JP2019122313A - Culture apparatus for microscopic observation - Google Patents

Abstract

To provide a culture apparatus for microscopic observation capable of easily medicating cells received in a well plate using a dispenser or the like and yet ensuring excellent airtightness within the well plate.SOLUTION: The culture apparatus for microscopic observation includes: an XY stage unit 1 for moving a plate holder in XY directions; a well plate held by the plate holder to receive specimens now being cultured; and a top heater unit 2 and a bottom heater unit 3 provided to hold the XY stage unit 1 therebetween from upper and lower sides. The top heater unit 2 has an opening 21a formed to add a liquid drug to the specimens received within the well plate, and has a lid portion 6 openably/closably provided to cover the opening 21a.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a culture apparatus for microscopic observation.

  For example, in the field of cell research, in recent years, a method using living cells has become important in place of conventional fixed cells. In the case of such living cells, observing the phenomenon while culturing the cells for a long time is performed.

In general, in order to culture cells for a long period of time, it is preferable to maintain a suitable environment for the cells. For example, Patent Document 1 describes that it is preferable to maintain a temperature of 37 ° C., a humidity of 90%, and a CO 2 concentration of 5% as a general cell culture environment.

Further, in Patent Document 1, a top heater unit and a bottom heater unit are provided to sandwich the XY stage unit from above and below, and the XY stage unit is provided with a plate holder for holding a well plate containing a sample being cultured. A culture apparatus for microscopic observation is described. Further, in Patent Document 1, the XY stage unit can be largely driven in either the X or Y direction so that the well plate can be attached and detached, and the plate holder is heated by a heating heater or CO ₂ gas from the outside. It is described that a CO ₂ port for supply is provided, and that a flow path is formed in the bottom heater unit and the inside of the plate holder can be humidified.

  Further, Patent Document 2 proposes a device having a configuration in which a lid is provided to each cell culture container in a cell culture device for simultaneously culturing cells using a plurality of cell culture containers (well plates).

JP, 2015-090374, A JP 2012-524527 gazette

  By the way, as a method of observing while culturing living cells, for example, observation by a method such as a calcium reaction or the like is carried out while observing the reaction of cells immediately after dosing or immediately after dosing. . In this case, the dosing of the living cells is performed by a dispenser or the like.

In the culture apparatus for microscopic observation described in Patent Document 1, in order to realize both functions of maintenance of an environment suitable for cell culture and operability of dispensing by a dispenser, the dispenser is provided on the top heater unit by a dispenser. A hole for medication is formed. However, in Patent Document 1, because of its hole in the top heater unit is formed, since is possible to ensure the airtightness of the cell culture environment difficult, it is necessary to mass supply of CO ₂ gas, CO ₂ gas There is a problem that the consumption of the

  Moreover, in the cell culture device described in Patent Document 2, the plurality of cell culture vessels are provided with lids, and the lids are covered with the lids, so the airtightness inside the containers is improved and environmental maintenance is easy. However, the outside of the cell culture vessel has a problem that the air tightness is low and it is difficult to maintain the environment. Further, in the case of the cell culture device described in Patent Document 2, since the container itself is covered with a lid, when dispensing using a dispenser, it is necessary to remove the lid each time, and it is inferior in workability. Since the entire lid was removed, there was a major problem that the internal environment decreased with each dose.

  The present invention has been made in view of the above problems, and cells accommodated in a well plate can be easily dosed using a dispenser or the like, and excellent airtightness in the well plate can be obtained. It is an object of the present invention to provide a culture apparatus for microscopic observation that can be secured.

In order to solve the above-mentioned subject, the present invention includes the following modes.
The culture apparatus for microscope observation according to one aspect of the present invention is a culture apparatus for microscope observation that performs microscope observation while culturing living cells, and includes an XY stage unit that moves a plate holder in the XY directions, and holding the plate holder And a well plate for containing a sample being cultured, and a top heater unit and a bottom heater unit provided so as to sandwich the XY stage unit from above and below, and the top heater unit is housed in the well plate While the hole for adding a chemical | medical solution to the said sample is formed, the cover part for covering this hole is provided so that opening and closing is possible.

  Further, the culture apparatus for microscope observation according to one aspect of the present invention is characterized in that the lid portion is slidably provided along a fixed axis provided in the top heater unit.

  Further, the culture apparatus for microscopic observation according to one aspect of the present invention is characterized in that the lid portion is provided so as to hold at least the well plate in an airtight manner when the hole is covered.

In the culture apparatus for microscope observation according to one aspect of the present invention, the plate holder is provided with at least one of a heater for heating and a CO ₂ port for supplying a CO ₂ gas from the outside. It is characterized by

  In the culture apparatus for microscope observation according to one aspect of the present invention, the bottom heater unit has a water flow path formed of a heater holder and a metal plate on one surface, and a bottom heater is provided on the other surface. It is characterized by

  According to the culture apparatus for microscopic observation according to one aspect of the present invention, when dispensing a sample using a dispenser or the like, the lid is opened and the hole is opened to allow the sample contained in the well plate to be contained. Thus, it is possible to easily dispense with a dispenser or the like. On the other hand, when culturing cells as a sample, the lid portion is closed to close the hole for adding the drug solution, whereby the airtightness in the whole well plate and hence in the whole plate holder can be secured. Therefore, it becomes possible to achieve both the securing of the workability of the dosing of the sample and the securing of the environment suitable for culture in the well plate.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which illustrates typically the culture apparatus for microscope observation which is one Embodiment of this invention, and is a perspective view which shows the whole structure of an apparatus. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which illustrates typically the culture apparatus for microscopes which is one Embodiment of this invention, and is the schematic which shows the whole structure of the apparatus containing the dispenser for dispensing to the sample accommodated in the well plate. It is a figure which illustrates typically the culture | cultivation apparatus for microscope observation which is one Embodiment of this invention, and is perspective schematic drawing which shows the XY stage unit for moving the well plate hold | maintained at the plate holder.

  Hereinafter, the culture apparatus for microscope observation which is one Embodiment to which this invention is applied is demonstrated, referring FIGS. 1-3 suitably. FIG. 1 is a perspective view showing the whole constitution of the culture apparatus 10 for microscope observation of the present embodiment, and FIG. 2 shows the whole constitution including a dispenser 7 for dispensing the sample contained in the well plate 5. FIG. 3 is a schematic perspective view showing an XY stage unit 1 for moving the well plate 5 held by the plate holder 4.

  In the drawings used in the following description, in order to make features of the present invention intelligible, the features that are the features may be enlarged and shown for convenience, and the dimensional ratio of each component is assumed to be the same as the actual There is no limit. In addition, the materials and the like exemplified in the following description are merely examples, and the present invention is not limited to them, and can be appropriately changed and implemented without changing the gist of the invention.

<Configuration of culture apparatus for microscopic observation>
Hereinafter, the configuration of the culture apparatus for microscopic observation (hereinafter, may be abbreviated as culture apparatus) 10 of the present embodiment will be described in detail.
The culture apparatus 10 of the present embodiment is an apparatus that performs microscopic observation while culturing a sample such as a living cell (not shown). As shown in FIGS. 1 to 3, in the culture apparatus 10 of the present embodiment, an XY stage unit 1 for moving the plate holder 4 in the XY directions and a well plate held by the plate holder 4 and containing a sample being cultured 5 and a top heater unit 2 and a bottom heater unit 3 provided so as to sandwich the XY stage unit 1 from above and below. In the culture apparatus 10 of the present embodiment, the top heater unit 2 is formed with the hole 21a for adding a chemical solution to the sample contained in the well plate 5, and a lid for covering the hole 21a. The part 6 is configured to be openable and closable.

  As described above, the XY stage unit 1 moves the plate holder 4 holding the well plate 5 in the XY direction. The XY stage unit 1 has an X table 11 driven in the X direction by an actuator (not shown) and a Y table 12 driven in the Y direction, and is configured to be able to drive the well plate 5 in the XY direction.

In addition, the XY stage unit 1 moves the plate holder 4 outside the position covered by the top heater unit 2 described later by largely driving in the Y direction, and the well plate 5 held by the plate holder 4 is moved. It is configured to be removable.
Further, a seal rubber (not shown) is provided on the bottom surface of the Y-table 12 so as to keep airtight with the bottom heater unit 3 described later.

  In the culture apparatus 10 of the present embodiment, as the XY stage unit 1, one conventionally used in this field can be adopted without any limitation.

  As described above, the top heater unit 2 is disposed so as to sandwich the XY stage unit 1 between the top heater unit 2 and the bottom heater unit 3 from above and below. That is, the top heater unit 2 is disposed above the XY stage unit 1 as shown in FIGS. 1 and 2.

  The top heater unit 2 includes a transparent heater panel 21, a heater holder 22 for holding the heater panel 21, and a fixing plate 23 for fixing these. Further, at the center of the heater panel 21, a hole 21a for adding a chemical solution is provided. Furthermore, although the details will be described later, the top heater unit 2 is provided with a lid 6 capable of covering the well plate 5 so as to be able to open and close.

The bottom heater unit 3 is disposed on the opposite side to the top heater unit 2 with respect to the XY stage unit 1, that is, below the XY stage unit 1.
The bottom heater unit 3 includes a heater holder 31 and heater frames 32 a and 32 b for fixedly supporting both ends of the heater holder 31. A sheet-like bottom heater (not shown) is attached to the lower surface of the heater holder 31 so that the heater holder 31 can be heated. Further, an open hole (not shown) is provided at the center of the heater holder 31, and a bellows (not shown) to be described later is fixed.

Moreover, although illustration is abbreviate | omitted in FIG.1 and FIG.2, the opening hole is provided in the center part of the bottom heater unit 3, and the illustration lens not shown is arrange | positioned in this opening hole. The objective lens is configured to be drivable in the optical axis direction by an actuator (not shown). Further, the above-mentioned bellows is provided in the peripheral edge portion of the objective lens and the opening hole of the bottom heater unit 3 described above, and when the objective lens is lifted in the optical axis direction, the space between the bellows and the opening hole By sealing the inside of the apparatus, in particular, in the well plate 9, the environment such as temperature, humidity, and CO ₂ can be maintained. Although details are omitted, an image of cells in the well plate 5 captured by an objective lens is configured to be captured by an optical system and a camera (not shown).

In the surface (upper surface) of the heater holder 31, the peripheral portion of the above-mentioned opening hole is processed so as to be slightly lower than the outer peripheral portion of the peripheral portion so as to form a flow path.
Moreover, the water supply hole and the drainage hole which are not shown in figure are provided in the surface of the heater holder 31, and it is comprised that water supply and drainage can be performed from the port of a bottom face.

  Furthermore, although illustration is abbreviate | omitted in FIG.1 and FIG.2, the water flow path which consists of the heater holder 31 and a metal plate is formed in the surface (upper surface: one surface) side in the bottom heater unit 3, A bottom heater (not shown) is provided on the side of the surface (lower surface: the other surface). That is, in the bottom heater unit 3, a metal plate is attached to the periphery of the above-mentioned opening formed on the surface of the heater holder 31 via a metal mesh (not shown) processed so as to fit in the periphery. The flow path is formed by the heater holder 31 and the metal plate. Further, in a predetermined region of the metal plate, a plurality of minute holes are formed in a zigzag shape at predetermined diameters and intervals. Thereby, in the culture apparatus 10 of this embodiment, although the details will be described later, it is possible to supply the water evaporated from the metal mesh into the well plate 5 (in the plate holder 4) and adjust the internal humidity. Become.

  The plate holder 4 is attached to the XY stage unit 1 as described above, and holds the well plate 5. In the example shown in FIG. 3, the plate holder 4 is formed in a concave shape, and the well plate 5 can be accommodated and held therein.

The plate holder 4 can be provided with, for example, at least one of a heating heater (not shown) and a CO ₂ port for supplying a CO ₂ gas from the outside. As described above, by providing the heater for heating the plate holder 4, it is possible to effectively heat the sample in the well plate 5 held by the plate holder 4. Further, by providing a CO ₂ port in the plate holder 4, it becomes possible to externally supply a CO ₂ gas adjusted to, for example, about 5% by volume to the well plate 5. A seal rubber (not shown) is provided at the opening of the plate holder 4 provided in the culture device 10 of the present embodiment, and is configured to keep the space between the plate holder 4 and the heater panel 21 airtight.

  The well plate 5 is a container for containing and culturing a sample such as a living cell as a culture object in the inside. The well plate 5 is configured to be movable in the XY direction by the above-described XY stage unit 1 by being held by the plate holder 4. As in the example shown in FIG. 3, the well plate 5 is provided with a plurality of concavely formed sample holders 51, and each of them is configured to be able to accommodate a sample.

  In the culture apparatus 10 of the present embodiment, any well plate conventionally used in this field can be adopted as the well plate 5 without any limitation.

  As described above, the lid 6 is provided so as to be able to open and close the hole 21 a on the upper surface side of the top heater unit 2 so as to cover the hole 21 a formed in the top heater unit 2. Further, the lid 6 in the illustrated example is slidably attached to a fixed shaft 61 provided on the upper surface side of the top heater unit 2 along the fixed shaft 61, and the slide movement allows the hole 21a to be formed. It is configured to be openable and closable.

The lid 6 is made of, for example, a transparent material such as a resin, and is configured to be able to observe the sample contained in the well plate 5 with transmitted light.
Further, the lid 6 in the illustrated example is provided so as to be able to airtightly maintain the inside of the well plate 5 when the hole 21a is covered and closed.

  The lid 6 is opened when the sample contained in the well plate 5 is subjected to a predetermined treatment, that is, when a treatment for adding a chemical solution to the sample (cells) is performed. That is, when the dispenser 7 as shown in FIG. 2 is used, the lid 6 is opened to make it possible to add the chemical solution to the sample, and when the dispenser 7 is not used, the lid 6 is closed. 21a is closed to ensure confidentiality within the well plate 5 and maintain the internal environment in a properly managed state.

  The dispenser 7 adds the drug solution from the dropping unit 72 to the sample (living cells) contained in the well plate 5. The dispenser 7 of the example shown in FIG. 2 is attached to a movable shaft 71 attached to a fixed part (not shown).

  The dispenser 7 is configured such that when the chemical solution is added to the sample contained in the well plate 5, the dropping portion 72 can be brought close to the sample by sliding in the axial direction, that is, in the vertical direction. ing. The dispenser 7 is configured to be able to suction the chemical solution (compound) stored in the chemical solution storage unit (not shown) through a pipe and drop it from the dropping unit 72 onto the sample in the well plate 5.

In the culture apparatus 10 of the present embodiment, as described above, the top heater unit 2 is formed with the holes 21a for adding a chemical solution to the sample contained in the well plate 5, and covers the holes 21a. A cover 6 is provided so as to be openable and closable. Thus, the culture apparatus 10 opens the lid 6 when adding the chemical solution to the sample in the well plate 5 and closes the lid 6 when not adding the chemical solution, thereby making the inside of the well plate 5 airtight. Hold. Further, in the present embodiment, as in the example shown in FIG. 3, the well plate 5 is accommodated in the concave plate holder 4 so that only the plurality of sample holders 51 provided in the well plate 5 can be obtained. The entire inside of the plate holder 4 is airtightly held. Furthermore, since the hole 21a is configured to be freely opened and closed by the lid 6, when the chemical solution is added, it is possible to minimize deterioration of the internal environment when the lid 6 is opened.
Therefore, according to the culture apparatus 10 of the present embodiment, it is possible to ensure both the dispensing workability for the sample and the environment in the well plate.

<How to use the culture apparatus for microscopic observation>
Below, it observes, culture | cultivating the living cell which is a sample, using the culture apparatus 10 for microscope observation mentioned above, and demonstrates the usage method in the case of adding a chemical | medical solution suitably with reference to FIGS.

  First, the well plate 5 in which living cells (sample for observation) are accommodated in the sample holder 51 is set in the plate holder 4 attached to the XY stage unit 1. At this time, the plate holder 4 is moved to the outside of the position covered by the top heater unit 2 by largely driving the XY stage unit 1 in the Y direction, and the well plate 5 containing the sample is moved to the plate holder 4. set. Thereafter, the XY stage unit 1 is moved in the opposite direction in the Y direction to return to the observation position.

  In this state, by moving the objective lens (not shown), the sample accommodated in the well plate 5 can be observed by the optical system and the camera. Here, the sample may be irradiated with light from above the top heater unit 2 through the transparent heater panel 21 by a lamp (not shown), or light may be transmitted from inside the objective lens from the optical system side. You may irradiate. Further, the desired sample holder 51 in the well plate 5 can be observed by driving the XY stage unit 1 and moving the well plate 5 appropriately in the X direction or Y direction.

  The temperature for culturing the sample may be, for example, a heater panel 21, a heater (not shown) attached to the plate holder 4, and a bottom heater (not shown) provided in the bottom heater unit 3 to heat and drive a thermocouple Temperature detection and control are performed using the control unit. Thereby, for example, the heating temperature by each heater is adjusted so that the inside of the well plate 5 becomes about 37.degree.

In addition, for the CO ₂ supplied into the well plate 5 (in the plate holder 4), for example, a mixed gas whose CO ₂ concentration is adjusted to about 5% is introduced from the outside of the apparatus, and the illustration is omitted Supply from the CO ₂ port of As a result, the CO ₂ concentration in the vicinity of the well plate 5 in the culture apparatus 10, that is, in the plate holder 4 can be set to about 5%.

  Further, the humidity in the well plate 5 (in the plate holder 4) can be adjusted, for example, by supplying pure water from a water supply hole (not shown) provided in the bottom heater unit 3. The pure water flows into a thin flow path formed by the heater holder 31 and the metal plate (not shown) by capillary action. As described above, since a metal mesh (not shown) is disposed in the flow path, the capillary phenomenon is more prominent when the flow path is narrowed, so that pure water is efficiently contained in the flow path. Led.

  Further, in the present embodiment, the supply amount of pure water from the water supply hole and the drainage amount of pure water from the drainage hole are adjusted so that the pure water does not overflow from a large number of micro holes provided in the metal plate. Is preferred. Here, when the diameter of the minute hole is reduced to, for example, about 0.5 mm, surface tension acts, so that it is possible to effectively prevent the pure water from overflowing from the minute hole, and it is possible to make the pure water more efficiently. Water can be introduced into the flow path.

  In addition, the layer of pure water in the flow path is very thin, and the volume is also a minute amount, so it can be easily heated by the heater (not shown). The evaporation of pure water is effectively promoted. As a result, the humidity in the well plate 5 (in the plate holder 4) can be efficiently raised. In addition, the metal mesh (not shown) disposed in the flow path also plays a role of transmitting the heat to the pure water more efficiently, so the evaporation of the pure water from the minute holes is more effectively promoted. Ru.

  Further, by setting the heating temperature of the heater to about 38.5 ° C., which is slightly higher than 37 ° C. which is the environmental temperature in the well plate 5, the distance between the heater and the metal plate on which the pure water is evaporated is short. In combination with this, the temperature of this portion is heated to approximately the same temperature (38.5 ° C.) as the heater. As a result, the temperature of the saturated vapor formed by pure water evaporation becomes higher than 37 ° C., so convection occurs and the saturated vapor convects inside the apparatus, and the non-saturated gas convects in the vicinity of the metal plate. From this, the next evaporation is also promoted. Each of these actions makes it possible to build a high humidity environment more efficiently than usual.

Then, when the chemical solution is added to the sample contained in the well plate 5, first, the lid portion 6 provided in the culture apparatus 10 is slide-moved to be in the open state, whereby the dispenser 7 and the well plate 5 are provided. The accommodated sample is opposed to each other through the hole 21a.
Next, the dispenser 7 is slid in the vertical direction to bring the dropping part 72 close to the sample.

  Next, the chemical solution stored in the chemical solution storage unit (not shown) is sucked by the dispenser 7 through a pipe and dropped onto the sample in the well plate 5. At this time, the well plate 5 is appropriately moved in the X direction and the Y direction by the XY stage unit 1 to repeat the operation of dropping the chemical solution onto each of the samples in the sample holder 51 provided in plurality in the well plate 5. .

Then, after the dripping (addition) of the drug solution on the sample in the well plate 5 reaches a predetermined amount, the dispenser 7 stops the suction and dropping of the drug solution and ascends in the axial direction to return to the standby position.
At this time, the lid 6 is slid along the fixed shaft 61 to move to a position covering the hole 21 a, thereby making the inside of the well plate 5 airtight again.

  According to the above-described procedure, living cells can be cultured and observed using the culture apparatus 10 of the present embodiment, and a drug solution can be added.

<Function effect>
As described above, according to the culture apparatus 10 for microscope observation of the present embodiment, the top heater unit 2 is formed with the holes 21 a for adding the chemical solution to the sample contained in the well plate 5. A configuration is adopted in which a lid 6 for covering the hole 21a is provided so as to be openable and closable. Thus, when dispensing the sample using the dispenser 7, the lid portion 6 is opened to open the holes 21a, thereby facilitating dispensing of the sample contained in the well plate 5 by the dispenser 7. It will be possible to do. On the other hand, when culturing cells as a sample, the lid portion is closed to close the hole for adding the drug solution, whereby airtightness in the entire well plate 5 and hence in the entire plate holder 4 can be secured. Therefore, it becomes possible to make compatible the securing of the workability of the administration to the sample (cells) and the securing of the environment suitable for culture in the well plate.

Furthermore, according to the culture apparatus 10 of the present embodiment, by keeping the inside of the well plate 5 airtight, it becomes possible to minimize the environmental change such as temperature, humidity, CO _{2 and the} like inside the well plate 5. It also becomes possible to reduce the flow rate of CO ₂ gas used.

  The culture apparatus for microscopic observation of the present invention can achieve both the operability of the treatment for cells and the excellent airtightness in the well plate, so that it is accurate and accurate while maintaining an environment suitable for cell culture. It becomes possible to perform cell observation with good workability. Therefore, the culture apparatus for microscopic observation of the present invention is very useful in the research of the cell field where observation is performed for a long time using living cells.

DESCRIPTION OF SYMBOLS 1 ... XY stage unit 11 ... X table 12 ... Y table 2 ... Top heater unit 21 ... Heater panel 22 ... Heater holder 23 ... Fixed plate 3 ... Bottom heater unit 31 ... Heater holder 32a, 32b ... Heater frame 4 ... Plate holder 5 ... Well Plate 51 ... Sample holder 6 ... Lid part 61 ... Fixed shaft 7 ... Dispenser 71 ... Movable shaft 72 ... Dropping part 10 ... Culture device for microscope observation (Culture device)

Claims (5)

A culture apparatus for microscopic observation which performs microscopic observation while living cells are cultured,
An XY stage unit that moves the plate holder in the XY directions;
A well plate held by the plate holder and containing a sample being cultured;
A top heater unit and a bottom heater unit provided so as to sandwich the XY stage unit from above and below,
The top heater unit is formed with a hole for adding a chemical solution to the sample stored in the well plate, and a lid for covering the hole is provided so as to be openable and closable. The culture apparatus for microscope observation which is characterized.   The culture apparatus for microscopic observation according to claim 1, wherein the lid portion is provided so as to be slidable along a fixed axis provided to the top heater unit.   The culture apparatus for microscopic observation according to claim 1 or 2, wherein the lid part is provided to hold at least the well plate in an airtight manner when the lid part covers the hole. 4. The plate holder according to claim 1, wherein at least one of a heating heater and a CO ₂ port for supplying a CO ₂ gas from the outside is provided. The culture apparatus for microscopic observation according to any one of the above.   The water flow path which consists of a heater holder and a metal plate is formed in one side of the bottom heater unit, and the bottom heater is provided in the other side. The culture apparatus for microscope observation as described in 1 item.

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