JP5024777B2 - Cell dynamics observation device - Google Patents

Description

  The present invention relates to an apparatus for observing cell dynamics for observing a change in a state of a cell or a response of a cell to a drug or the like in the field of biological, medical, or biotechnology research.

  In the fields of conventional biology, pharmacy, and medicine, in order to observe changes in the state of cells or the response of cells to drugs, etc., a culture container (also referred to as a culture chip) having a chamber for containing cells is used. Observation on a sample stage (stage) of a microscope is performed.

  In this culture chip, in order to maintain the cell culture, it is necessary to monitor the culture environment such as temperature and the state of the medium that changes due to the metabolism of the cultured cells. If the culture environment is not kept constant, the reproducibility of the experiment is low, and reliable data cannot be obtained. In addition, when the composition in the medium is depleted or waste is accumulated, the division rate of the cultured cells decreases. A cultured cell having a reduced cell division rate is not suitable for research because the metabolic capacity of the cultured cell is greatly inferior to a cultured cell in a favorable culture state.

  Therefore, in order to culture and observe cells for a long time, a culture chip provided with a medium exchange means has been conventionally used.

  For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 2004-113092) is made of a transparent plate having a size that can be placed on a sample stage of a microscope, has a well inside, and a liquid supply port to the well. In addition, a cell culture chip having a sensor for detecting a medium in a well is disclosed in a cell culture chip having a liquid discharge port.

  However, the cell culture chip disclosed in Patent Document 1 has a drawback that the image quality of the obtained image is inferior because cells in the well are observed with a microscope through a transparent member that is a bottom plate of the chip. In addition, such a cell culture chip has a drawback that an oil immersion lens cannot be used.

  That is, an inverted microscope in which an objective lens protrudes upward from below the stage is usually used for observation of animal tissues and cultured cells. When using an oil immersion type lens with this inverted microscope, it is necessary to set the cover glass so that it comes to the lens side (due to restrictions such as the working distance of the objective lens). In the cell culture chip, since the transparent member of the chip is located on the lens side, an oil immersion lens cannot be used.

  In Patent Document 2 (Japanese Patent Laid-Open No. 10-28576), a transparent heat-generating cover glass that can be controlled in temperature is arranged vertically to form a space between them, and the periphery of the cover glass is sealed to form a sealed container In order to maintain the humidity in the space, a constant temperature culture container in which an evaporating dish is arranged in the container is disclosed.

  However, in this container, since the transparent heat generating cover glass is formed by joining the heat generating glass and the cover glass with an adhesive, there is a disadvantage that the image quality is inferior in order to observe the microscope through the transparent heat generating cover glass. is there. Furthermore, the production cost of the constant temperature culture container is also high.

  In Patent Document 3 (Japanese Patent Laid-Open No. 2002-153260), a cell culture part is formed by providing a number of well-shaped holes on a substrate, and a semipermeable membrane is disposed on the cell culture part. An apparatus has been disclosed in which a cell culture container having a medium exchanging section is arranged on the cell culture medium supply means to the cell culture container, and the cells in the cell culture section are observed with a microscope for a long period of time.

However, the apparatus disclosed in Patent Document 3 also has the disadvantages that the image quality is inferior and that an oil-immersed lens cannot be used because cells are observed through a substrate through a microscope.
JP 2004-113092 A JP-A-10-28576 JP 2002-153260 A

  An object of the present invention is to solve the above-mentioned drawbacks, and to provide a cell dynamics observation apparatus that can control the state of a medium such as temperature and pH and can perform high-resolution microscopic observation.

  Another object of the present invention is to provide a fluid (medium, etc.) chamber that enables continuous observation of cells such as living cells with a high-resolution oil immersion lens for a long period of time, and observation of a reaction over a long period of time to a stimulus. The object is to provide an apparatus for observing cell dynamics.

  Still another object of the present invention is to maintain a culture environment for a long period of time, to enable microscopic observation of cells for a long period of time, and to obtain more reliable data in biological, pharmaceutical and biotechnology research. An object of the present invention is to provide an apparatus for observing cell dynamics capable of performing the above.

  Still another object of the present invention is to provide a cell dynamics observation apparatus capable of accurately and reliably recording a long-term response of a cell to a specific substance, a series of behaviors of the specific substance on cultured cells, and the like. There is.

The observation device for cell dynamics of the present invention has a chamber for containing cells, and is a device for observing cell dynamics in the chamber, comprising a thermally conductive substrate and the substrate provided on the substrate. A chamber, and a culture medium reservoir for supplying a culture medium to the chamber. The chamber includes a wall portion of an opening formed in the substrate, and an upper end opening of the opening. And a cover glass capable of closing the lower end opening of the opening , and cells are cultured on the cover glass. The cover glass has a silicone-based grease on the lower surface around the opening. or removably attached by a double-sided adhesive tape, a flow path from the medium reservoir may supply the medium to the chamber are formed through the substrate, the discharge flow for discharging the medium supplied to the chamber There are formed through the substrate, the above-mentioned object can be achieved.

  In one embodiment, a silicone treatment agent is applied around the cover glass.

  In one embodiment, a Teflon (registered trademark) tape is attached around the cover glass.

  In one embodiment, the transparent plate is fixed around the upper part of the opening of the substrate.

  In one embodiment, the transparent plate is a glass plate or an acrylic plate.

  In one embodiment, the culture medium storage part is formed of a frame material arranged on the upper surface of the substrate.

  In one embodiment, the flow path is formed through the substrate, and the culture medium reservoir and the chamber are communicated with each other by the flow path.

  In one embodiment, the inlet of the flow path and the outlet of the discharge flow path in the chamber are located diagonally.

  In one embodiment, the cell is a living cell, an immobilized cell, a microorganism, or an animal or plant tissue.

  In one embodiment, the substrate can be mounted on a device capable of controlling temperature.

The method for observing cell kinetics of the present invention is a method for observing cell kinetics in a chamber using an apparatus having a chamber for containing cells, the apparatus comprising: a substrate; and A chamber for supplying a culture medium to the chamber; and the chamber closes a wall of an opening formed in the substrate and an upper end opening of the opening. A transparent plate and a cover glass capable of closing the lower end opening of the opening, and cells are cultured on the cover glass. removably attached by adhesive tape, a flow path from the medium reservoir may supply the medium to the chamber are formed through the substrate, to discharge the medium supplied to the chamber discharging A step in which a path is formed through the substrate and an injection needle attached to an injection syringe containing a drug is inserted into the chamber from the entrance of the flow path, and the drug is added to the target location in the observation field in the chamber , Thereby achieving the above objective.

  In one embodiment, a nozzle attached to the tip of a bottle containing a washing medium is inserted into the chamber from the inlet of the flow path, and the medium is ejected from the nozzle into the chamber, and at the same time, the medium in the discharge flow path. Increasing the flow rate.

  According to the present invention, a substrate having excellent thermal conductivity is used, and a cover glass is attached to the lower surface of the opening provided in the substrate to form a chamber for culturing cells in the opening. In addition, the medium is supplied from the medium reservoir to the chamber, and the medium in the chamber is discharged.

  Therefore, it is possible to maintain a culture environment for a long period of time, and it is possible to observe cells for a long period of time with a microscope. Further, high-resolution observation with an optical microscope can be performed for a long time. Furthermore, by using the chamber of the present invention, it is possible to continuously observe an instantaneous to long-term response to stimulation of living cells such as cultured cells, immobilized cells, microorganisms, or animal and plant tissues. Become. For example, it is possible to accurately and reliably record a long-term response response of a cell to a specific substance, a series of behaviors of the specific substance on cultured cells, and the like. As a result, more reliable data can be obtained in biological, pharmaceutical, and biotechnology research.

Furthermore, the present invention has the following effects.
(1) Since a cover glass in which cells are cultured is attached, observation with a high-magnification / high-resolution objective lens can be handled. In particular, it is desirable to use a cover glass having a silicone coat around it.
(2) By simply applying silicone grease to the cover glass bonding surface of the device, adhesion to the cover glass on which the cells are cultured is maintained, and the material of the substrate of the device is made of aluminum, so the temperature conductivity is good. It reaches a certain temperature quickly, and the subsequent temperature control is very good.
(3) Since it is possible to maintain the pH of the medium by strict temperature control and constant exchange of the medium, it is possible to perform observation for a long time while maintaining the cells in a good state while being a simple apparatus.
(4) Since the culture medium storage part is mounted on the substrate of the apparatus, when this culture medium storage part is filled with a culture medium, continuous observation is possible without replenishing the culture medium. For example, continuous observation for about 40 minutes was possible with the apparatus of the example shown below.
(5) Produces a slow flow in the diagonal direction (flow rate 20 μl / min) in the chamber and constantly supplies fresh medium to maintain the pH of the medium and maintain the cells in good condition for a long period of time. Is possible.
(6) Since the flow path for supplying the culture medium from the culture medium storage part to the chamber is formed, the drug can be administered to the cells or the cleaning liquid can be supplied using this flow path. Thereby, high-resolution observation of cell dynamics by adding a small amount of drug is possible. In particular, by increasing the flow rate of the liquid on the diagonal line in the chamber, it is possible to quickly wash the administered drug and observe the cell dynamics with respect to the drug administration with high resolution.
(7) In the method for observing cell dynamics of the present invention, an injection needle attached to an injection syringe filled with a drug to be administered is inserted into the chamber from the entrance of the flow path, and the target position of the observation visual field in the chamber is set. A step of adding a drug, so that a small amount of the drug can be reliably added to a target portion of the observation visual field in the chamber.

  Furthermore, a nozzle attached to the tip of a bottle containing a fresh medium for washing is inserted into the chamber from the entrance of the flow path, and the medium is sprayed from the nozzle into the chamber. Excess drug present in the chamber can be quickly removed (that is, rapid cleaning is possible). Thereby, high-resolution observation of cell reaction by adding a small amount of drug is possible.

  The present invention is described in detail below.

  As shown in FIGS. 1 to 3, the cell dynamics observation device of the present invention includes a substrate 2 having excellent thermal conductivity, a chamber 4 for accommodating cells provided on the substrate 2, and the substrate 2. And a medium storage section 6 for supplying the medium to the chamber 4.

  The board | substrate 2 can be formed with the metal excellent in heat conductivity, such as aluminum, iron, copper, nickel, brass. As the shape of the substrate 2, as shown in FIG. 3, it can be formed of a rectangular plate material.

  A chamber 4 is provided at a substantially central portion of the substrate 2. The chamber 4 includes a wall 10 of the opening 8 formed in the substrate 2, a transparent plate 12 that closes the upper end opening of the opening 8, and a cover glass 14 that can close the lower end opening of the opening 8. , Is formed by.

  The opening 8 is formed through the substrate 2. The planar shape of the opening 8 may be any shape such as a quadrangle, a triangle, an ellipse, or a circle. The transparent plate 12 is formed of a transparent plate material such as a glass plate or a plastic plate such as an acrylic plate, and is disposed on the substrate 2 so as to close the opening 8, and the periphery of the transparent plate 12 is the opening of the substrate 2. It is fixed to the periphery of 8 in a liquid-tight manner. As shown in FIG. 2, a recess may be provided over the entire periphery of the opening 8 and the end of the substrate 2 may be fitted into the recess.

  The cover glass 14 is formed of a transparent glass thin material. As the cover glass 14, a commercially available cover glass having a refractive index of about 1.52 and a thickness of 0.17 mm ± 0.02 mm can be typically used. Commonly used sizes are 18x18mm, 18x24mm, 24x24mm, 24x40mm, of essentially any size and shape (may be circular) as long as they do not affect the optical field of view Can be used.

  In addition, it is preferable that a silicone-based treatment agent is applied around the cover glass 14 or a fluororesin-based tape is attached to the cover glass 14 because the liquid does not leak. As this fluororesin tape, a thin tape having a thickness of about 0.08 mm can be used. Examples of the fluororesin include polytetrafluoroethylene. Cells are cultured on the cover glass 14.

  The cover glass 14 is detachably attached to the lower peripheral surface of the opening 8 of the substrate 2. The cover glass 14 is removably attached to the lower peripheral surface of the opening 8 by applying silicone grease around the cover glass 14 and bonding the grease. Alternatively, the cover glass 14 is detachably attached to the lower peripheral surface of the opening 8 with a double-sided adhesive tape.

  A culture medium reservoir 6 is provided on one end side of the substrate 2. The medium storage unit 6 is formed by arranging a frame member 16 on the upper surface of the substrate 2. The frame member 16 is fixed to the surface of the substrate 2 with a fixing tool such as a screw.

  A flow path 18 is formed from the bottom of the medium reservoir 6 (the upper surface of the substrate 2) toward the chamber 4. The flow path 18 is formed through the substrate 2, and the culture medium storage unit 6 and the chamber 4 are communicated with each other by the flow path 18.

  Further, a discharge flow path 20 is formed through the substrate 2, and the inside of the chamber 4 and the medium discharge tube 22 are communicated with each other through the discharge flow path 20. A pump (not shown) for sucking the culture medium is connected to the tip of the tube 22.

  As shown in FIG. 3, the positions of the inlet 18a of the flow path 18 formed in the chamber 4 and the outlet 20a of the discharge flow path 20 formed in the chamber 4 are arranged almost diagonally in a plan view. The medium sent from the inlet 18a of the flow path 18 into the chamber 4 is discharged from the outlet 20a of the discharge flow path 20 without stagnation.

  In the figure, reference numeral 24 denotes a presser for fixing the tube 22 to the substrate 2, and is fixed to the other end of the substrate 2 with a fixing tool such as a screw. The substrate can be provided with a temperature sensor and a pH sensor. A known pH sensor can be used, and examples thereof include a LAPS type pH sensor and an ISFET type sensor.

  The apparatus of the present invention having the above configuration is set on an inverted microscope stage capable of temperature control as follows.

  Cells are placed on the cover glass 14, the cover glass 14 is attached to the lower surface of the opening 8 of the substrate 2, and the chamber 4 is formed by the cover glass 14, the wall 10 around the opening 8, and the transparent plate 12. . The medium is supplied from the medium storage unit 6 to the chamber 4. Alternatively, the substrate 2 shown in FIGS. 1 to 2 is turned upside down so that the opening of the chamber 4 faces upward. In this state, the medium is filled into the chamber 4, and then the cover glass 14 is attached to the opening 8. Also good. Thereafter, after the substrate 2 is turned upside down again, the medium storage section 6 is filled with the medium.

  Then, the apparatus of the present invention is set on the inverted microscope stage.

  When the medium is filled in the chamber 4, the medium is gradually discharged from the discharge channel 20 by sucking the medium in the chamber 4 with a pump, so that a fresh medium is supplied from the medium reservoir 6 to the chamber 4. The

  Here, since the substrate 2 is thermally conductive, when the apparatus is placed on a temperature-controlled stage, heat is transferred to the substrate 2 and the medium in the chamber 4 and the medium in the medium reservoir 6 are heated. Is done. Therefore, when a pump is attached to the distal end of the tube 22 extending from the fluid suction port 20a and the medium is slowly sucked, the chamber 4 has a fluid kept at a constant temperature (for example, fresh and kept at the culture temperature). Medium) is supplied from the reservoir 6.

  In this way, the dynamics of cells without drugs can be monitored (evaluated). That is, in order to observe living cells for a long time, it is necessary to sequentially replace the medium with a fresh one in order to prevent fluctuations in medium components accompanying cell temperature control and cell metabolism. In addition, high-resolution observation of living cells is often performed for the purpose of observing a response to cell stimulation. For that purpose, it is required that a drug or the like in an amount as required is added to the space in which the cells on the microscope are enclosed without moving the cells from the microscope.

  According to the present invention, an injection needle attached to an injection syringe or the like is inserted from the fluid inlet 18 of the reservoir 6 into the chamber 4, and the drug is accurately added to the target location on the observation field. Insert a nozzle or the like attached to the tip of a bottle or the like into the chabar 4 from the inlet 18 of the flow path, spray a fresh medium for washing, and simultaneously increase the flow rate to quickly remove excess chemicals after addition. It becomes possible to do.

  As the injection syringe, any known syringe can be used as long as it can add a drug, and the injection needle has an outer diameter smaller than the inner diameter of the flow path 18. Moreover, the conventionally well-known thing can also be used for the said nozzle and the nozzle attached to the front-end | tip.

  Therefore, administration and washing of drugs and the like to cells can be performed freely on a microscope. High-resolution continuous observation of living cells becomes possible. In addition, it is possible to observe the reaction from the moment to the long-term to the stimulation of the cell with high resolution.

  With such a mechanism, when observing a culture medium containing tissue, the chamber 4 can maintain the freshness of the culture medium while maintaining the culture temperature on the microscope stage on which the high-resolution objective lens is set. Further, by adjusting the degree of suction by the pump, for example, the medicine can be removed by increasing the injection rate of the medium into the chamber 4.

  As described above, according to the present invention, it is possible to exchange, recirculate, add, and control the temperature of a fluid (such as a culture medium). For example, a pump is connected to the tube, the medium can be aspirated over about 40 minutes, and the dynamics of cells without drugs can be captured and analyzed as an image with a cooled CCD camera or the like. Or increase the injection rate to remove the drug.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

Example 1
Temperature stability in the chamber of the cell dynamics observation device of the present invention The cell dynamics observation device of the present invention is installed on the temperature control plate (shifted from 20 ° C setting to 38 ° C setting), and the temperature of the cover glass 14 surface Change was tracked.

  The apparatus stored at 20 ° C. showed a temperature rise corresponding to the temperature rise of the temperature control plate, and after reaching the set temperature, stable temperature control was possible within a range of ± 0.2 ° C. Temperature data was captured every 2 seconds. The outside temperature was 28 ° C.

  The result is shown in FIG.

(Example 2)
High-resolution photography of living animal cultured cells with oil immersion objective lens Using cultured cells derived from the ovary of Chinese hamsters (CHO-K1, Riken Cell Bank), which are commonly used for studies at the cellular level of animals, While maintaining the physiological condition of the cells on the microscope, high-resolution micrographs of live cells were performed using an oil immersion objective lens.
A. In order to keep cells alive in a small sealed container on a microscope for a long time, it is necessary to keep the temperature at about 37 ° C. In addition, the acidity of the medium by extracellular discharge of acidic waste products In order to prevent oxidization, it is necessary to replace the medium with a fresh one.

  Therefore, in this example, an apparatus as shown in FIGS. 1 to 3 was produced.

  The apparatus used was an aluminum substrate 2. The substrate 2 is provided with an opening (observation window) 8 for adhering a cover glass 14 of 24 mm × 24 mm at the center thereof, and the cells to be observed are put in the substrate 2 and observed by bringing the cover glass 14 into close contact therewith. Is possible.

For medium flow and drug addition, insert an injection needle attached to an injection syringe or the like through the inlet of the flow path, add the drug directly to the target location in the field of view, or use a suction tube (slow and stable speed) This can be done by connecting to a suction pump. A culture medium is added to the culture medium storage section 6 formed of an acrylic frame material, and the start of suction and the control of the suction speed are all performed by a suction pump.
B. Attaching the cover glass to the chamber After the medium is filled in the chamber, the cover glass 14 to which the CHO cells cultured on the cover glass that has been coated with silicone is adhered to the opening so that the cells are in contact with the medium. Covering and pressing the peripheral edge against the substrate side completed the mounting of the cover glass 14. By mounting the cover glass 14, the medium can be flowed.

  The apparatus of the present invention having the above-described configuration is placed on a sample stage (stage) of an inverted microscope in which the objective lens protrudes upward from the bottom of the stage, and the living cell height is increased using a 100 × oil immersion objective lens. Observation with a resolution microscope (using a photographing apparatus having a computer image analysis function based on a highly sensitive cooled CCD camera) was performed.

  The results are shown in FIG. 5 and FIG.

  FIG. 5 is a phase difference image immediately after the cover glass is mounted on the substrate, and FIG. 6 is a phase difference image 60 minutes after the cover glass is mounted.

  From these figures, it can be seen that the movement of minute tubes and granules in the cell is clearly captured. Further, even after 60 minutes, the cells remained in a good state, and the same movement was observed immediately after wearing. Moreover, it turns out that the cell environment is kept favorable by the good temperature control and the constant replacement | exchange of a culture medium.

(Example 3)
Using the same apparatus as shown in Example 2, after adding fluorescently labeled denatured LDL (DiD-AcLDL: ligand) to cells expressing a receptor that recognizes denatured LDL, excess ligand is added. Removed and observed its uptake process.

  7 to 13 show continuous images of this living cell (taken images by a cooled CCD camera).

  FIG. 7 is a phase difference image.

  FIG. 8 is a fluorescence image before ligand administration. It is dark because there is no fluorescent material.

  FIG. 9 is a fluorescence image immediately after ligand administration. Since there is a large amount of ligand in the medium, the image is entirely white.

  FIG. 10 shows an image 5 seconds after injecting fresh medium at once with a nozzle and at the same time increasing the flow rate and starting to wash off excess ligand. The excess ligand in the medium begins to be removed, and the ligand image bound to the cells becomes clear.

  FIG. 11 is an image 15 seconds after the start of washing. Excess ligand is completely removed, and only the ligand image bound to or taken up by the cells can be clearly captured.

  FIG. 12 is a fluorescence image 120 seconds after ligand administration. Compared to FIG. 11, the movement of the ligand can be tracked.

  FIG. 13 is an image 12 minutes after ligand administration. The cells remain healthy and it is observed that the ligand is accumulated around the cell nucleus.

  Thus, according to the apparatus of the present invention, an image immediately after addition of a fluorescently labeled ligand that can be taken up by cells, an image after washing, and an image obtained by tracking the process of taking up the ligand by cells with high resolution over time are obtained. Obtainable.

Example 4
Using the apparatus shown in Example 2, cells cultured on the cover glass 14 not subjected to the silicone coating treatment were mounted on the lower surface of the opening of the apparatus and observed with a 100-times oil immersion lens.

  The phase difference images are shown in FIGS.

  FIG. 14 is an image immediately after the cover glass is mounted, and FIG. 15 is an image 60 minutes after the mounting.

  From these figures, it is possible to use the cover glass 14 that has not been subjected to silicone treatment, but the image quality may be inferior because of poor adhesion. Also, a temperature change of less than one may occur, so that cells may react.

It is a perspective view of the observation device of cell dynamics of one example of the present invention. It is sectional drawing of the observation apparatus of the cell dynamics of one Example of this invention. It is a top view of the observation apparatus of the cell dynamics of one Example of this invention. It is a graph which shows the temperature stability in the chamber using the observation apparatus of the cell dynamics of this invention. FIG. 5 is a photomicrograph immediately after the cover glass is attached to the substrate. FIG. 6 is a photomicrograph 60 minutes after mounting. FIG. 7 is a photomicrograph of a cell phase contrast image. FIG. 8 is a photograph of a fluorescent image of a continuous image of cells before ligand administration. FIG. 9 is a photograph of a fluorescence image immediately after administration of a ligand to a cell. FIG. 10 is a photograph of cells 5 seconds after injecting fresh medium at once with a nozzle and at the same time increasing the flow rate and starting to wash off excess ligand. FIG. 11 is a photograph of the cells 15 seconds after the start of washing. FIG. 12 is a photograph of a fluorescence image 120 seconds after ligand administration. FIG. 13 is a photograph of cells 12 minutes after ligand administration. FIG. 14 is a photograph of a cell immediately after mounting of a cover glass of a phase difference image. FIG. 15 is a photograph of the cells 60 minutes after mounting the cover glass of the phase contrast image.

Explanation of symbols

2 Substrate 4 Chamber 6 Medium storage part 8 Opening part 10 Wall part 12 Transparent plate 14 Cover glass

Claims (11)

An apparatus for observing cell dynamics in the chamber, the chamber containing cells;
A thermally conductive substrate, the chamber provided in the substrate, and a culture medium reservoir provided in the substrate for supplying a culture medium to the chamber;
The chamber is formed and the wall of the opening formed in the substrate, and a transparent plate which closes the upper end opening of the opening, and a cover glass capable of closing the lower end opening of the opening, by the cover Cells are cultured on the glass, the cover glass is removably attached to the lower peripheral surface of the opening with silicone-based grease or double-sided adhesive tape, and a flow path capable of supplying the medium from the medium reservoir to the chamber is provided. An apparatus for observing cell dynamics in a chamber, wherein a discharge channel formed through the substrate and discharging the medium supplied into the chamber is formed through the substrate. The apparatus according to claim 1, wherein a silicone-based treatment agent is applied around the cover glass. The apparatus according to claim 1, wherein a fluororesin tape is attached around the cover glass. The apparatus according to claim 1, wherein the transparent plate is fixed around an upper portion of the opening of the substrate. The apparatus according to claim 1, wherein the transparent plate is a glass plate or an acrylic plate. The apparatus according to claim 1, wherein the culture medium storage unit is formed of a frame member disposed on the upper surface of the substrate. The apparatus of claim 1, wherein an inlet of the flow path and an outlet of the discharge flow path in the chamber are located diagonally. The device according to claim 1, wherein the cell is a living cell, an immobilized cell, a microorganism, or an animal or plant tissue. The apparatus of claim 1, wherein the substrate is mountable on an apparatus capable of controlling temperature. A method for observing cell dynamics in a chamber using an apparatus having a chamber containing cells,
The apparatus includes a substrate, the chamber provided in the substrate, and a medium reservoir for supplying a medium to the chamber,
The chamber is formed and the wall of the opening formed in the substrate, and a transparent plate which closes the upper end opening of the opening, and a cover glass capable of closing the lower end opening of the opening, by the cover Cells are cultured on the glass, the cover glass is removably attached to the lower peripheral surface of the opening with silicone-based grease or double-sided adhesive tape, and a flow path capable of supplying the medium from the medium reservoir to the chamber is provided. A discharge channel formed through the substrate and discharging the medium supplied into the chamber is formed through the substrate;
A method for observing cell dynamics, comprising a step of inserting an injection needle attached to an injection syringe containing a drug into the chamber from the entrance of the flow path and adding the drug to a target location in an observation field in the chamber. Further, a step of inserting a nozzle attached to the tip of a bottle containing a washing medium into the chamber from the entrance of the flow path, and ejecting the medium from the nozzle into the chamber, while simultaneously increasing the flow rate of the medium in the discharge flow path, The method of claim 10 comprising .