KR20190070096A - Functional well plate system for electro-mechanical stimulation and analysis of live cells - Google Patents

Abstract

The present invention relates to a system that, while culturing cells in real time through functional well plates capable of electro-mechanical stimulation and an incubator, enables analysis on changes in deformation, contraction, and pulsation in cells in response to electro-mechanical stimulation by using a microscope and a laser displacement sensor. More specifically, the present invention applies electro-mechanical stimuli to cardiac muscle cells cultured on a diaphragm made of polydimethylsiloxane (PDMS) to mature the cells and stains particular proteins in the cells to confirm the location and distributions thereof. Thus, the cell analysis system of the present invention enables analysis on maturation of the cells and enables real-time measurement of changes in contraction and heart beat count of the cardiac muscle cells by using laser displacement sensors integrated in an upper part of the microscope. The electro-mechanical stimulation and analysis system according to the present invention enables cellular morphologies, contraction, and pulsation of cardiac muscle cells in response to electro-mechanical stimuli to be measured in real time using a microscope and laser displacement sensors and can effectively optimize the electro-mechanical stimulation. Further, the electro-mechanical stimulation and analysis system may be made of three wells in total and used to analyze drug responses using cells matured through electro-mechanical stimulation by using a microscope and laser displacement sensors. Additional well establishment may be possible through structure modifications.

Description

(Functional well plate system for electro stimulation and analysis of live cells)

The present invention enables mechanical stimulation and electrical stimulation (hereinafter referred to as "stimulation") by using a functional well plate and maintains temperature, humidity, and CO ₂ concentration constantly while observing the cells using a microscope and an incubator By using the motorized stage, it is possible to analyze the contraction force and the pulse number of the cell through accurate and rapid movement of the multi position, the cell shape analysis through the microscope, and the laser displacement meter. In addition, To a functional well plate system for the stimulation and analysis of live cells.

Heart disease is one of the most important health problems in the world today, and the heart problem is that it is rarely recovered by itself, and heart muscle cells are very poorly regenerated when damaged by disease or the like.

For cardiac regeneration to be successful, these cardiac cells must respond to the signal and fuse with surrounding cardiac muscle. However, myocardial cells produced from stem cells are immature and irregularly beating, which limits the availability for regenerative medicine or biology research. However, recent studies have shown that cardiomyocytes made from human stem cells can be stimulated to enhance development and function.

However, when mechanical stimulation and electrical stimulation are simultaneously performed, a system for analyzing morphology and contractility of myocardial cells is needed.

Accordingly, the present inventors have developed a functional well plate capable of stimulating the cell during cell culture on a diaphragm designed with polydimethylsiloxane (PDMS), and measured the temperature, humidity, CO2 incubator and multi well plate, a motroized stage for precise and rapid positional movement during analysis, and a microscope and a laser displacement meter for cell analysis to enable the stimulation and analysis of live cells. .

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a functional multiwell plate capable of being stimulated, The present invention aims at solving the problems of the conventional inspection method by providing an inspection device capable of analyzing the shape of other cells and analyzing the contraction force and the heart rate.

According to an aspect of the present invention, there is provided a diaphragm comprising: a diaphragm plate accommodating cells and having one or more regions having fine groove patterns; Electrodes provided on both sides of the outside of the diaphragm plate to give electrical stimulation; And a laser displacement gauge reflector at the center of the area where the cells are accommodated.

Further, the present invention is characterized in that it further includes an array of microelectrodes (MEAs), which are located in a space where the cells on the diaphragm plate are accommodated.

The present invention also relates to an upper well plate; A lower well plate; An array of diaphragms positioned between the upper well plate and the lower well plate and having flexibility; And a pressure application regulator, wherein the diaphragm is swollen when pressure is applied to impart mechanical stimulation to the cell. It is also possible to give an electric stimulus by forming an electric field in the area where the cells are cultured.

The present invention also relates to the above-mentioned well plate; And an incubator for culturing the cells, and includes a motorized stage capable of moving in the two-dimensional directions of the X-axis and the Y-axis, a laser vibrometer, An electrical stimulation generator, and a mechanical stimulation generator. ≪ Desc / Clms Page number 2 >

The incubator includes a top incubator having a connecting pin for stimulating cells and a bottom incubator having a heating line for maintaining the temperature.

The present invention provides a method for measuring the toxicity of a drug to cells using a well plate system for analyzing the mechanism of the stimulus, comprising the steps of: (1) administering a drug to a cell on a well plate system for stimulation analysis; (2) acquiring a shrinkage value from a well plate system for an electromotive force analysis using a laser displacement meter; (3) comparing the obtained shrinkage value with a reference table to determine the toxicity of the diagnostic drug, and (3) a method of measuring the toxicity of the drug to a cell.

The systematic stimulation and analysis system according to the present invention is capable of real time measurement of the shape, contraction force, and heart rate of a cardiomyocyte according to the mechanical stimulation of heart muscle cells using a microscope and a laser displacement meter, and is effective in deriving the stimulus optimization.

Also, the stimulation and analysis system according to the present invention is composed of single or multi wells, and it is possible to analyze the reaction according to drug through a microscope and a laser displacement meter through a matured cell through an electrostimulation.

1 is a block diagram of a well plate system for analyzing electromechanical stimulation of the present invention.
FIG. 2 is a schematic diagram of a well plate system for analyzing electromechanical stimulation, which includes an actual instrument, a constitution diagram for a core part (an electrophoresis controller, a stage top incubator, a incubator controller, a functional well plate,
Fig. 3 is an enlarged view of the motorized stage of Fig. 2, showing a motorized stage in the XY direction for measuring and moving multiple positions accurately and quickly.
Figure 4 shows the incubator cover and body appearance without the well adapter
Fig. 5 is a view showing the stage top incubator (Fig. 4) being fastened from a component
6 shows a functional generator for generating electric stimulation and a pressure pump for generating mechanical stimulation. In addition, a pressure sensor and an electromechanical stimulation controller (SMPS) Fig.
7 is a specific image of each part constituting a well plate for electrophoresis analysis.
FIG. 8 shows a final assembled image of the electroplate stimulation well plate (FIG. 7), with the top photograph showing the Pogo Pin generating electrical stimulation and the bottom photograph showing the appearance of the pressurized expansion diaphragm producing mechanical stimulation show.
9 is a view showing a process of manufacturing a PDMS diaphragm structure in one well constituting a well plate for electrophoresis analysis.
10 shows a diaphragm structure with a fine groove pattern on a PDMS diaphragm structure, showing the relative position of a laser displacement gage reflector and an electrode generating an electrical stimulus.
FIG. 11 is a photograph and a graph showing the difference in retraction of cardiac cells according to the radial micro groove formed / formed on the PDMS diaphragm and thus the contractive force. FIG.
FIG. 12 is a graph showing a deformation image and a real-time displacement of a PDMS diaphragm by periodically increasing / decreasing a pressure by using a syringe pump.
FIG. 13 is a graph showing changes in cell contraction force and pulse number according to a change in the frequency of electrical stimulation through a laser displacement meter. FIG.
14 shows the results of DAPI nuclear protein (blue), Cx-43 protein (green) and F-actin (red) through fluorescence staining after matured by electrical stimulation for 5 days starting from the third day after cell culture. Photos taken after dying.
FIG. 15 is a graph showing changes in cardiac contractility according to treatment of a drug (Verapamil). FIG.
Figure 16 is a conceptual diagram and analysis data of a microelectrode array integrated on a diaphragm to analyze the electrical activity of the cultured cells.

In order to achieve the above object, the present invention provides a diaphragm plate comprising: a diaphragm plate accommodating cells and having one or more regions having fine groove patterns; Electrodes provided on both sides of the outside of the diaphragm plate to give electrical stimulation; And a laser displacement gauge reflector at the center of the area where the cells are accommodated.

The present invention may further include an array of microelectrodes (MEAs) positioned in the space where the cells on the diaphragm plate are accommodated.

The present invention also relates to an upper well plate; A lower well plate; An array of diaphragms positioned between the upper well plate and the lower well plate and having flexibility; And a pressure application regulator, wherein the diaphragm is swollen when pressure is applied to impart mechanical stimulation to the cell. A gasket may be applied between the upper well plate and the lower well plate to prevent leakage when pressure is applied and to induce deformation of the diaphragm. On the other hand, it is also possible to give an electric stimulus by forming an electric field in the region where the cells are cultured. The number of wells in the plate is not definite, and adding a well is possible by changing the structure.

The PDMS diaphragm-based functional multiwell plate that can stimulate the mechanism is composed of a bottom well plate which can uniformly apply pressure to each diaphragm constituting the array by using a mechanical pump such as a syringe pump, A top plate on which the culture medium and the cells can be confined, and a PDMS diaphragm where the cells are cultured. The lower and upper well plates, except the PCB and PDMS structures, can be fabricated using Polystyrene (PS).

The present invention also relates to the above-mentioned well plate; A motorized stage capable of moving in the two-dimensional directions of the X-axis and the Y-axis, a laser vibrometer, an electric stimulation generator, and a mechanical stimulation generator Lt; RTI ID = 0.0 > cell / cell < / RTI > A functional multiwell plate based on a PDMS diaphragm capable of stimulating the stimulation, an incubator for maintaining the temperature and humidity of the CO2 concentration, a motorized stage for precise position control of the multi position, a microscope for confirming the shape of the cells, The function can be used by adding a laser displacement meter to measure the number.

In the case of a laser displacement meter for measuring the retraction force and pulse rate of a cell, the measuring range may be 50 ± 5 mm, the resolution may be 0.01 μm, and the repeatability may be ± 0.1 μm. The motorized stage is driven by X-axis and Y-axis. The working distance is more than 150x100mm and it is configured to facilitate observation of cells at every position in the well plate. The maximum speed is 20mm / sec and the repeatability is ± 3μm. An incubator capable of continuously culturing cells may be part of the composition by adding a controller to control each functional element such as temperature, humidity, and CO2 concentration during cell culture.

The cell may be a cardiac cell. In addition, since this system can measure changes in cells due to an electrical stimulation, it can be used in various cells such as muscle cells, cartilage cells and neurons in addition to cardiac cells.

The incubator may include a top incubator having a connecting pin for stimulating cells and a bottom incubator having a heating line for maintaining the temperature.

The incubator is composed of a top incubator and a bottom incubator. The upper incubator keeps the temperature inside the incubator by preventing the condensation by heating the FTO glass and circulating the hot water at the bottom.

In addition, an adapter was installed inside the incubator to make it possible to apply the electrophoresis to the well plate mounted inside the incubator. The internal adapter is divided into a single well plate and a multi well plate. The upper adapter is provided with a pogo pin for electrical connection, and the lower adapter is designed to allow mechanical stimulation using the position fixation and pressure of the well plate.

The present invention provides a method for measuring the toxicity of a drug to cells using a well plate system for analyzing the mechanism of the stimulus, comprising the steps of: (1) administering a drug to a cell on a well plate system for stimulation analysis; (2) acquiring a shrinkage value from a well plate system for an electromotive force analysis using a laser displacement meter; (3) comparing the shrinkage value of the obtained laser displacement gauge with a reference table to determine the toxicity of the diagnostic drug, and (3) a method for measuring the toxicity of the drug to a cell.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention in the drawings, parts not related to the description are omitted.

Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise. Also, the terms " part, "" ... "," module ", and the like described in the specification mean units for processing at least one function or operation, and may be implemented by hardware or software or a combination of hardware and software .

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments of the present invention with reference to the accompanying drawings.

 (FIG. 1) is a configuration diagram of a well plate system for analyzing electromechanical stimulation according to the present invention. Specifically, a functional generator for generating electrical stimulation and a syringe pump for generating mechanical stimulation, (Cardiac, muscular, chondrogenic, and neuronal cells) using a laser vibrometer, and analyzing the cell shape using a CCD monitor. System.

(Fig. 2) is a schematic diagram of a well plate system for analyzing electromechanical stimulation, which is composed of an actual instrument and has a schematic diagram of a core part (electrophoresis controller, stage top incubator, incubator controller, functional well plate, , And an electromechanical stimulation controller, a stage top incubator, and an incubator controller are shown around a single well plate. Concretely, we can check the interrelationship of components such as Incubator, Motorized Stage, Controller and Microscope equipped with CCD camera which can shoot.

(Fig. 3) is a further enlarged view of the motorized stage of Fig. 2, and is a view of a motorized stage in the XY direction for measuring and moving multiple positions accurately and quickly . The structure consists of an XY axis component and an incubator jig. The working distance is more than 150x100 mm. It is configured to facilitate observation of cells at every position in the well plate. The maximum speed is 20mm / sec. repeatability) can be made to ± 3 μm.

(FIG. 4) is an incubator cover and a body without a well adapter. This is the appearance of the top incubator and the bottom incubator constituting the incubator inside the incubator jig. Humans and CO2 concentrations of the functional well plate in which the cells are cultured are incubated in an incubator. The top incubator is capable of measuring the contractile force of cardiac cells using a Leaser vibrometer, In order to prevent condensation due to the difference in temperature between the outside and the outside, the FTO glass is heated to prevent condensation, and the bottom incubator is designed to circulate hot water to maintain the temperature inside the incubator and to inject CO 2 gas.

(Fig. 5) is a state in which a stage top incubator of Fig. 4 is fastened from a component, and includes an adapter cover including a plurality of wells, an adapter body, It consists of two parts and is fastened together. Pogo pins and air injection tubes are provided for position fixation, electrical stimulation and air supply of the electrophoresis assay well plate. This allows electrical stimulation and air supply to the well plate inside the incubator using an external stimulation controller. Mechanical stimulation is also possible using position fixation and pressure of the well plate.

(Fig. 6) includes a function generator for generating an electric stimulus and a pressure pump for generating a mechanical stimulus, and a pressure sensor and an electromotive stimulation controller Electromechanical stimulation controller). This allows electrical stimulation, mechanical stimulation, or mechanical stimulation while the cells are cultured in a well plate for electrophysiological analysis.

(Fig. 7) shows a specific image of each part constituting the well plate for electrophysiological analysis. In addition, since the components are displayed in the order of their relative positions, they are also an assembly configuration. The entire structure consists of cover, top well plate, gasket 2, PDMS material diaphragm, gasket 1 and bottom well plate.

(Fig. 8) shows the final assembled image of the electrophoresis analysis well plate (Fig. 7), the upper right picture shows the Pogo Pin generating electrical stimulation, the lower right picture shows the pressurized expansion diaphragm I can see a picture of the state of. The electrical stimulus forms an electric field using a function generator. The mechanical stimulus is stretched through the deformation of the PDMS diaphragm using a pressure pump.

(FIG. 9) is a process diagram for manufacturing a PDMS diaphragm structure in one well constituting a well plate for electrophoresis analysis, and the detailed manufacturing process is divided into a PET film portion and a transparent substrate portion. In the PET film part, a PUA mold with a micro / nano groove pattern is formed on PET film, and PDMS is patterned on it by coating and shadow mask. Next, the shape of the PDMS diaphragm is defined using a pinnacle die. The transparent substrate is metal patterned using a shadow mask on a transparent substrate and holes are formed through a sandblasting process. The fabricated PET film and transparent substrate are bonded by oxygen plasma process and the PET film and PUA mold are removed.

(FIG. 10) shows a diaphragm structure having a fine groove pattern (micro groove pattern and a Nano groove pattern) on a PDMS diaphragm structure, in which a relative position of a laser displacement gauge reflector and an electrode for generating an electrical stimulus is indicated. The fine groove pattern on the left is an enlarged view of the diaphragm portion on the right photo. A micro groove or a nano groove is formed on a glass substrate on which a hole is formed and a reflector for laser vibrometer and an electrical stimulation electrode are disposed. In this process, a PDMS diaphragm is fabricated through a plasma bonding process between a PDMS membrane formed with a laser displacement gauge reflector and a micro groove, and a glass formed with an electrical stimulation electrode and a hole. The picture on the right is the actual prototype.

Example 1: Confirmation of changes in laser displacement according to presence or absence of microgrooves

(Fig. 11) are photographs and graphs showing the differences in cardiac cell alignment and concomitant force according to the radial micro groove grooves formed on the PDMS diaphragm. The photo and graph on the left show the growth of cardiac cells in a PDMS diaphragm without micro grooves, and the graph on the right shows the growth of cardiac cells in a PDMS diaphragm with micro grooves. Microscopic examination of the alignment of cardiac cells in the direction of the grooves showed that the displacement of the diaphragm was 4 to 4.5 μm without groove, To 17 ~ 18 ㎛ (with groove). With this increase in displacement, even if the same concentration of the substance is treated, the change in the contraction force of the cardiac cell can be more easily confirmed as to how the substance of the concentration affects the contractility of the cardiac cell.

Example 2 The changes in the contraction force and the number of beats of the cells were measured using a laser displacement meter and confirmed by photographs

(FIG. 12) is a graph of deformed images and real-time displacements of the PDMS diaphragm by periodically increasing / decreasing the pressure using a syringe pump. As a result of measuring the displacement of the PDMS diaphragm with a periodic pressure change using a laser displacement sensor, the diaphragm is deformed by cyclic stretching and the cells cultured on the diaphragm are stretched.

(Fig. 13) is a graph obtained by measuring a change in the contractive force and the number of beats of a cell with a laser displacement meter according to a frequency change of an electric stimulus. The heart rate of myocardial cells is synchronized with electrical stimulation, and it can be confirmed that the number of beats increases as the frequency of stimulation increases from 0.5 Hz to 3 Hz.

(Blue), Cx-43 protein (green), and F-actin (blue) through fluorescence staining, after which cells were matured by electrical stimulation for 5 days starting from the third day after cell culture Red). Comparing the photographs with (a) no electrical stimulation (ES) and (b) with electrical stimulation (ES), cell- It can be seen in the photograph that this is strengthened.

Example 3: Effect of change of cardiac contractility according to drug treatment concentration

(Fig. 15) is a graph showing changes in cardiac contractility after treatment with the drug Verapamil. Using the well plate system for electrophysiological analysis of the present invention, the concentration of Verapamil, a Ca ²⁺ channel inhibitor, was changed to differentiate into cardiac cells. As a result, it was confirmed that the contraction force decreased with increasing concentration. However, it was confirmed that contraction of myocardial cells always occurs constantly at the positions of 0.5 sec and 2.5 sec. Therefore, it was confirmed that the electric pulse stimulates the heartbeat of a certain heart cell.

Example 4. Microelectrode array (MEA) integration for electrophysiology analysis

(Fig. 16), a microelectrode array (MEA) integrated on a diaphragm was added to analyze the electrical activity of the cultured cells, and the diagram and result data of the diaphragm structure are shown. (B) is a view of a diaphragm structure integrated with a microelectrode array (MEA), and (A) is an enlarged view of an MEA on a diaphragm. (C) is an example of data on the field potential measured using an integrated MEA, and (D) is an example showing the change in the data of the conduction velocity measured value according to the concentration of the drug administered to the cell. The integrated MEA can measure the heart rate, depolarization amplitude, field potential duration, and conduction velocity of the myocardial cells through field potential measurements.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, And all changes to the scope that are deemed to be valid.

Claims (6)

A diaphragm plate in which cells are accommodated and one or more regions having fine groove patterns are formed;
Electrodes provided on both sides of the outside of the diaphragm plate to give electrical stimulation; And
And a laser displacement gauge reflector at the center of the area in which the cells are received.
The method according to claim 1,
And a microelectrode array disposed in a space where the cells on the diaphragm plate are accommodated. 2. The diaphragm of claim 1,
An upper well plate;
A lower well plate;
An array of diaphragms according to any one of the preceding claims, which is located between the upper well plate and the lower well plate and has flexibility. And
And a pressure application regulator, wherein when the pressure is applied, the diaphragm swells to impart mechanical stimulation to the cell.
A well plate according to claim 3; And
An incubator for culturing the cells,
A mechanism for cells, which further comprises one or more devices in the group consisting of a motorized stage, a laser vibrometer, an electrical stimulus generator, and a mechanical stimulus generator, which can be moved in two dimensions in the X and Y axes Well plate system for stimulus analysis.
5. The method of claim 4,
In the incubator,
Characterized in that it comprises a top incubator with a connecting pin for stimulation to cells and a bottom incubator with a heating line for temperature maintenance. Well plate system.
A method for measuring the toxicity of a drug to cells using a well plate system for an induced stimulation assay,
(1) administering a diagnostic drug to a cell on a well plate system for electrophysiological analysis;
(2) acquiring a shrinkage value from a well plate system for an electromotive force analysis using a laser displacement meter;
(3) comparing the shrinkage value of the obtained laser displacement gauge with a reference table to determine the toxicity of the diagnostic drug,
A method for measuring the toxicity of a drug to a cell.

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