21 shows a calibration curve of the impedance sensor of FIG.

20 and 21, the impedance sensor may include interdigitated electrodes. The impedance sensor may also function as an electrical stimulator that provides electrical stimulation to the cell by providing voltage or current.

FIG. 22 shows a heater and a temperature sensor according to an embodiment of the present invention, and FIG. 23 shows a calibration curve of a heater and a temperature sensor of FIG.

The heater (heater) may include a meandering line-shaped metal pattern. The heater can linearly change the temperature from 27 캜 to 43 캜. The temperature sensor may include a line-shaped metal pattern. The temperature sensor may be disposed under the heat generator to measure the temperature of the heat generator. The resistance of the temperature sensor linearly changes from 25 占 폚 to 50 占 폚 and can cope with rapid temperature changes quickly.

FIG. 24 shows a pH sensor according to an embodiment of the present invention, FIG. 25 shows a calibration curve of the pH sensor of FIG. 24, FIG. 26 shows a potassium ion sensor according to an embodiment of the present invention, 26 shows a calibration curve of the potassium ion sensor of Fig.

24-27, the pH sensor and the potassium ion sensor measure the open circuit potential of H + and K + at 4 to 10 pH and 1 to 32 mM K + concentration, respectively, and measured the open circuit potential at 7.94 to 6.94 pH and 0.125 to 2 mM K + Sensitively sensing in the range of change.

28 to 33 show long term stability of the sensor and the heater according to an embodiment of the present invention.

28 to 33, both the impedance sensor, the electric stimulator, the heater, the temperature sensor, the pH sensor, and the potassium ion sensor can stably function in the medium for a long period of time.

Figure 34 shows the impedance curves of human umbilical vein endothelial cells (HUVEC), human dermal fibroblast (hDFB), cardiac muscle cells (HL-1), and C2C12 myoblasts .

Referring to Fig. 34, in all types of cells, the impedance increases when the cells proliferate and cover the electrode. Muscle cells are differentiated and form myotubes, so they have conductivity, so impedance decreases with time.

Fig. 35 shows fluorescence images of C2C12 myoblasts, which were subjected to thermal stimulation and electrical stimulation, and Fig. 36 shows impedance curves of cells according to the types of stimulation.

Referring to FIG. 35, C2C12 myoblasts were shown to be enhanced by thermal stimulation (TS) and / or electrical stimulation (TS). The combination of the two stimuli greatly improved the formation of canaliculus cells.

Referring to Figure 36, the enhanced effect of stimulation on C2C12 myoblast can be sensed and recorded by an impedance sensor.

Figure 37 schematically shows a cell culture platform wirelessly connected to monitoring software.

37, a cell culture platform may be integrated with and controlled for signal conditioning, amplification, and wireless data transmission using a printed circuit board and an Arduino-based wireless communication system, and may include signal processing, analog-to- Digital-to-analog (DA) conversion. Whereby the sensors and stimulators (heaters) disposed on the cell culture substrate of the cell culture platform can be controlled and managed wirelessly.

38 to 41 schematically show the wireless control of the sensor and the heater.

Referring to Figures 38-41, for impedance measurement, the generated alternating signal is passed through the impedance sensor array continuously using a 1:16 multiplexer (MUX). This signal is amplified by a transimpedance amplifier (TIA), processed by a microcontroller unit (MCU), and transmitted to a Bluetooth module for wireless data transmission. For electrical stimulation, the circuit for impedance measurement can be used as is with an additional bypass switch. For pH and potassium ion concentration measurements, it is desirable to use a voltage buffer to accurately measure the open circuit voltage due to the high impedance of the pH and potassium ion sensors. By controlling the multiplexer wirelessly, the heaters and electrical stimulators can selectively stimulate the cultured cells to promote cell proliferation and differentiation.

Figure 42 shows an impedance monitoring calibration for C2C12 myoblasts in a cell culture platform comprising a laminated cell culture substrate according to an embodiment of the present invention and Figure 43 shows the radio impedance monitoring of C2C12 myotube cells under normal growth conditions , Figures 44 and 45 show the radio impedance monitoring of C2C12 myoblasts in growth using stimulation.

Referring to Figure 43, the impedance color map shows the cultivation of C2C12 in steady state without stimulation. Referring to FIG. 44, the upper left position of the first layer Ll is thermally and electrically excited, and referring to FIG. 45, the upper right portion of the second layer L2 and the lower left portion of the fourth layer L4 Was stimulated. Proliferation and differentiation of stimulated C2C12 were promoted at the site of stimulation.

Hereinafter, specific embodiments of the present invention have been described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

1. An array of impedance sensors for cell culture, which is disposed on a cell culture substrate for culturing cells to monitor the culture condition of the cells,
And a plurality of impedance sensors disposed on the cell culture substrate. The method according to claim 1,
Wherein the cell culture substrate includes N cell culture regions from a first cell culture region to an Nth cell culture region,
The cell culture impedance sensor array includes N impedance sensors from the first impedance sensor to the Nth impedance sensor,
Wherein the first to N-th impedance sensors correspond to the first to N-th cell culturing regions at a ratio of 1: 1, and one impedance sensor is disposed in one cell culturing region. 3. The method of claim 2,
In the impedance array for cell culture,
Wherein the culture condition of each cell culture area is monitored sequentially from the first impedance sensor to the Nth impedance sensor. 3. The method of claim 2,
Wherein the impedance sensor is electrically connected to a wireless communication system disposed adjacent to the cell culture substrate,
Wherein the wireless communication system wirelessly communicates with an external device that controls the impedance sensor. A method for confirming the state of a cell using an impedance sensor array for cell culture, the cell being disposed on a cell culture substrate for culturing the cell,
Wherein the cell culture substrate includes N cell culture regions from a first cell culture region to an Nth cell culture region,
The cell culture impedance sensor array includes N impedance sensors from the first impedance sensor to the Nth impedance sensor,
Wherein the first to Nth impedance sensors correspond to the first to Nth cell culture regions at a ratio of 1: 1, and one impedance sensor is disposed in one cell culture region,
And monitoring the cell culture state of each cell culture area sequentially from the first impedance sensor to the Nth impedance sensor.

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