JP2010239916A - Spacer for culturing container, culturing apparatus equipped with spacer, and observation method using culturing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a spacer for a culturing container without using a special apparatus, only by applying it to a generally used culturing container such as a multi-well plate with a lid body, capable of observing the effects of an objective substance contained in a gas phase to a living body sample by real time, evaluating in a good reliability and also without being limited by the kind of objective substances, a culturing apparatus including the spacer, and a method of observation by using the culturing apparatus. <P>SOLUTION: This spacer 10A for the culturing container is provided by being attached between a culturing container body 21 of the culturing apparatus and a lid body 22 being covered on the culturing container body 21, and while separating the culturing container body 21 and lid body 22, air-tightly connecting them. By the culturing apparatus equipped with the spacer 10A, while culturing the living body sample, it is possible to observe the effects of the substance contained in the gas phase in the culturing apparatus to the living body sample in real time. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a culture container spacer mounted on a biological sample culture container, a culture apparatus provided with the spacer, and an observation method using the culture apparatus.

In recent years, it has been pointed out that substances contained in the atmosphere, such as vaporized organic solvents, nitrogen oxides, and suspended particulates, can affect the respiratory system of animals and cause diseases. On the other hand, it is also widely attempted to promote health by actively utilizing volatile substances such as essential oils and substances that emit trees, such as aromatherapy and forest baths.
In this way, substances contained in the gas phase have a great influence on living organisms. Therefore, methods for observing and evaluating the influence of such substances (hereinafter sometimes referred to as target substances) in vitro have been studied.

 For example, when culturing cells in a culture dish, there is a method of observing or evaluating the influence of the target substance by adding a culture solution in which the target substance is dissolved, as in the usual in vitro method. . However, in such a method, the target substance is added in the state contained in the liquid phase, and not added in the state contained in the gas phase. Cannot be evaluated accurately. Furthermore, since it is not always possible to dissolve a plurality of target substances existing in the gas phase in the culture solution at the same concentration as in the gas phase, also in this respect, the effect when these target substances are included in the gas phase, It was difficult to accurately observe and evaluate.

Therefore, it has been studied to accurately observe and evaluate the influence when the target substance is contained in the gas phase by actually bringing the gas phase containing the target substance into contact with the cells.
For example, by moving the culture dish containing the culture solution up, down, left and right in the gas phase, the cells are exposed from the culture solution, thereby bringing the cells into contact with the gas phase, and observing the state of the cells at that time for evaluation How to do is being studied. However, since the liquid has surface tension, even when the cells are exposed, the liquid remains on the cells, so that accurate evaluation is difficult.
In addition, a method of observing and evaluating a cultured cell from which the culture solution has been removed is placed in a gas phase containing a target substance. However, this method has a problem that accurate evaluation is difficult because the time during which the cell activity continues after the culture solution is removed is limited.
Furthermore, a method for observing and evaluating the influence of the target substance contained in the gas phase by culturing cells on a membrane that allows gas to permeate but not permeate liquid has been studied. The culture itself was difficult on a thick film.

Therefore, a method of observing and evaluating in real time the effect of a substance contained in the gas phase on the cell by contacting the cell with the gas phase containing the target substance while culturing the cell has been studied. Document 1 discloses a method using a special apparatus.
In addition, devices that can bring a gas phase containing a target substance into contact with the cells while culturing the cells are also commercially available (for example, Shibata Kagaku Co., Ltd., cell exposure experiment apparatus “SIS-CUL”).

JP 2002-101897 A

However, in the method described in Patent Document 1, a special support equipped with a support for supporting cells, a culture solution storage tank for supplying a culture solution to the cells, a gas storage unit containing a target substance, a water storage unit, and the like. It was necessary to use an apparatus, and it was not possible to easily observe and evaluate.
In addition, the commercially available cell exposure experimental apparatus illustrated above has a configuration in which an opaque lid is covered on the upper part, and further, a water bath is provided on the lower part, and the distance from the lens of the microscope to the cell is increased. Since it is much longer than the working distance of the lens, real-time microscope observation was difficult.

Further, for example, a method of applying a multiwell plate 50 with a lid 51 as shown in FIG. The multi-well plate 50 is a commercially available 6-hole type in which six wells (2 × 3) are formed, and is made of transparent plastic.
Specifically, after the biological sample 60 is placed on the medium 61 in the well W, the multi-well plate 50 is covered with the lid 51. Here, a gas supply port 52 for supplying gas and a gas discharge port 53 for discharging gas are formed in the lid 51 in advance. Then, after covering the lid 51 on the multi-well plate 50, a gas containing the target substance is supplied from the gas supply port 52 into the multi-well plate 50 and discharged from the gas discharge port 53. Then, the state of the biological sample 60 at that time is observed and evaluated with a microscope or the like.
According to such a method, the space in the multiwell plate 50 is replaced by the supplied gas, the biological sample 60 and the gas come into contact with each other, and the influence of the target substance contained in the gas on the biological sample 60 is measured in vitro. It is expected to be able to observe and evaluate in real time.

However, in such a multiwell plate 50 with a general lid 51, when the lid 51 is covered, as shown in FIG. 17B, the multiwell plate 50 is placed between the multiwell plate 50 and the lid 51. Almost no gaps are formed.
Therefore, even if gas is supplied from the gas supply port 52 into the multi-well plate 50, it is difficult to move the gas between the wells W, so that the supplied gas sufficiently replaces the space in each well W. Instead, leakage occurs from a slight gap between the multiwell plate 50 and the lid 51 mainly around the multiwell plate 50. In order to prevent leakage of gas from such a gap, there is no gap between the multiwell plate 50 and the lid 51 even if the gap between the multiwell plate 50 and the lid 51 is sealed with packing or the like. Since the state has not changed, the movement of the gas between the wells W was still difficult. For this reason, when the gas supply rate is increased, the internal pressure of the container increases and the temperature and humidity in the container change, and there is a problem that such a change in atmospheric pressure affects the biological sample 60. Further, in the method of connecting the pressure reducing means to the gas supply port 52 and replacing the air in the multiwell plate 50 by the suction method, if the gas suction speed is increased, the internal pressure of the container is lowered, and the same problem occurs. .

That is, by simply applying such a commercially available multi-well plate 50 with a lid 51, gas cannot be efficiently diffused and replaced in the multi-well plate 50 in a short time. Therefore, it takes a long time from the start of the gas supply until the gas in contact with the biological sample 60 disposed in the well W is sufficiently replaced, and is contained in the gas. It is difficult to observe the effect of the target substance in real time. In addition, since the time required for sufficiently replacing the space in the well W differs for each well W, even if the evaluation is performed, variation in data obtained between the wells W is large. High evaluation is not possible.
In addition, in the commercially available multi-well plate 50 with the lid 51, the supplied gas leaks into the atmosphere from the gap between the lid 51 and the multi-well plate 50 as described above. Accordingly, the types of gas that can be supplied are limited, and it is difficult to supply a gas containing a toxic substance, for example.

  The present invention has been made in view of the above circumstances, and the target substance contained in the gas phase can be obtained only by applying it to a culture container such as a multiwell plate with a general lid without using a special apparatus. Observation of the influence on the biological sample in real time, reliable evaluation, and a culture vessel spacer that does not limit the type of target substance, a culture device equipped with the spacer, and the culture device The objective is to provide an observation method.

The spacer for a culture container according to the present invention is mounted between a culture container main body of a culture apparatus and a lid that covers the culture container main body, and separates the culture container main body and the lid from each other and connects them in an airtight manner. It is characterized by doing.
It is preferable that at least one opening that communicates the inside and the outside of the culture apparatus is formed.
Examples of the culture container spacer of the present invention include a configuration in which the culture container is attached to a culture container main body formed with a plurality of wells.
In this case, the culture vessel spacer includes an intermediate plate portion in which a plurality of holes corresponding to each well are formed, and the peripheral edge portion of each hole and the open peripheral edge portion of each well are in close contact with each other during the mounting. The form that is to be preferred.
In addition, a mode in which a separating means for separating the wells is provided is also preferable.
The culture apparatus according to the present invention includes the culture container main body, the lid, and the culture container spacer mounted between the culture container main body and the lid.
The biological sample observation method of the present invention is characterized by observing the influence of a substance contained in a gas phase in the culture apparatus on the biological sample while culturing the biological sample with the culture apparatus.

  According to the present invention, the effect of a target substance contained in a gas phase on a biological sample can be measured in real time only by applying it to a culture vessel such as a general multiwell plate with a lid without using a special device. It is possible to provide a culture container spacer, a culture apparatus provided with the spacer, and an observation method using the culture apparatus, which can be evaluated with reliability and can be evaluated with high reliability and the type of target substance is not limited.

It is a partially broken perspective view which shows the culture container spacer of the example of 1st Embodiment. It is sectional drawing which follows the I-I 'line | wire of the spacer for culture vessels of FIG. It is sectional drawing which shows a mode that the spacer for culture containers of FIG. 1 was mounted | worn to the culture container. It is a disassembled perspective view which shows a mode that the spacer for culture containers of FIG. 1 was mounted | worn to the culture container. It is a schematic block diagram which shows the culture apparatus provided with the spacer for culture vessels of FIG. It is a schematic plan view which shows the example of arrangement | positioning of the gas supply port formed in the spacer for culture vessels, and a gas exhaust port, Comprising: (a) The example which formed three sets of the gas supply port and the gas exhaust port, (b) Gas supply This is an example in which four sets of ports and gas discharge ports are formed. It is the (a) schematic top view which shows the example of arrangement | positioning of the gas supply port formed in the culture container spacer, and a gas exhaust port, (b) Side view, (c) The front view seen from the gas supply port side. It is sectional drawing which shows a (a) perspective view which shows the spacer for culture containers of 2nd Example, and a mode that the spacer for culture containers of (b) (a) was mounted | worn to the culture container. It is sectional drawing which shows a mode that the culture container spacer of (a) which shows the spacer for culture containers of 3rd Embodiment is mounted | worn with the culture container spacer of (b) and (a). It is sectional drawing which shows a mode that (a) perspective view which shows the spacer for culture vessels of the example of 4th Embodiment, and the culture vessel spacer of (b) (a) was mounted | worn to the culture vessel. It is the schematic which shows the example of the division | segmentation of the space in a culture apparatus using the spacer for culture vessels of FIG. 10, Comprising: (a) The example without a division | segmentation, (b) The example of 2 division | segmentation, (c) 3 division | segmentation It is an example. It is a schematic plan view which shows the example of arrangement | positioning of the gas supply port formed in the circular culture container spacer of 5th Example, and a gas exhaust port, Comprising: (a) One set of the gas supply port and the gas exhaust port was formed. An example and (b) an example in which two sets of gas supply ports and gas discharge ports are formed. It is sectional drawing which shows a mode that the culture container spacer of the example of 5th Embodiment was mounted | worn with the culture container. In Experiment 1, it is a graph which shows the relationship between exposure time and cilia motion frequency (CBF). 6 is a schematic configuration diagram showing a configuration of a culture apparatus used in Test Example 2. FIG. In Experiment 2, it is a graph which shows the relationship between exposure time and cilia movement frequency (CBF). (A) exploded perspective view and (b) side view schematically showing a method of observing the influence of substances contained in a gas phase on a biological sample while culturing the biological sample in a commercially available multi-well plate with a lid It is.

Hereinafter, the present invention will be described in detail.
[First Embodiment]
1 and 2 are a partially broken perspective view and a sectional view showing a culture vessel spacer 10A of the first embodiment. 3 and 4 are a cross-sectional view and an exploded perspective view showing the culture apparatus 30. The culture apparatus 30 is a culture vessel 20 for culturing a biological sample such as a cell, and a figure attached to the culture apparatus 20. 1 and the culture vessel spacer 10A shown in FIG.

  A culture container spacer (hereinafter simply referred to as a spacer) 10A in this example is mounted on a commercially available culture container 20 as shown in the figure, and has a rectangular frame shape with an open top and bottom in the figure. Here, as a commercially available culture vessel 20, a culture vessel main body 21 composed of a 6-well type square multi-well plate in which six wells (2 × 3) W1 to W6 are formed, and a lid body 22 to be covered therewith. Are provided. The multi-well plate and the lid 22 in this example are both made of transparent plastic and can be observed with a microscope or sterilized.

As shown in the figure, the spacer 10A is mounted between the culture vessel main body 21 and the lid 22 to separate the culture vessel main body 21 and the lid 22 and between them, the culture vessel main body 21 is interposed therebetween. A space S having a height h from the upper end of the culture vessel to the lid body 22 is formed, and the culture vessel main body 21 and the lid body 22 are hermetically connected.
The spacer 10 </ b> A in this example includes a rectangular frame-shaped upper frame 11 that fits with the lid body 22, and a rectangular frame-shaped lower frame 12 that fits with the culture vessel body 21. 11 is fitted inside the lid 22, and the lower frame 12 is fitted outside the culture vessel main body 12. For this reason, the upper frame 11 is formed smaller than the lower frame 12, and the upper frame 11 is formed on the outer peripheral side, and the lower frame 12 is formed on the inner peripheral side. When the packing 13 is provided and the spacer 10A is attached to the culture vessel 20, the upper frame 11 is connected to the lid 22 in an airtight manner, and the lower frame 12 is connected to the culture vessel main body 21 in an airtight manner. .

  Further, in this example, one opening is provided in each of the center of the pair of short sides forming the lower frame 12 of the spacer 10A so as to communicate the inside and the outside of the culture device 30 provided with the spacer 10A. Is formed. One of the openings is a gas supply port 14 to which a gas supply means (not shown) that supplies gas into the culture apparatus 30 is connected. The other opening is a gas discharge port 15 through which gas is discharged from the inside of the culture apparatus 30 and, if necessary, gas recovery such as bags and containers for collecting the discharged gas so as not to leak into the atmosphere. Means are connected. A backflow prevention valve for preventing a backflow of gas may be provided between the gas supply port 14 and the gas supply unit or between the gas discharge port 15 and the gas recovery unit. Further, the gas recovery means may be connected to a decompression means such as a suction pump so that the gas can be sucked.

  In this example, the sensor insertion port 16 is formed as an opening on the short side of the lower frame 12 where the gas supply port 14 is formed, and the temperature, humidity, gas composition, etc. in the culture apparatus 30 are adjusted. Sensors (not shown) for measurement can be inserted from the outside to the inside.

  Here, the height H of the spacer 10A is such that the gas supplied from the gas supply port 14 into the culture device 30 diffuses uniformly into the culture device 30 efficiently in a short time, and the whole culture device 30 is used for microscopic observation. In such a case, the range is determined so that the culture apparatus 30 and the condenser of the microscope do not interfere with each other. Further, when the gas supply port 14, the gas discharge port 15, and the sensor insertion port 16 are formed in the spacer 10A as in the illustrated example, the points are also taken into consideration so that these are formed at appropriate positions. Thus, the height H is determined.

Specifically, depending on the number and size (volume) of the wells W1 to W6 formed in the culture vessel 30 and the height of the culture vessel 20, a general multiwell plate with a lid is used as the culture vessel 20. When the gas supply port 14, the gas discharge port 15, and the sensor insertion port 16 are not formed in the spacer, it is preferable that the height h of the space S is determined in a range of 3 to 30 mm. More preferably, the height h of the space S is in a range of 5 to 20 mm. Within this range, gas can be diffused efficiently and uniformly in the culture apparatus 30 in a short time. In addition, the height of the entire culture device 30 becomes too large, which hinders microscopic observation for each culture device 30, the volume in the culture device 30 becomes too large, and the gas replacement efficiency decreases. Is not wasted.
When the gas supply port 14, the gas discharge port 15, and the sensor insertion port 16 are formed in the spacer 10 </ b> A as in the illustrated example, the height h of the space S may be determined within a range of 10 to 30 mm. Preferably, it is determined within a range of 15 to 20 mm.

The material of the spacer 10A may be any material as long as it does not cause inconvenience such as corrosion by the supplied gas, has low toxicity with respect to the biological sample, and can be sterilized. Plastics such as polystyrene, polypropylene, polycarbonate, polyethylene, polyethylene terephthalate, metals such as stainless steel, etc. can be used, and they can be produced by a known molding method.
Examples of the sterilization treatment include a gamma ray irradiation method, a dry heat sterilization method, a method using an autoclave, and 70% ethanol spraying.

A specific example of observing and evaluating the influence of a target substance on a biological sample by placing the target substance in the gas phase in the culture apparatus 30 while placing and culturing the biological sample in such a culture apparatus 30 The method will be described below with an example.
As shown in FIG. 5, first, after the culture medium 31 is provided in the well W5 of the multi-well plate that is the culture vessel main body 21, a cell-shaped culture insert 32 having a bottom made of a membrane is placed in the well W5. Then, the biological sample 33 is placed so as to be in contact with the culture medium 31 through the membrane and cultured. In this way, so-called gas-liquid interface culture is possible in which the lower side of the biological sample 33 is in contact with the culture medium 31 and the upper side is in contact with a gas supplied later. Further, at this time, a rubber packing 34 made of natural rubber, silicone rubber or the like is inserted and arranged between the outer periphery of the culture insert 32 and the inner periphery of the well W5 above the medium 31, It is preferable to prevent the gas supplied later from the gas supply port 14 into the culture apparatus 30 from coming into contact with the culture medium 31 and adversely affecting the culture medium 31.
In FIG. 5, a 6-well type multi-well plate is used as the culture vessel main body 21, but details in the wells other than the well W <b> 5 are not shown.

Next, the lower frame 12 of the spacer 10A is fitted to the multi-well plate, the upper frame 11 is fitted to the lid 22, and the spacer 10A is mounted between the culture vessel body 21 and the lid 22.
By mounting in this manner, the multi-well plate and the lid body 22 are separated from each other, a space S is formed between them, and the multi-well plate and the lid body 22 are hermetically sealed by the action of the packing 13 of the spacer 10A. Connecting.
Next, a bag (gas recovery means) 35 for collecting the discharged gas is connected to the gas discharge port 15 of the culture apparatus 30, and necessary sensors are inserted into a sensor insertion port (not shown) in FIG. Then, the entire culture apparatus 30 is placed on a microscope stage (not shown). At this time, if necessary, the culture apparatus 30 may be covered with a heat insulating sheet within a range that does not hinder observation with a microscope. Reference symbol M in the figure is a microscope capacitor.

Next, an injection cylinder (gas supply means) 36 containing a gas containing the target substance is prepared, and the injection cylinder 36 is connected to the gas supply port 14 via a tube 37. Then, the piston of the syringe barrel 36 is pushed in, gas is supplied into the culture apparatus 30, and the biological sample 33 is continuously observed with a digital camera (not shown) connected to a microscope.
And the influence which the target substance contained in gas has on the biological sample 33 is evaluated by analyzing data obtained by such observation.
The used spacer 10A can be repeatedly used after being appropriately washed and sterilized. Further, the packing 13 may be detachably provided from the spacer 10A, and only the packing 13 may be removed, washed, sterilized and used repeatedly, or only the packing 13 may be replaced each time.

When such a culture apparatus 30 is used, a spacer 10A is disposed between the multiwell plate that is the culture vessel body 21 and the lid body 22 to separate them, and a space S is formed. The gas supplied from the gas supply port 14 into the culture apparatus 30 diffuses efficiently and uniformly in the multi-well plate in a short time, and the spaces in the wells W1 to W6 are sufficiently replaced and disposed therein. It takes no long time for the gas to contact the biological sample 33. Therefore, the influence of the target substance contained in the gas can be observed and evaluated in real time. In addition, since the time required for sufficiently replacing the spaces in the wells W1 to W6 does not vary from well to well, it is possible to perform highly reliable evaluation without data variation between wells. it can.
Further, since the spacer 10A connects the lid 22 and the multiwell plate in an airtight manner, the supplied gas does not leak from between the lid 22 and the multiwell plate. Therefore, the type of gas that can be supplied is not limited, and for example, a gas containing a toxic substance can be supplied.

  In addition, in the spacer 10A of this example, a gas supply port 14, a gas discharge port 15, and a sensor insertion port 16 are formed in the lower frame 12 as openings that communicate the inside and outside of the spacer 10A. There are no particular restrictions on the number and position of the swords, and they may be formed on the upper frame 11. In addition, for example, as shown in FIGS. 6 and 7 for a 12-well type multi-well plate, the number and arrangement of wells are set so that gas can be supplied to the wells W1 to W12 of the multi-well plate without variation. Accordingly, a plurality of gas supply ports 14 and gas discharge ports 15 can be formed at appropriate positions of the spacer.

Moreover, the gas supply port 14, the gas discharge port 15, and the sensor insertion port 16 do not necessarily need to be formed. For example, when using a volatile substance as a target substance and observing and evaluating its influence, after putting the volatile substance in a well different from the well on which the biological sample is placed, immediately place the lid on the multi-well plate. The volatile substance can also be supplied into the gas phase by the method of attaching the spacer.
Further, if a gas supply port and a gas discharge port are used to supply and discharge gas from here, even if the gas supply port and gas discharge port are not formed in the spacer, Good.

Even when a liquid volatile substance is used as the target substance, the volatile substance can be supplied from the gas supply port 14 using the spacer 10A in which the gas supply port 14 is formed. In that case, it is preferable to insert a syringe containing a volatile substance from the gas supply port 14 and drop the volatile substance into a well different from the well on which the biological sample is placed.
Moreover, there is no restriction | limiting in particular also in the kind of the gas supply means connected to the gas supply port 14, the gas exhaust port 15, and a gas collection | recovery means.

In addition, since the spacer 10A of this example is used for a multi-well plate with a 6-hole type quadrangular lid, a rectangular frame-shaped upper frame 11 fitted to the lid 22 and a culture A rectangular frame-like lower frame 12 fitted to the vessel main body 21 is provided, and the shape thereof can be appropriately determined according to the shape of the culture vessel 20 or the like. For example, some commercially available multi-well plates have some of their shapes changed, such as rounding two of the four corners so that the order of each well in culture is correct. The shape of the spacer can be appropriately designed so as to correspond to such a multi-well plate. In this example, the upper frame 11 is fitted inside the lid 22 and the lower frame 12 is fitted outside the culture vessel main body 21. On the contrary, the upper frame is placed outside the lid. It may be configured to be fitted and the lower frame to be fitted inside the culture vessel main body.
As the culture vessel, other wells such as a 12-well type (3 × 4) and a 24-well type (4 × 6) may be used. A circular petri dish or the like may be used.

[Second Embodiment]
FIG. 8A is a perspective view showing the spacer 10B of the second embodiment, and FIG. 8B is a cross-sectional view showing a state in which the spacer 10B is mounted on a commercially available culture vessel 20. FIG. Here, as a commercially available culture vessel 20, as shown in FIG. 4, a culture vessel main body 21 comprising a 6-well type square multi-well plate in which six wells (2 × 3) W1 to W6 are formed. And what comprises the cover body 22 covered on this is illustrated.
The spacer 10B of this example is different from the spacer 10B of the first embodiment in that the spacer 10B has a horizontal middle plate portion 17 that is fixed by being laid over the inner peripheral side of the lower frame 12. Yes. In the middle plate portion 17 of this example, six holes 17a to 17f are formed so as to correspond to the six wells formed in the culture vessel main body 21, and as shown in FIG. When the example spacer 10B is mounted on the culture vessel 20, the peripheral edge portions of the holes 17a to 17f and the open peripheral edge portions of the upper ends of the wells W1 to W6 are in close contact with each other.

According to the spacer 10B of the second embodiment as described above, by attaching the spacer 10B to the culture vessel 20 as shown in the figure, the gap portion P formed between the wells is formed from above by the intermediate plate portion 17. Can be covered and closed. When the space P is thus closed, the gas (the space in each of the wells W1 to W6 and the space above the space) in the culture device 30 is short-lived by the gas supplied into the culture device 30 from the gas supply port 14. Can be replaced efficiently and sufficiently. This is because when the gap portion P is closed in this way, it is not necessary to replace the gap portion P with gas, and the turbulence of the air flow at the time of replacement is reduced.
Furthermore, although illustration is omitted, by closing any one or more of the holes 17a to 17f of the middle plate portion 17 with a transparent plate such as a transparent film or a sheet, the well plate corresponding to the hole is introduced into the well. The supply of gas may be shut off. The well in which the gas supply is cut off in this way can be used as a control well for a control experiment that is not affected by the gas. Moreover, blocking in this way also reduces an extra space in the culture apparatus 30 and also leads to prevention of turbulence in the air flow during replacement.

[Third Embodiment]
FIG. 9A is a perspective view showing a spacer 10C of the third embodiment, and FIG. 9B is a cross-sectional view showing a state in which the spacer 10C is mounted on a commercially available culture vessel 20. Here, as a commercially available culture vessel 20, as in the case shown in FIG. 4, a culture vessel main body 21 composed of a 6-well type square multi-well plate in which six wells (2 × 3) W1 to W6 are formed. And what comprises the cover body 22 covered on this is illustrated.
The spacer 10C of this example includes a middle plate portion 17 having the same configuration as that of the second embodiment, and further vertically extends from the middle plate portion 17 so as to span between a pair of long sides forming the spacer 10C. Two partition plates 18a and 18b are provided upright in the direction. Each partition plate 18a, 18b is formed at a height such that the upper end reaches the lid body 22, and is configured so that the space in the culture device 30 can be divided into three zones by mounting the spacer 10C. ing. In each of the three divided zones, a pair of gas supply ports 14 and a gas discharge port 15 are provided in the lower frame 12 so that gas can be supplied and discharged independently.

As described above, the spacer 10C according to the third embodiment includes the middle plate portion 17 in which the holes 17a to 17f are formed, and the two partition plates 18a and 18b erected in the vertical direction from the middle plate portion 17. Separation means is provided, so that the space in the culture apparatus 30 can be divided into three zones by separating the wells.
Therefore, according to the spacer 10C of this example, by attaching the spacer 10C to the culture vessel 20 as shown in the figure, different types of gases can be supplied independently for each zone divided by the partition plates 18a and 18b. It is possible to discharge, and it is possible to conduct experiments of multiple systems, such as observing the effects of multiple gases simultaneously.
Also in this example, if necessary, any one or more of the holes 17a to 17f of the middle plate portion 17 are closed with a transparent plate, and the supply of gas into the well corresponding to the hole is shut off. May be.
In this example, the six wells W1 to W6 are divided into two zones and divided into three zones. However, as long as the three wells are divided into a plurality of zones, there are no restrictions on the division method and the number of divisions.

[Fourth Embodiment]
FIG. 10A is a perspective view showing a spacer 10D of the fourth embodiment, and FIG. 10B is a cross-sectional view showing a state in which this spacer 10D is mounted on a commercially available culture vessel 20. Here, as a commercially available culture vessel 20, as in the case shown in FIG. 4, a culture vessel main body 21 composed of a 6-well type square multi-well plate in which six wells (2 × 3) W1 to W6 are formed. And what comprises the cover body 22 covered on this is illustrated.
The spacer 10D in this example includes six cylinders 19a to 19f that are vertically arranged so as to correspond to the six wells formed in the culture vessel main body 21, and that both ends are open. When the spacer 10D of this example is mounted on the culture vessel 20 as shown in FIG. 10 (b), the lower peripheral edge of each cylinder 19a-19f and the open peripheral edge of each well upper end are in close contact with each other. The upper end peripheral part of the bodies 19a to 19f and the lid body 22 are in close contact with each other. In addition, as shown in FIG. 11, the spacer 10D of this example includes seven cylinder communication pipes 41a to 41g that connect adjacent cylinders 19a to 19f, and each cylinder 19a to 19f and the outside of the spacer 10D. 10 external communication pipes 42a to 42j that communicate with each other. In this example, the cylinder communication pipes 41a to 41g and the external communication pipes 42a to 42j also serve as fixing means for fixing the cylinders 19a to 19f to the lower frame 12 of the spacer 10D. Further, each of the cylinder communication pipes 41a to 41g and the external communication pipes 42a to 42j can be easily closed by a plug made of rubber or the like.

As described above, the spacer 10D according to the fourth embodiment is vertically arranged so as to correspond to the six wells formed in the culture vessel main body 21, and the six cylinders 19a to 19f opened at both ends. An isolating means is provided, so that the wells can be separated from each other. Further, seven cylindrical communication pipes 41a to 41g and ten external communication pipes 42a to 42j are provided, and these can be easily closed by a plug.
Therefore, according to the spacer 10D of this example, the spacer 10D is attached to the culture vessel 20 as shown in the figure, and by appropriately closing any cylindrical communication pipes 41a to 41g and external communication pipes 42a to 42j, As shown in 11 (b) and (c), the space in the culture apparatus 30 can be divided into a plurality of zones. In FIG. 11B and FIG. 11C, a portion “CLOSE” is a portion closed by a plug.
Therefore, different types of gas can be supplied and discharged independently for each of the divided zones, and it becomes possible to conduct experiments of a plurality of systems such as simultaneously observing the influence of a plurality of gases.
In the example of FIG. 11 (b), six wells W1 to W6 are divided into two zones to form two zones, and in the example of FIG. 11 (c), two six wells W1 to W6 are arranged in two. Although three zones are formed by dividing into three, there is no limitation on the division method and the number of divisions as long as it can be divided into plural zones.

[Fifth Embodiment]
In the first to fourth embodiments described above, a multi-well plate in which a plurality of wells are formed is exemplified as a culture vessel body of a commercially available culture vessel. In the following embodiment, a case where a commercially available petri dish composed of a circular petri dish main body and a lid that closes it is used as a commercially available culture container will be described.
When a circular petri dish is used, as shown in FIG. 12, a circular spacer 10E formed in a shape corresponding to the petri dish is adopted, and a set of a gas supply port 14 and a gas discharge port 15 is formed in the spacer 10E. It can be provided (FIG. 12A) or two sets can be provided (FIG. 12B).

Furthermore, as shown in FIG. 13, a culture insert 32 can be locked inside a spacer 10E used in a commercially available circular petri dish 60 composed of a circular petri dish body 61 and a lid 62 for closing the main body 61. While providing a horizontal locking plate 23 having a single locking hole 23a and maintaining the culture insert 32 at an appropriate height so that the bottom of the culture insert 32 does not contact the bottom of the petri dish body 61, It is also preferable to adopt a form that can be locked to the spacer 10E. Specifically, it is preferable that the gap between the bottom of the culture insert 32 and the bottom of the petri dish body 61 is about 0.8 to 1 mm. Further, a packing 24 is formed on the entire periphery of the locking hole 23a so that the culture insert 32 and the locking hole 23a are airtightly connected.
In this case, the height h ′ of the space formed by the spacer 10E is preferably the height described in the first embodiment, and the height of the spacer 10E is also set so as to ensure such a height. It is preferable to design. Further, the upper frame 11 has a lid 62 and the lower frame 12 can be fitted with the petri dish body 61. The height of the upper frame is preferably 3 to 27 mm, and the height of the lower frame is preferably about 3 to 25 mm.

In this way, so-called gas-liquid interface culture is possible in which the lower side of the biological sample 33 is in contact with the medium 31 and the upper side is in contact with the supplied gas, and the influence of the target substance existing in the gas phase on the biological sample 33 is possible. It can be observed and evaluated using the petri dish 60 and the culture insert 32 locked thereto.
In this example, the number of the locking holes 23a is one, but a plurality of the locking holes 23a may be provided. By doing so, a plurality of culture inserts 32 can be locked, and the multi-well type can be used.

  In each embodiment described above, the target substance whose influence on the biological sample is observed and evaluated may be various gases, liquids contained in the gas phase, solids, volatile substances, and the like. For example, particulate matter discharged from automobiles, solid particulates such as dust and pollen; volatile organic substances such as toluene, benzene, chlorofluorocarbons, dichloromethane, alcohols, aldehydes; There is no limitation as long as it can be supplied and supplied into the culture apparatus. These target substances may be supplied alone or in combination of two or more. Further, the target substance may be supplied together with a gas suitable for maintaining the culture environment (humidity 100%, 37 ° C., air 95 vol%, carbon dioxide 5 vol%).

  Examples of biological samples include cells derived from animals and plants, tissue pieces, organs, microorganisms, and the like. In particular, as cells, cells existing in the respiratory system that are easily affected by substances present in the gas phase (pulmonary airways and alveolar cells, trachea cells, nasal cells, etc.), skin cells, Examples thereof include cells constituting the cornea, hair cells, plant callus, nematodes, and hyphae.

In addition, various methods can be employed to appropriately maintain various conditions in the culture apparatus 30 during observation.
For example, for humidity control, water can be placed between wells where the biological sample 33 is not placed or between wells. At that time, condensation may be prevented by a treatment such as coating the inside of the lid 22 with a hydrophilic substance so that observation from the outside can be facilitated. Specifically, for example, a method of applying a small amount of a solution of a hydrophilic polymer such as gum arabic and drying, a method of applying an aqueous surfactant solution (diluted kitchen detergent) and drying, described in JP-A-5-15362 For example, a method of applying an antifogging film obtained by saponifying a cellulose resin can be used.

  For temperature control, in addition to the above-described method using a heat insulating sheet, a method using a microscope having a stage with a heat retention function or a microscope with a heat retention / humidification chamber in the case of microscopic observation can be employed. Furthermore, a method of giving a heat generation function to the lid and the spacer is also effective. For example, a method of providing a conductive film having a temperature control function connected to at least a part of the lid or the spacer and causing a heat to flow through the conductive film can be exemplified.

Hereinafter, a method for observing and evaluating the influence of substances contained in the gas phase in the culture apparatus on the biological sample while culturing the biological sample in the culture apparatus will be specifically described.
[Evaluation Example 1]
Rabbit (Japan SCC Co., Ltd .: New Zealand White, 16 weeks old (male) -derived trachea is cut into half vertical with sterile physiological saline, and the mucosal layer on the tracheal lumen surface is peeled off from tissues including outer cartilage did. The mucous membrane layer was cut into pieces with a scalpel to obtain a mucosal piece.
On the other hand, as shown in FIG. That is, first, as a culture vessel 20, a multiwell plate with a 2 × 3 6-hole lid (SUMILON MS-80060, made of polystyrene, outer dimensions 127.6 mm (L) × 85.8 mm (W), main body 20.2 mm, well depth 17.0 mm, well diameter 35.6 mm, culture area 9.2 cm ² / well, well volume 16 mL), and medium 31 was placed in well W5. A cell-shaped culture insert 32 having a bottom made of a membrane was disposed in the well W5. Then, the mucosal piece previously fragmented was placed as a biological sample 33 so as to come into contact with the culture medium 31 through the membrane, and cultured. For medium 31, penicillin-streptomycin added, Dulbecco's MEM, and 10% fetal calf serum (0.5 mL) were used. A rubber packing 34 was inserted between the outer periphery of the culture insert 32 and the inner periphery of the well W5 above the medium 31. Then, the spacer 10 </ b> A shown in FIGS. 1 and 2 was mounted between the multiwell plate and the lid 22. The height h of the space S formed thereby was 17 mm. Next, in FIG. 5, sensors (temperature sensor and humidity sensor are inserted into a sensor insertion port not shown in the figure so that these conditions can be monitored. The sample was placed on a heat retaining stage of an interference microscope (Olympus IX70, objective lens 20 times).
Next, an injection cylinder 36 containing sulfur dioxide gas (air containing sulfur dioxide as a target substance at a concentration of 100 ppm) is prepared, and this injection cylinder 36 is connected to the gas supply port 14 of the spacer 10 via a tube 37 for injection. The gas in the culture apparatus 30 was supplied by pushing the piston of the cylinder 36. On the other hand, a bag 35 is attached in advance to the gas discharge port 15 of the spacer 10 as a gas recovery means, and the excess gas due to the supply of gas is recovered, so that the pressure in the culture apparatus 30 can be kept constant. I did it.
Then, using a digital camera (not shown) connected to a microscope (Aqua Cosmos: manufactured by Hamamatsu Photonics), subarray 64 × 64 pixels, burst uptake, and cilia part on the surface of the mucous membrane at a frequency of about 70 times / second continuously. 1024 images are taken and observed, and for each of the 25 areas of the cilia part, the luminance change with time is Fourier transformed to calculate the cilia movement frequency (CBF), and the effect of sulfur dioxide gas on the mucous membrane pieces evaluated.
The results are shown in FIG.
In the graph, the horizontal axis represents the exposure time (min), the vertical axis represents CBF (Hz), and the exposure time 0 min is the time when the supply of sulfur dioxide gas from the syringe barrel 36 is started.
As shown in FIG. 14, immediately after the supply of sulfur dioxide gas was started, it became clear that CBF decreased, that is, cilia movement decreased, and real-time observation and evaluation could be performed.

[Evaluation Example 2]
The same mucosal piece used in Evaluation Example 1 was prepared.
On the other hand, the culture apparatus 30 shown in FIG. 15 was configured. That is, a multi-well plate with the same lid as that used in Evaluation Example 1 is prepared as the culture container 20, the medium 31 is placed in the well W5 in the same manner as in Evaluation Example 1, the culture insert 32 is disposed, and the biological sample is prepared. A mucosal piece was placed as 33 and cultured. Further, a rubber packing 34 was inserted and disposed between the outer periphery of the culture insert 32 and the inner periphery of the well W5 in the same manner as in Evaluation Example 1. Then, the same spacer 10 used in Evaluation Example 1 was mounted between the multiwell plate and the lid body 22 to form a space S having the same height h. However, a three-way cock 38 was connected to the gas supply port 15 of the spacer 10 via a tube 37 having a length of 1 m. One of the remaining two sides of the three-way cock 38 is connected to an air box 39 adjusted to be supplied with air at a temperature of 37 ° C. and a humidity of 100%, and the other is connected at a temperature of 25 ° C. and a humidity of 40%. It was opened indoors, and either the air in the air box 39 or the indoor air was selected so that it could be supplied to the culture apparatus 30. On the other hand, a suction pump 41 was connected to the gas discharge port 15 of the spacer 10 via a trap 40 for preventing pulsation, and the air in the air box 39 was continuously taken into the culture apparatus 30. Then, the same sensors as in Evaluation Example 1 are inserted into the sensor insertion port (not shown), and the culture apparatus 30 and the heat insulation stage of the inverted differential interference microscope with the heat insulation stage (not shown) used in Evaluation Example 1 are used. Placed.
Then, with a digital camera connected to a microscope (Aqua Cosmos: manufactured by Hamamatsu Photonics), images of cilia on the mucosal surface are continuously captured and observed in the same manner as in Evaluation Example 1, and cilia motion frequency (CBF) is obtained in the same manner. ) Was calculated, and the influence of humidity (water) on the mucous membrane piece was evaluated.
The results are shown in FIG.
In the graph, the horizontal axis represents the exposure time (min), the vertical axis represents CBF (Hz), and the exposure time 0 min is the time when the supply of air was started.
As shown in FIG. 16, when air with a temperature of 37 ° C. and humidity of 100% was supplied, CBF did not change before and after the supply, but when air with a temperature of 25 ° C. and humidity of 40% was supplied. It has been clarified that CBF gradually decreases immediately after the start of supply, and cilia movement decreases due to air drying. As described above, according to this example, it was possible to perform observation and evaluation in real time.

10A to 10E Spacers 20, 60 Culture vessel 21, 61 Culture vessel body 22, 62 Lid 30 Culture device

Claims (7)

  A culture vessel mounted between a culture vessel main body of a culture apparatus and a lid that covers the culture vessel main body, wherein the culture vessel main body and the lid are separated from each other and airtightly connected. Spacer.   The culture container spacer according to claim 1, wherein at least one opening that communicates the inside and the outside of the culture apparatus is formed.   The culture container spacer according to claim 1 or 2, wherein the culture container spacer is attached to a culture container main body formed with a plurality of wells. Provided with a middle plate portion formed with a plurality of holes corresponding to each well,
4. The culture container spacer according to claim 3, wherein the peripheral edge portion of each hole and the open peripheral edge portion of each well are in close contact with each other.   4. The culture container spacer according to claim 3, further comprising isolation means for separating the wells.   The culture vessel main body, the lid, and the culture vessel spacer according to any one of claims 1 to 5 mounted between the culture vessel main body and the lid. Culture device.   A method for observing a biological sample, comprising observing the influence of a substance contained in a gas phase in the culture device on the biological sample while culturing the biological sample with the culture device according to claim 6.

Images