JP2004113092A - Cell culture chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture chip enabling cells to be observed for a long period and recorded by photographing or using video technique through monitoring the condition of a culture liquid and exchanging it at proper timing. <P>SOLUTION: The cell culture chip 10 includes a sensor 18 for detecting the pH, temperature or the like of the culture liquid in a well. The sensor 18 to be used is e.g. a pO<SB>2</SB>sensor, NO sensor, CO<SB>2</SB>sensor. By using a pH sensor for example, timing of exchanging the culture liquid can be grasped accurately. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a cell culture chip that enables long-term microscopic observation by measuring a culture environment and exchanging a culture solution at an appropriate timing in a research field of biology, medicine, or biotechnology.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, observation of a culture state of a cell in a biological, medical, or biotechnology research field is mainly performed by liquid culture. In order to directly observe the state of the cultured cells, a culture vessel (culture chip) that can be mounted on a sample stage (stage) of a microscope is used (for example, see Patent Document 1). Further, in order to culture and observe cells for a long period of time, a culture chip provided with a culture solution exchange means is also used (for example, see Patent Document 2).
[0003]
[Patent Document 1]
JP-A-10-28576 ([0006], FIG. 2)
[Patent Document 2]
JP-A-2002-153260 ([0015], FIG. 1)
[0004]
[Problems to be solved by the invention]
In order to maintain the cultured cells, it is necessary to constantly monitor the culture environment such as temperature and the state of the culture solution that changes due to the metabolism of the cultured cells. If the culture environment is not kept constant, the reproducibility of the experiment will be low and reliable data cannot be obtained. When the composition in the culture solution is depleted, the division rate of the cultured cells decreases. Cultured cells with a reduced cell division rate have a much lower metabolic capacity than cultured cells in good culture conditions, and are not suitable for research.
[0005]
However, with a cell culture chip large enough to be mounted on a microscope sample stage, the amount of culture solution that can be held is small, and the composition in the culture solution is quickly depleted.
[0006]
The present invention has been made in order to solve such problems, the purpose of which is to monitor the state of the culture solution, by performing the replacement of the culture solution at an appropriate timing, for a long time An object of the present invention is to provide a cell culture chip that enables recording of observation and photographing (video).
[0007]
[Means for Solving the Problems]
The present invention has been made to solve the above-mentioned problems. The present invention is made of a transparent plate material large enough to be placed on a sample stage of a microscope, has a well therein, a liquid supply port to the well, and a liquid drainage. In a cell culture chip having an outlet, a sensor for detecting a culture solution in a well is provided.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
In the cell culture chip according to the present invention, since the sensor for detecting the culture solution in the well is provided, the state of the culture solution inside the well can be detected.
[0009]
For example, in order to grasp the state of cells in a culture solution, it is sufficient to mainly monitor substances metabolized by the cells. Metabolites vary depending on the type of cultured cells (eg, plant cells, animal cells, fungi, etc.), but by using sensors that detect the presence and / or abundance of metabolites according to each type, Can be grasped. By collating the state of the cultured cells detected in this way with the results of microscopic observation (images, videos, etc.), detailed knowledge about the cells can be obtained.
[0010]
Even when the metabolite is unknown or there is no sensor for detecting the metabolite, the state of the cultured cells can be detected by using the pH sensor. That is, in almost all cultured cells, the pH of the culture solution can be an appropriate index indicating the state of the cells. Therefore, in the culture environment of the cells to be cultured, the optimum pH range is examined, and the pH value at which the culture solution should be replaced is determined. By doing so, the timing for replacing the culture solution can be accurately grasped by monitoring the pH of the culture solution.
[0011]
A variety of sensors can be used to monitor pH. LAPS-type pH sensors (see Japanese Patent Application Laid-Open No. 9-203722) and ISFET-type sensors (see Japanese Patent Application Laid-Open No. -203721).
[0012]
It is also possible to monitor the pH of the culture by adding a dye such as phenol red whose color changes due to a change in pH to the culture and measuring the culture during the culture spectroscopically.
[0013]
The pO ₂ sensor and CO ₂ sensor can monitor the activity of cells in the culture chip by measuring the increase / decrease. Further, according to the measurement result, the amounts of O ₂ and CO _{2 in} the culture solution supplied to the culture chip may be adjusted.
[0014]
The NO sensor is effective not only as an indicator of the metabolism of nitrate nitrogen concentration when culturing plant cells in the composition in the culture solution, but also as an effective indicator of the response of the cell to stimulation by a specific substance. Become.
[0015]
A temperature sensor monitors temperature, which is one of the important factors in the cell culture environment. As the temperature sensor, it is desirable to use a thermocouple because of its small size.
[0016]
The temperature sensor can also be used as a sensor for controlling the temperature of the culture environment. In this case, as means for changing the temperature of the culture solution in the culture chip, heating means include hot water circulation, an infrared heater (infrared irradiation), a hot air fan (hot air blowing), and a cooling means. Examples include cold water circulation. By using a Peltier element, both heating and cooling can be performed.
[0017]
【The invention's effect】
By using the cell culture chip according to the present invention, a culture environment can be maintained for a long period of time, and microscopic observation of cells can be performed for a long period of time. This makes it possible to obtain more reliable data in research on living organisms, medicines, biotechnology, and the like. For example, it is possible to accurately and reliably record a long-term response reaction of a cell to a specific substance, a series of behaviors of a specific substance to a cultured cell, and the like, which could not be investigated until now.
[0018]
Embodiment 1
FIG. 1 shows the configuration of a cell culture chip according to one embodiment of the present invention. As shown in FIG. 1 (B), the cell culture chip 10 of the present embodiment is composed of three transparent plates of a well layer 11, a channel layer 12, and a sensor layer 13. These three transparent plates are bonded by optical contacts.
[0019]
The well layer 11 has a well 14 for holding cultured cells, the channel layer 12 has channels 15a and 15b connecting the well 14 to the culture solution supply port 16 and the discharge port 17, and the sensor layer 13 has a well 14 A sensor 18 for monitoring various parameters of the culture solution therein, and a culture solution supply port 16 and a discharge port 17 are provided. The tip of the sensor 18 is provided at a position where the culture solution in the well 14 can be detected. Note that the sensor 18 is desirably arranged so that its tip is as close to the ceiling of the well 14 as possible so as not to interfere with the cells. Further, in order to detect the culture solution after the interaction with the cells, it is desirable to arrange the culture solution as close to the culture solution outlet 17 as possible.
[0020]
FIG. 2 shows a configuration for observing cultured cells using the cell culture chip 10 of the present embodiment. First, the target cells 20 are put into the well 14 together with the culture solution from the supply port 16 or the discharge port 17 of the cell culture chip 10. The cell culture chip 10 containing the cells is placed on the stage 21 of the microscope, and the cell culture chip 10 is fixed by the holder 22. Then, the tube from the culture solution tank 23 is connected to the supply port 16 of the cell culture chip 10, and the discharge port 17 is connected to the drainage tank 25. A peristaltic pump 24 is provided in the middle of the tube from the culture solution tank 23. Note that, instead of the culture solution tank 23 and the pump 24, a syringe and a syringe pump may be used. A dedicated terminal unit 19 is attached to the terminal of the sensor 18 protruding outside the cell culture chip 10, and is connected to the monitor system 27.
[0021]
The cells in the well 14 are observed by the objective lens 26 arranged below the microscope stage 21. The image captured by the objective lens 26 is converted into an electric signal in real time by the CCD, and is similarly sent to the monitor system 27.
[0022]
The monitor system 27 is a personal computer operated by a dedicated program. The monitor system 27 processes a detection signal from the sensor 18 of the cell culture chip 10 and an observation video signal. 21 is also controlled.
[0023]
The observed image is sent to the monitor system 27 in real time as described above, and is displayed on a monitor screen 28 connected thereto. Further, it is possible by switching the switch in the monitor system 27, the value of the detected pH and pO ₂ such parameters may also be displayed on the monitor screen 28 in the sensor 18. In addition, these detection data and images can be recorded in a storage device 29 such as an HDD.
[0024]
A change (deterioration) in the culture conditions of the cells 20 can be detected by the sensor 18. For example, if a pH sensor is included in the sensor 18 and the pH of the culture solution detected thereby exceeds a predetermined value, it can be determined that the culture solution has deteriorated. In this case, the culture medium is supplied to the well 14 by driving the pump 24, and the culture medium is exchanged by pushing out the old culture medium from the outlet 17.
[0025]
The pH of the culture solution can also be detected by the following method. A pH indicator dye such as phenol red is put into the culture solution and supplied to the cell culture chip 10. When the pH value of the culture solution increases due to the metabolism of the cultured cells, phenol red discolors. As shown in FIG. 6, this color change is detected as an absorbance of a specific wavelength by a spectrophotometer 63 and an optical fiber guide (61 is for transmitting light, 62 is for receiving light), and is detected by an absorbance (Abs) -pH converter 64. Convert to pH value. By comparing this value with a predetermined value, it is possible to detect deterioration of the culture solution.
[0026]
By continuing the culturing and the observation for a long time in this way, a series of response reactions of the cultured cells can be observed from both sides of a microscope image and changes in the culture solution composition.
[0027]
In order to keep the culture temperature constant, a temperature control means may be provided on the microscope stage 21 or the holder 22. Examples of such a temperature control means are shown in FIGS. In the example of FIG. 4, the infrared light source 43 is used as a heating unit, and in the example of FIG. 5, the sheet heater 53 attached to the cell culture chip 10 is used as a heating unit. In any case, temperature sensors 41 and 51 are included in the sensor 18, and the temperature of the culture solution in the well detected by the feedback is fed back to the controllers 42 and 52 to perform temperature adjustment.
[0028]
Embodiment 2
A tandem cell culture chip according to a second embodiment of the present invention will be described. By using this cell culture chip, the response of the cultured cells to the fluorescently labeled substance P can be continuously observed using a fluorescence microscope.
[0029]
The tandem cell culture chip 30 shown in FIG. 3 is one in which two wells 31a and 31b having the same shape are provided in parallel in one chip. Each of the wells 31a and 31b is provided with channels 32a and 32b, supply ports 33a and 33b, discharge ports 34a and 34b, and sensors 35a and 35b, respectively, as in the embodiment of FIG. In the cell culture chip of the present embodiment, second supply ports 36a and 36b are provided for each well 31a and 31b.
[0030]
In the cell culture chip 30 of the present embodiment, the same cells are put in the two wells 31a and 31b, and the cells are cultured in parallel at the same time. However, only one of the wells (the upper 31a in FIG. 3) is added with the fluorescently labeled substance P through the supply port 36a to form an experimental section. Cultured cells in the well 31b to which the substance P is not added are treated as a control for cell response.
[0031]
Microscopic observation is performed on the experimental section (well 31a) to which the fluorescently labeled substance P has been added. Since the fluorescence-labeled substance P is excited by the excitation light of the fluorescence microscope, the behavior of the substance P on the cultured cells can be observed by observing the fluorescence.
[0032]
Further, by comparing the detection data obtained by the sensors 35a and 35b in the experimental section (well 31a) in which the fluorescent-labeled substance P was added to the culture solution and the control section (well 31b) in which the fluorescent-labeled substance P was not added, the culture cells against the substance P were compared. The response response can be accurately grasped. Since both experimental sections were cultured in the same chip, the difference in the detection data of the sensors 35a and 35b between the experimental section and the control section was due to the reliability of the response of the cultured cells to the pure substance P. Can be treated as high data.
[Brief description of the drawings]
FIG. 1 is a plan view (A) and a cross-sectional view (B) of a cell culture chip according to a first embodiment of the present invention.
FIG. 2 is a schematic configuration diagram when a cultured cell is observed under a microscope using the cell culture chip of the first embodiment.
FIG. 3 is a plan view of a tandem cell culture chip according to a second embodiment of the present invention.
FIG. 4 is a configuration diagram showing an example of a temperature control unit using an infrared light source.
FIG. 5 is a configuration diagram showing an example of temperature control means using a seat heater.
FIG. 6 is a configuration diagram showing an example of pH detection using phenol red.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 10 ... Cell culture chip 11 ... Well layer 12 ... Channel layer 13 ... Sensor layer 14 ... Wells 15a and 15b ... Channel 16 ... Culture solution supply port 17 ... Culture solution outlet 18 ... Sensor 19 ... Sensor terminal unit 20 ... Cell 21 ... Microscope stage 22 ... Chip holder 23 ... Culture buffer 24 ... Culture buffer 25 ... Drain tank 26 ... Objective lens 27 ... Monitor system 28 ... Monitor screen 29 ... Storage device

Claims (4)

In a cell culture chip having a transparent plate member of a size that can be placed on a sample stage of a microscope, having a well therein, and having a liquid supply port to the well and a liquid discharge port,
A cell culture chip provided with a sensor for detecting a culture solution in a well. The cell culture chip according to claim 1, further comprising a channel between the well and a liquid supply port or a liquid discharge port. The cell culture chip according to claim 1, wherein the sensor is one or a combination of a pH sensor, a pO ₂ sensor, a NO sensor, a CO ₂ sensor, and a temperature sensor. The cell culture chip according to any one of claims 1 to 3, wherein a temperature control means is provided in addition to the sensor.

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