KR20080030297A - Cell culture environment and chemotaxis measuring device - Google Patents

Abstract

The cell culture and chemotaxis measuring device having a built-in sensor according to the present invention is formed on a transparent substrate, the first well for accommodating the cell culture, the first well on the substrate is formed spaced apart from the cell culture medium and the cell reactant A second well for receiving and a channel connecting the first well and the second well to each other, and at least one sensor for sensing a culture environment of the cell or sensing a cell culture solution in the first well or the second well; Built-in, one side of the second well further comprises an outlet for the discharge of the cell culture medium and the cell reactant.

Therefore, the cell culture and chemotaxis measuring device according to the present invention is easy to measure the movement according to the chemotaxis of the cell at the same time as the real-time observation of the cell, can be cultured for a long time, the cell culture solution using a sensor built in the well Characterization of and control of the surrounding environment during cell culture has an easy effect.

Description

Sensor-inserted apparatus for incubating cells and measuring chemotaxis

1 is a cross-sectional view of the chemotaxis measuring apparatus of the vertical channel structure according to the prior art,

2a and 2b is a cross-sectional view of the chemotaxis measuring device of a conventional horizontal channel structure,

Figure 3 is a three-dimensional view of the cell culture and chemotaxis measuring device of the horizontal channel structure according to the present invention,

Figure 4a to 4d is a three-dimensional view of the cell culture and chemotaxis measuring device of the horizontal channel structure according to an embodiment of the present invention.

<Description of the symbols for the main parts of the drawings>

300: cell culture and chemotaxis measuring apparatus 310: first well

320: second well 330: channel

340: substrate 360: inlet

370: outlet 410: contact temperature sensor

420 and 470: pH sensor 430: electrode wiring

440: electrode pad 450: non-contact temperature sensor

460, 480: wiring

The present invention relates to a cell culture and chemotaxis measuring device with a sensor inserted, and more specifically, a well in which the cell culture solution is injected, a temperature sensor for cell culture, and a well in which the chemotactic material is injected, a pH sensor It relates to a built-in device.

As a structure for real-time observation of cell migration, a cell chamber having a horizontal structure is required to measure chemotaxis at the same time as cell culture. In the form of a structure that can track the movement path of these cells in real time, enabling measurement and control of temperature and pH during the real-time movement measurement of cells in its horizontal chamber (chip) minimizes human direct handling and standardized data. To get

Conventionally, the most commonly used for measuring chemotaxis of cells is a boyden chamber assay, which is a cell vertical movement structure. FIG. 1 illustrates a Boyden chamber assay 100 according to the prior art, in which a factor for inducing cell migration, that is, a cheatable factor, is injected into a lower chamber 120, and a cell is injected into the upper chamber 110. When the suspension is injected, the cell 140 moves in the direction of the chemotaxis factor through the minute holes present in the filter 130.

After the cells have been incubated to move in the direction of chemotaxis factors for a certain time, the filter 130 is removed and the number of cells 140 stuck in the holes is determined to determine the movement by the chemotaxis of the cells. do.

However, in the boyden chamber assay (100), the vertical structure consisting of the upper chamber 110 and the lower chamber 120 makes it impossible to observe the cells in real time, so that the cell temperature and the pH characteristics of the chemotaxis factor are observed at the same time. There is a disadvantage that analysis is impossible.

Next, there is a chemotaxis detection structure by horizontal movement of cells.

As shown in FIG. 2A and FIG. 2B, the chemotaxis detection structure by the horizontal movement of cells is injected into one well 210 of two wells 210 and 220 formed horizontally, and the other well. In 220, after the chemotaxis factor is injected, the cells move in the direction of the chemotaxis factor. In addition, the channel 230 may be provided with a barrier 240 configured as a groove through which cells can pass to perform a role as a filter of a boyden chamber assay.

Experimental methods using the same generally follow the same method as shown in Figure 2c.

However, such a chemotaxis detection structure by horizontal movement of the cell should be maintained for a long time the concentration gradient of the chemotactic material in order to measure the migration by the chemotaxis of adherent cells, the conventional structure is difficult to maintain this.

In addition, it is difficult to analyze the temperature of the cell and the pH concentration of the chemotaxis factor, as in the boyden chamber assay.

It is an object of the present invention to provide an apparatus for easily measuring chemotaxis at the same time as culturing cells.

Another object of the present invention is to provide a cell culture and chemotaxis measuring device equipped with a sensor inside the chamber.

Another object of the present invention is to provide a cell culture and chemotaxis measuring apparatus capable of real-time tracking (monitoring) of a cell's migration route.

It is another object of the present invention to provide a cell culture and chemotaxis measuring device capable of measuring the characteristics of the cell culture during the real-time tracking of cell movement and controlling the surrounding environment during cell culture.

The cell culture and chemotaxis measuring device having a built-in sensor according to the present invention is formed on a transparent substrate, the first well for accommodating the cell culture, the first well on the substrate is formed spaced apart from the cell culture medium and the cell reactant A second well for receiving and a channel connecting the first well and the second well to each other, and at least one sensor for sensing a culture environment of the cell or sensing a cell culture solution in the first well or the second well; It is built.

Preferably, one side of the first well includes an inlet for input of the cell culture solution, and one side of the second well further includes an outlet for discharging the cell culture medium and the cell reactant to enable long-term culture of the cells. The inlet and outlet are formed so as to be located from the center of the well height.

The channel is preferably formed to be narrower in contact with the second well than the width in contact with the first well, the width of the channel adjacent to the first well is 10 μm or more, and the width of the channel adjacent to the second well is 1 μm to 10 micrometers.

The sensor is patterned on a substrate with a temperature sensor to form a contact or contactlessly formed on top of the first well.

The temperature sensor is connected with electrode wirings and electrode pads patterned on the substrate.

The temperature sensor is connected to a temperature control unit.

The sensor is a pH sensor which is formed as a surface mount element type on a substrate or as a surface mount element type on one side wall of the second well.

The pH sensor is connected to the electrode wiring and the electrode pad patterned on the substrate, and immersed in the cell culture medium and the cell reactant.

The first well, the second well and the channel are formed of a transparent biocompatible material, which corresponds to PDMS, PMMA, glass-based materials, transparent plastics, and the like.

In the foregoing, the terms or words used in the present specification and claims should not be construed as being limited to the ordinary or dictionary meanings, and the inventors properly define the concept of terms in order to explain their own invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

Therefore, the embodiments described in the specification and the drawings shown in the drawings are only the most preferred embodiment of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be replaced at the time of the present application It should be understood that there may be equivalents and variations.

Hereinafter, with reference to the accompanying drawings as follows.

3 is a cell culture and chemotaxis measuring apparatus 300 according to the present invention, has a horizontal channel structure.

At this time, the first well 310 into which the cell culture is injected to incubate the cells and the well for accommodating the cell reaction material, that is, the second well 320 into which the chemotactic material is injected are formed on the glass substrate or the transparent substrate 340. And channels connected to each other between the first well and the second well.

In this case, when viewed from the top, the channel 330 has a triangular shape, and forms a narrower width in contact with the second well than a width in contact with the first well.

This is to maintain the properties for a long time by forming a concentration gradient when the chemotactic material present in the second well moves to the first well through the channel.

In an embodiment of the present invention, the width of the channel adjacent to the first well is formed to be 10 μm or more, and the width of the channel adjacent to the second well is reduced to 1 μm to 10 μm by narrowing the width of the channel adjacent to the second well. To maintain the concentration gradient of the chemotactic material.

On the other hand, by forming an inlet 360 for perfusion of the culture solution at the half h (h) of the height of the first well or the upper portion thereof to allow long-term culture of the cells. The size of the inlet is selected to a suitable size for insertion of a pump (not shown) for spreading the culture medium, it is preferable to always maintain the appropriate amount of culture medium and not directly affect the cells.

An outlet 370 for discharging the culture solution and the chemotactic material is formed at or above the height half point h of the second well.

As such, the structure 350 including the first well 310, the second well 320, and the channel 330 formed on the substrate 340 is a polymer made of a transparent material and has a flexible characteristic of polydimethylsiloxane (PDMS). Can be formed using all transparent bio compatible materials such as poly methyl methcrylate (PMMA), glass-based materials, and transparent plastics, thereby real-time tracking (monitoring) of cell migration pathways. have.

In addition, the cell culture and chemotaxis measuring apparatus according to the present invention includes a sensor for detecting the culture environment of the cells in the first well or the second well or the cell culture solution.

Sensors for sensing the culture environment of the cell or for detecting the cell culture medium include a temperature sensor, a pH sensor, a carbon dioxide sensor, an oxygen sensor, and at least one embedded in the first well for accommodating the cell culture, or the first well and At least one or more may be incorporated in each of the second wells.

In a specific embodiment of the present invention, a sensor is provided in each of the first well and the second well, which will be described in detail with reference to the accompanying drawings.

Embedded sensors in the first well and the second well, respectively, are implemented through the following embodiments.

[First Embodiment]

Figure 4a shows a first embodiment of the cell culture and chemotaxis measuring apparatus 300 according to the present invention, the first well 310 is produced by patterning a contact temperature sensor 410 on a glass substrate or The formed contact type temperature sensor is bonded to the glass substrate, and the second well 320 is formed by bonding the pH sensor 420 to the substrate as a surface mount device type.

As such, when the temperature sensor is provided in the first well, the temperature sensor may be connected to the temperature control unit so as to appropriately control the internal temperature of the first well, that is, the temperature of the cell culture solution.

The temperature sensor and the pH sensor allow direct contact with the culture medium and the chemotactic material, respectively, and particularly the pH sensor 420 can be immersed in the chemotactic material.

The electrode wire 430 and the electrode pad 440 connected to the temperature sensor and the pH sensor are formed on the glass substrate.

Second Embodiment

4B illustrates a second embodiment of the cell culture and chemotaxis measuring apparatus 300 according to the present invention. In the first well 310, a non-contact temperature sensor 450 is formed on the upper portion of the first well 310, and the wiring 460 is provided. The silver is separately removed from the sensor, and the second well 320 is formed by bonding a pH sensor 420 to the substrate as a surface mount device type.

In this case, the pH sensor 420 may be immersed in a chemical substance, and the electrode wiring 430 and the electrode pad 440 connected to the pH sensor are formed on the glass substrate.

Third Embodiment

Figure 4c shows a third embodiment of the cell culture and chemotaxis measuring apparatus 300 according to the present invention, the first well 310 has the same contact temperature sensor 410 as the first embodiment of the present invention Is fabricated by patterning on a glass substrate, or formed by bonding the fabricated contact temperature sensor to the glass substrate. In the second well 320, a pH sensor 470 is placed on one wall of the well, and the wiring 480 is connected It is formed into a structure to be taken out from.

The first well and the second well may be formed in a quadrangular structure as shown in the drawing, and may be formed in other polygonal structures.

[Example 4]

Figure 4d shows a fourth embodiment of the cell culture and chemotaxis measuring apparatus 300 according to the present invention, the first well 310 is provided with the same non-contact temperature sensor 450 as the second embodiment of the present invention It is formed on the upper side, and the pH sensor 470 is formed on the wall of one side of the well, as in the third embodiment of the present invention.

At this time, the wires 460 and 480 of the non-contact temperature sensor 450 and the pH sensor 470 are separately pulled out from the sensor.

As described above, the cell culture and chemotaxis measuring apparatus according to the present invention is easy to measure the characteristics of the cell culture medium and control the surrounding environment during cell culture using a sensor built in the well, transparent on a transparent glass substrate It is formed using a bio-compatible material, so it is easy to measure the movement according to the chemotaxis of the cell and at the same time real-time observation of the cell.

In addition, when the cell culture apparatus is used, it is possible to incubate for a long time by having an inlet and an outlet in consideration of perfusion of cell culture solution, and attaching a surface-mount element type pH sensor to a substrate or to a wall inside a well. Applying the method has the advantage of simplifying the wiring.

Although the present invention has been shown and described with reference to the preferred embodiments as described above, it is not limited to the above embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

Cell culture and chemotaxis measuring apparatus according to the present invention is formed by using a transparent bio-affinity material on a transparent glass substrate, it is easy to measure the movement according to the cell chemotaxis and at the same time real-time observation of the cell.

In addition, when the cell culture apparatus is used, an inlet and an outlet for considering the perfusion of the cell culture can be provided for a long time and can be cultured for a long time. It is easy to control the environment, and it is possible to solve the wiring simply by incorporating a surface-mount element type pH sensor.

Claims (18)

A first well formed on the transparent substrate for receiving cell culture; A second well formed on the substrate, the second well being spaced apart from the first well to receive a cell culture solution and a cell reactant; And A channel connecting the first well and the second well to each other Including, Cell culture and chemotaxis measuring device is built-in sensor in which at least one sensor for sensing the culture environment of the cell or the cell culture solution in the first well or the second well. The method of claim 1, One side of the first well includes an inlet for the introduction of cell culture, One side of the second well is a cell culture and chemotaxis measuring device is built-in sensor further comprises an outlet for discharging the cell culture medium and cell reactants. The method of claim 1, And the channel has a width narrower in contact with the second well than a width in contact with the first well. The method of claim 1, The environmental sensor is a cell culture and chemotaxis measuring device is a sensor built-in temperature sensor. The method of claim 1, The characteristic detection sensor is a cell culture and chemotaxis measuring device is built-in sensor which is a pH sensor. The method of claim 4, wherein The temperature sensor is a cell culture and chemotaxis measuring device is embedded with a patterned sensor on the substrate. The method of claim 4, wherein The temperature sensor is a cell culture and chemotaxis measuring device is built in a non-contact sensor formed on top of the first well. The method of claim 5, wherein The pH sensor is a cell culture and chemotaxis measuring device is built in the sensor formed in the surface-mount device type on the substrate. The method of claim 5, wherein The pH sensor is a cell culture and chemotaxis measuring device having a sensor formed in the side wall of the second well formed in the surface-mounted device type. The method of claim 1, The first well, the second well and the channel is a cell culture and chemotaxis measuring device is embedded with a sensor formed of a transparent biocompatible material. The method of claim 2, The inlet and the outlet is a cell culture and chemotaxis measuring device is built-in sensor located above the center of the height of the first well and the second well. The method of claim 3, wherein Cell culture and chemotaxis measuring device width of the channel adjacent to the first well is 10㎛ or more. The method of claim 3, wherein Cell culture and chemotaxis measuring device width of the channel adjacent to the second well is 1㎛ 10㎛. The method according to claim 6 or 7, The temperature sensor is a cell culture and chemotaxis measuring device is built-in sensor connected to the temperature control. The method of claim 8, The pH sensor is a cell culture and chemotaxis measuring device embedded with a sensor connected to the electrode wiring and the electrode pad patterned on the substrate. The method of claim 14, The temperature sensor is a cell culture and chemotaxis measuring device is embedded with a sensor connected to the electrode wiring and the electrode pad patterned on the substrate. The method according to claim 9 or 15, The pH sensor is a cell culture and chemotaxis measuring device is built in the sensor is immersed in the cell culture medium and the cell reactant. The method of claim 10, The biocompatible material is any one of PDMS, PMMA, glass-based material, transparent plastic cell culture and chemotaxis measuring device.

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