KR20150042316A - Monitering and control system for cultivating cell in sample using microfluidic channel device - Google Patents

Abstract

The present invention relates to an apparatus for monitoring and controlling cell culture, comprising a microfluidic channel device; a measurement unit for measuring the state of the microfluidic channel device; and a control unit for controlling the microfluidic channel device, wherein the microfluidic channel device comprises a microfluidic channel element including a cell culture channel, a nerve cell growth factor channel, and a nerve cell growth channel disposed therebetween; and an electrode substrate including electrodes.

Description

TECHNICAL FIELD [0001] The present invention relates to a monitoring and control system for a cell culture in a sample using a microfluidic channel device,

The present invention relates to a cell culture monitoring and control system in a sample using a microfluidic channel device.

The regeneration of neurons in the central nervous system is extremely limited, and studies on the growth and regeneration of nerve cells are highly valued in neuroscience and brain science. The study also covered mechanisms of neuronal cell development and networking of neuronal cells, as well as applications and pharmacology of neurodegenerative diseases such as degenerative brain diseases and neurodegenerative diseases, It is utilized in various fields. In the study of neurons, the length and number of neurons are a measure of growth. Accurate analysis and quantification of neurons is a fundamental and important step.

Currently, image analysis is the main method of confirming the growth of neurons. It takes a series of steps to identify and quantify the growth of neurons using this method. First, the cells are cultured for several days in a culture dish, and the cells are fixed with PFA solution. Then, the cells are stained with immunostaining. Subsequently, the cells should be photographed using a microscope and analyzed over a long period of time using image processing software based on the photographed images. With the exception of the image processing software, all the processes are manually performed, making it difficult to analyze accurately, and it takes a long time to analyze it due to the preprocessing steps until analysis. Above all, there is a limitation that it is impossible to monitor growth in real time and to control growth based on the characteristics of the experiment.

The present invention provides a system capable of real-time monitoring of cell culture using a microfluidic device for culturing cells in a sample.

The present invention also provides a system for controlling cell culture in real time based on the monitoring result using the microfluidic device.

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

A cell culture monitoring and control apparatus according to a first aspect of the present invention includes a microfluidic channel device; A measuring unit for measuring a state of the microfluidic channel device; And a control unit for controlling the microfluidic channel device, wherein the microfluidic channel device includes a cell culture channel, a neuron cell growth factor channel, and a nerve cell channel between the cell culture channel and the neuron cell growth factor channel A microfluidic channel element including a growth channel; And an electrode substrate including an electrode.

According to one aspect, the state of the microfluidic channel device may be at least one selected from the group consisting of the temperature in the microfluidic channel device, the pH in the microfluidic channel device, and the degree of differentiation and growth of the neuron .

According to one aspect, the measuring unit may include at least one selected from the group consisting of a temperature measuring unit, a pH measuring unit, and a neuron growth measuring unit.

According to one aspect of the present invention, the electrode includes a plurality of microelectrodes, and the plurality of microelectrodes includes a reference electrode and a plurality of measurement electrodes, And measuring the electrical change between the two electrodes generated while connecting the measurement electrodes, thereby measuring the degree of differentiation and growth of the neuron.

According to one aspect, the electrical change may include at least one selected from the group consisting of resistance, voltage, current, and capacitance.

According to one aspect of the present invention, the control unit may include at least one selected from the group consisting of a heater unit, a culture fluid injecting unit, and a nerve growth factor injecting unit.

According to one aspect of the present invention, the heater unit is driven according to a result of temperature measurement in a microfluidic channel, and the culture medium injecting unit and the nerve growth factor injecting unit measure the pH measurement result in the microfluidic channel, And may be driven according to the result.

According to one aspect of the present invention, there is provided an information processing apparatus comprising: a transmission unit that transmits a measurement result of the measurement unit; And a receiving unit receiving the control unit driving signal.

According to one aspect of the present invention, the transmitting unit may remotely transmit the measurement result to the terminal, and the receiving unit may remotely receive the driving signal from the terminal.

The cell culture monitoring and control apparatus according to the present invention provides a convenient experimental tool for quantifying neuronal cell growth in neuronal and neurogenic neuronal development and regeneration studies and provides real time monitoring of the neuronal cell growth, Can be controlled to provide an optimal environment for nerve cell growth, and can provide important experimental diagnostic and measurement / control devices in the treatment of degenerative brain diseases and neurological diseases and in the development of new drugs. In addition, quantitative and systematic analysis of neuronal growth can be used to develop 3D nerve cell growth assay and in vivo experiments.

FIG. 1 is a conceptual diagram of neuron cell growth measurement and control using a cell culture monitoring and control apparatus according to an embodiment of the present invention.
2 is a conceptual diagram illustrating operation of software interlocked with a cell culture monitoring and control apparatus according to an embodiment of the present invention.
3 is a plan view of a microfluidic channel device included in a cell culture monitoring and control apparatus according to an embodiment of the present invention.
4 is an enlarged plan view of a portion A in Fig.
5 is an enlarged perspective view of a portion A in Fig.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. Also, terminologies used herein are terms used to properly represent preferred embodiments of the present invention, which may vary depending on the user, intent of the operator, or custom in the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the contents throughout this specification. Like reference symbols in the drawings denote like elements.

Throughout the specification, when a part is referred to as being "connected" to another part, it includes not only "directly connected" but also "electrically connected" with another part in between .

Throughout the specification, when a member is located on another member, it includes not only when a member is in contact with another member but also when another member exists between the two members.

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.

Hereinafter, the cell culture monitoring and control apparatus of the present invention will be described in detail with reference to examples and drawings. However, the present invention is not limited to these embodiments and drawings.

The device for monitoring and controlling cell culture of the present invention comprises a microfluidic channel device; A measuring unit for measuring a state of the microfluidic channel device; And a control unit for controlling the microfluidic channel device, wherein the microfluidic channel device includes a cell culture channel, a neuron cell growth factor channel, and a nerve cell channel between the cell culture channel and the neuron cell growth factor channel A microfluidic channel element including a growth channel; And an electrode substrate including an electrode.

In the present invention, the term " growth of cells " is a concept including the growth of cell protrusions. That is, the growth of the protrusions of the cells may be a form of cell growth.

FIG. 1 is a conceptual diagram illustrating control and measurement of neuronal cell growth using a cell culture monitoring and control apparatus according to an embodiment of the present invention. FIG. 2 is a flow chart illustrating a method of controlling the cell culture monitoring and controlling apparatus according to an embodiment of the present invention. It is a concept of operation.

FIG. 3 is a plan view showing a microfluidic channel device included in a cell culture monitoring and controlling apparatus according to an embodiment of the present invention. FIG. 4 is an enlarged plan view of part A of FIG. 3, Fig.

3 to 5, a microfluidic channel device according to an embodiment includes a cell culture channel 112, a neuron growth factor channel 114, and a cell culture channel 112, A microfluidic channel device 110 including a neuron growth channel 116 disposed between channels 114, and an electrode substrate 120 including electrodes on a substrate.

The microfluidic channel device 110 can be manufactured through a photolithography process using, for example, PDMS, a photosensitive liquid, or a silicon wafer as a material.

The electrode substrate 120 is formed by patterning an electrode for sensing the growth of nerve cells on a substrate, and can be manufactured through a semiconductor manufacturing process such as a photolithography process and a deposition process on a glass wafer.

The microfluidic channel device 110 and the electrode substrate 120 may be bonded to each other by oxygen plasma treatment. The technique of joining by the oxygen plasma treatment is advantageous in that the joining is simple and the joining force is strong.

Wherein the cell culture channel (112) is a channel through which the cell is cultured, and the neuron cell growth factor channel (114) is a channel for injecting a growth factor with a concentration suitable for the cell to be cultured together with the culture solution, ) Is a channel for injecting a hydrogel suitable for the type of cell to be grown.

The culture medium suitable for culturing cells to be injected into the neuron cell growth factor channel 114 may diffuse in the direction (A) of the cell culture channel 112 through the hydrogel. The neuron may be grown on the electrode substrate 120 of the neuron growth channel 116 in the direction of the neuron growth factor channel 114 (B). As described above, the growth of the nerve cells may be the growth of nerve cell protrusions.

The nerve cell growth channel 116 may include at least one selected from the group consisting of a hydrogel, a scaffold, a porous material, and a micropillar array. The cells grow through the three-dimensional space, and the hydrogel, the scaffold, the porous material, and the fine column array structure can serve as a support for guiding the direction of cell growth.

The nerve cell growth channel 116 may include at least one hydrogel selected from the group consisting of collagen, fibrin, fibronectin, and matrigel.

The microfluidic channel device 110 may further include a cell adhesion material coating depending on the type of cells to be cultured. The cell adhesion material may be at least one selected from the group consisting of poly L lysine (PLL), poly D lysine (PDL), laminin, collagen, fibrin, fibronectin and matrigel One can be included. The cell adhesion material coating may be coated on the microfluidic channel walls to assist adhesion of the growth cells.

The electrode 122 may include a biocompatible material and may include at least one selected from the group consisting of gold, silver, platinum, titanium, aluminum, copper, nickel, palladium and ITO .

The electrode 122 may further include a plurality of fine electrodes. The plurality of microelectrodes can induce the growth of neurons by applying electrical stimulation to the cells, while sensing the growth of neurons. The plurality of microelectrodes may be formed above, below, or both of the nerve cell growth channels. The width of the plurality of microelectrodes and the width of the plurality of electrodes may be 2 to 1000 탆 in consideration of the electrode patterning process and the maximum growth length of the nerve cells. If the minimum line width of the mask required for the electrode patterning process is less than 2 탆, there is a problem that the mask type and the process method therefor are changed and the process cost increases.

The plurality of microelectrodes may further include a reference electrode 124 and a plurality of measurement electrodes 126.

The reference electrode 124 may be formed in contact with the left side of the interface between the nerve cell growth channel 116 and the cell culture channel 114. The reference electrode 124 may be formed in contact with the nerve cell growth channel 116 and the cell culture channel 114, As shown in FIG.

The measurement electrode 126 can be measured when one or more measurement electrodes other than one reference electrode 124 are present. As the number of the measurement electrodes 126 increases, the number of measurement electrodes can be several or more since the fine growth of the nerve cells can be measured. For example, the measuring electrode may be 1 to 250 electrodes.

The microelectrodes may be planar microelectrodes having a shape in which the microelectrodes are arranged in one direction on the substrate, a three-dimensional columnar shape on the substrate, or a three-dimensional tunnel-shaped microelectrode that forms a wall on the substrate. Such a three-dimensional microelectrode may serve as a cell growth channel by simultaneously providing space by the electrode itself.

A method of culturing cells in a sample according to one embodiment will be described.

The method for culturing cells in a sample according to one embodiment includes the steps of injecting hydrogel suitable for cells cultured in the nerve cell growth channel, injecting a sample containing cells into the cell culture channel, Injecting a culture medium suitable for the cells cultured with the growth factor channel, and culturing the cells by the culture medium.

First, a sample containing cells is injected into the cell culture channel.

Then, a culture medium suitable for the cells cultured with the neural cell growth factor channel is injected.

The culture solution may be diffused through the hydrogel in the cell culture channel direction.

Next, a suitable hydrogel is injected into the cells cultured in the neural cell growth channel. The nerve cell growth channel may be formed of a hydrogel, and the hydrogel may include at least one selected from the group consisting of, for example, collagen, fibrin, fibronectin and matrigel.

Then, the cells are cultured by the culture solution. The neuron of the cell may be grown in the growth factor channel direction on the electrode substrate of the neuron growth channel.

In addition, the step of culturing the cell may further include the step of analyzing the growth of the neuron. The step of analyzing the growth of the nerve cell may be to measure a measurement parameter between the two electrodes, wherein the nerve cell changes between the reference electrode and the measurement electrode. The measurement parameter may include at least one selected from the group consisting of resistance, voltage, current, and capacitance. The measurement parameter may be associated with the number and length of the nerve cells. Until the end of the analysis, the culture medium containing the cell culture medium and the nerve growth factor can be replaced at an appropriate time depending on the degree of neuron growth and the state of the cell.

As the growth of neurons becomes more active, the number of measurement electrodes can be increased and the measurement parameters of each electrode can be analyzed to determine the degree of neuronal growth.

The composition ratio of the cell culture liquid and the concentration of the nerve growth factor can be controlled according to the degree and condition of the nerve cell growth, and thus, a substantially similar amount of nerve cell growth body can be obtained. Therefore, it is possible to form a uniform amount of a neuron cell growth body in a large amount, and if the amount of the growth bodies is uniform, the error between the experimental groups can be reduced in an experiment using these cells. In particular, in the study of stem cell differentiation, the effects of the differentiation inducing agent and the culture medium can be precisely experimented and grasped.

Experiments have shown that as neurons grow over time, capacitances and currents in the electrical measurement parameters at the measuring electrodes increase, and as time passes, the nerve cells grow and thus the resistance And the voltage decreased, respectively.

The present invention relates to a neurite outgrowth analysis and a neurite outgrowth analysis, which can provide experimental information on the growth of the nerve protrusion by inputting the pH, temperature, and electrical measurement parameters measured from the microfluidic channel device and the microfluidic channel device, Cell culture channel monitoring and cell differentiation control software, neural projection growth control software to control the microfluidic device to regulate the growth of neurite through the information of growth of neurite outgrowth, And a microfluidic channel device control system for controlling the device.

Neurite outgrowth analysis and cell culture channel monitoring and cell differentiation control software, which receives signals in the form of potential and resistance through an electrode sensor measuring the pH and temperature integrated on the bottom of the cell culture channel, always monitors the environment of the cell culture channel And maintains and regulates the environment optimized for cell culture. If the environment of the cell culture channel exceeds a predetermined range based on the preset optimized temperature and pH level, information is transmitted to the user's terminal and the temperature is automatically controlled using the heating electrode or the culture liquid is replaced or injected into the new culture liquid . Also, if the rate of change of pH is constantly monitored over time for automatic cell differentiation control and the rate of change becomes larger than a preset level, the culture fluid of the cell culture channel is replaced with the culture solution for differentiation, and the nerve growth factor A neuronal differentiation sequence is initiated.

For example, you can set the control according to the following formula.

(R _{temp , set} : resistance set value for optimum temperature, R _{range, set} : resistance range set value for optimum temperature, Y _heat : heater electrode operation output, V _pHset1 : optimum pH Y _{C M: change} : output of culture fluid replacement, _{Δx 2} / t: pH change rate, V _{pHset 2} : pH change rate according to the differentiation conditions, Y _{D M change} : differentiation culture injection output for differentiation , Y _NGF : nerve growth factor injection output)

When the neurite of the neuron is differentiated using the microfluidic channel device of the cell culture monitoring and control device of the present invention, the value of the measurement parameter (resistance, voltage, current, capacitance, etc.) changes in the electrode channels of the device, Values are passed to the input of the neurite outgrowth signal processing software. In the microfluidic channel device, one reference electrode and the measuring electrode are constituted by four channels. However, the number of channels of the measuring electrodes, the width of the measuring electrodes, and the width between the measuring electrodes can be variably designed {E _i | E _i , 4 ≤ I ≤ 250}, {2 μm ≤E _w , W _E Lt; = 1000 [micro] m}. (E _w : Width of electrode, W _E : Distance between electrodes)

※ growth illustrates measurement (E _w: width of the electrodes, E _i: the number of electrodes, R _set: resistance set value, V _set: the voltage set value, V _set: the current setting value, C _set: capacitance settings, △ x _i : Change value of I-th resistance, N _L : length of neurite, N _GB : growth rate of neurite, t: time)

In the neurite outgrowth analysis software that receives the measured values transmitted from the channel device in the microfluidic device, the growth control value is generated using a single rule or pattern database as follows do. Here, the database can be expressed in various file structures such as a text file, a structured data structure file, and XML.

※ illustrates growth control (t: time, t _set: set time value, y _{1 -4:} an output control value of the parameter)

는 구조체 변수로 표현할 수 있는데 신경성장인자 주입 여부를 설정할 수 있는 변수, 신경성장인자 주입 농도를 설정할 수 있는 변수들의 값으로 구성할 수 있다.Here, the output value is not only a variable that stores a simple constant value but also a structure variable that can represent various data structures. For example

Can be expressed as a structure variable, and it can be constituted by a value for setting the nerve growth factor injection or a value for setting the nerve growth factor injection concentration.

In the case of growth promotion, if the amount of change in the measurement parameter for a specific time at the measurement electrode at a specific position is lower than the set value, it means that the neurite has not yet been grown to a specific length and a higher concentration of nerve growth factor And the nerve growth factor injection unit is driven. Thus, the growth of the nerve cells is promoted.

On the contrary, in the case of growth inhibition, if the change amount of the measurement parameter before the set time at the measurement electrode at the specific position is larger than the set value, it means that the growth of the neurite is fast, so that a signal for lowering the growth factor injection concentration is sent, To be reduced or stopped.

The microfluidic device control system, which receives the output signal from the neurite outgrowth control software, controls the growth of the neurite desired by the experimenter by controlling the composition of the culture fluid injected into the microfluidic channel device and the nerve growth factor concentration.

The neurite outgrowth signal processing software is capable of bi-directional information delivery, allowing neuron growth information to be delivered to the experimenter in real time, and the experimenter can also monitor neurite outgrowth at all times. The neuron and neurite growth information from the microfluidic device can be transmitted to the experimenter through an alarm signal. Based on this information, the experimenter can remotely control the microfluidic channel device Can be controlled.

For example, monitoring and control of the cell culture monitoring and control apparatus of the present invention by a terminal will be described in order.

1) The neuron cells are cultured in the microfluidic channel device of the prepared microfluidic channel device. 2) Neurons continue to proliferate until differentiated according to the experimenter's setting, and are optimized for optimal cell culture through pH and temperature monitoring during proliferation. 3) At the end of proliferation, when the differentiation begins, the culture is replaced with the controlled culture, and the culture solution is injected and the nerve growth factor is injected according to the set order. 4) Neurons differentiated by nerve growth factor grow neurites on the fabricated electrodes, which connect the two electrodes and cause electrical changes. 5) When the neurite grows up to a specific measuring electrode, it is possible to measure the degree of differentiation and growth of the neuron through the change of the electrical characteristic of the electrode, and this electrical change is transmitted through the transmitter, Analysis is done through analysis software and this information is communicated to the user. 6) Information sent from neurite growth analysis software is input to the neurite growth control software, and it delivers the information to be performed by the controller among the microfluidic channel devices according to the setting conditions of the experimenter. 7) The receiver receiving the information drives the heater part of the microfluidic channel device, the culture fluid injection part and the nerve growth factor injection part to control the composition of the culture fluid and the concentration of the nerve growth factor, .

The cell culture monitoring and control apparatus according to the present invention can provide a convenient experimental tool for quantifying neuronal cell growth in the development and regeneration of neurons in the neuroscience and brain science fields, It can provide important experimental and diagnostic tools in therapy and drug development research. In addition, quantitative and systematic analysis of neuronal growth can be used to develop 3D nerve cell growth assay and in vivo experiments.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those of ordinary skill 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. This is possible. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined by the equivalents of the claims, as well as the claims.

110: Microfluidic channel element
112: cell culture channel
114: Neuronal growth factor channel
116: Neuronal growth channel
120: electrode substrate
122: electrode
124: reference electrode
126: measuring electrode

Claims (9)

Microfluidic channel devices;
A measuring unit for measuring a state of the microfluidic channel device; And
A control unit for controlling the microfluidic channel device;
Lt; / RTI >
The microfluidic channel device comprising: a microfluidic channel device comprising a cell culture channel, a neuron growth factor channel, and a neuron cell growth channel disposed between the cell culture channel and the neuron cell growth factor channel; And an electrode; ≪ / RTI >
Cell culture monitoring and control devices.
The method according to claim 1,
Wherein the state of the microfluidic channel device is at least one selected from the group consisting of temperature in the microfluidic channel device, pH in the microfluidic channel device, degree of differentiation and growth of the neuron,
Cell culture monitoring and control devices.
The method according to claim 1,
Wherein the measuring unit comprises:
A temperature measuring unit, a pH measuring unit, and a nerve cell growth measuring unit.
Cell culture monitoring and control devices.
The method of claim 3,
Wherein the electrode comprises a plurality of microelectrodes,
Wherein the plurality of microelectrodes include a reference electrode and a plurality of measurement electrodes,
Wherein the neuron cell growth measuring unit comprises:
Wherein the degree of differentiation and the degree of growth of the neuron are measured by measuring an electrical change between the two electrodes generated while the neuron is grown and connecting the reference electrode and the measurement electrode.
Cell culture monitoring and control devices.
5. The method of claim 4,
Wherein the electrical change includes at least one selected from the group consisting of resistance, voltage, current, and capacitance.
Cell culture monitoring and control devices.
The method according to claim 1,
Wherein,
A heater, a culture fluid injecting part, and a nerve growth factor injecting part.
Cell culture monitoring and control devices.
The method according to claim 6,
Wherein the heater unit is driven according to a temperature measurement result in the microfluidic channel,
Wherein the culture liquid injecting unit and the nerve growth factor injecting unit are driven according to a result of pH measurement in the microfluidic channel,
Cell culture monitoring and control devices.
The method according to claim 1,
A transmission unit for transmitting a measurement result of the measurement unit; And
A receiving unit for receiving a control unit driving signal;
Further comprising a cell culture monitoring and control device.
9. The method of claim 8,
Wherein the transmitter transmits the measurement result to the terminal remotely,
Wherein the receiving unit remotely receives the driving signal from the terminal.
Cell culture monitoring and control devices.

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