JP4973155B2 - Airtight container for gas concentration regulator for cell culture - Google Patents

Description

  The present invention relates to a method for observing a cell culturing process under a microscope, and has a culture part in which a gas atmosphere provided with a transparent part is controlled and a gas concentration regulator that controls the atmosphere, and is placed on a microscope stage. The present invention relates to an airtight container for microscopic observation that can be observed while culturing a sample in a general culture container.

Cell cultures carried out in biological, reproductive or biotechnology research or industrial fields require a gas environment that is different from the atmosphere.
When living cell observation is performed continuously for a long time, it is necessary to keep the living cells in the culture solution constant at the normal culture temperature and also to keep the pH of the culture solution constant. In this case, for example, the condition for maintaining the pH of the culture solution at the same pH 7.4 as the normal state of blood is that the atmospheric carbon dioxide concentration is 5%.

  In addition, hypoxic culture of cells has attracted attention in many research fields. Specifically, by culturing cells in a hypoxic atmosphere similar to that in vivo, genes such as hypoxia inducing factor (HIF) related to biochemical reactions and angiogenesis similar to those in vivo are induced, Has been confirmed to promote the growth and differentiation of potato. In addition, in an ischemia reperfusion experimental model that reproduces organ failure due to blood flow cessation, the mechanism in the cell is studied in detail by placing cells and tissues under anoxic for a certain period of time. Furthermore, a drug metabolism test is carried out by producing a reducing atmosphere in the absence of oxygen in a drug sensitivity test using human cells.

In such cell culture, a predetermined gas environment is created by using a CO ₂ incubator or a multi-gas incubator, and cell culture is performed in a normal culture vessel having air permeability. In this case, a multi-well plate, a flask, a petri dish, a chamber slide, a culture bag, or the like is generally used as the culture container.

  In order to record cell culture conditions or record photographs in a gas environment different from the atmosphere, remove the culture vessel from the incubator that forms the specified gas environment, place it in the microscope, and observe it. Or photography is performed. At this time, in a microscope installed in the atmosphere, the gas concentration of the medium changes during cell observation, and a predetermined gas environment cannot be maintained. Therefore, there is a problem that the state of the cultured cells changes during transfer and observation.

  As a means for solving this, a multi-gas control chamber can be installed in the laboratory, and culture and microscopic observation can be performed in the chamber. Specifically, an in vivo environment experiment device ViVox-TC Bio (manufactured by ViVox) is commercially available. However, these multi-gas control chambers are very expensive and use a large amount of replacement gas. Therefore, the installation location is limited in consideration of the safety of the experimenter.

  Therefore, many experimenters use a method in which the microscope stage is surrounded by a transparent plastic and the gas concentration is controlled with a gas cylinder or the like to observe the microscope. However, since this method has a large gas replacement space and is always observed while leaking gas, the amount of gas consumed is large and it takes time to reach a predetermined gas concentration.

  In the method of storing observation equipment such as a microscope in a commonly used gas control incubator or multi gas control chamber, the system functions normally due to dew condensation on the equipment due to high humidity (almost 100%) in the system. do not do. Therefore, it is difficult to observe the sample stably for a long time.

On the other hand, in recent years, culture microscope apparatuses have been commercialized in order to observe a large number of biological specimens.
An apparatus (Patent Document 1) in which an imaging unit is provided in a sample chamber in which temperature, humidity, and gas concentration are controlled, a culture chamber in which temperature, humidity, and gas concentration are controlled, and a microscope observation chamber that is isolated from the culture chamber are provided. A culture microscope (Patent Document 2) and the like have been developed.

  In the case of a culture microscope apparatus having a culture chamber, it is necessary to maintain the culture sample chamber at high humidity and the microscope chamber under dryness. In order to ensure the airtightness of each of the culture sample chamber and the microscope chamber, there is a problem that the structure of the apparatus becomes complicated. In addition, since only one type of gas concentration can be set for the entire sample chamber, if the required gas concentration differs for each sample, it is necessary to perform gas replacement for the entire sample chamber for each measurement sample. Gas control is not possible.

  A small culture chamber dedicated to microscopic observation is provided on a microscope stage, and the temperature, humidity, and gas concentration are controlled, and one type of cultured cell is continuously microscopically observed or photographed for a long period (Patent Document 3) There is. This method also requires a gas cylinder and a gas controller. In addition, assuming gas circulation, when the circulation gas is not a saturated water vapor gas, the composition is likely to change due to evaporation of water from the medium, which may adversely affect the culture results. In addition, when using saturated steam gas, it is essential to handle without contamination of bacteria and viruses.

  In bacterial culture, a transparent hermetic container that controls gas by adding a gas concentration regulator has been proposed (Patent Document 4), but when observing with a microscope, the upper and lower surfaces are optical so that a clear image can be obtained. When viewing with an inverted microscope, the bottom surface is too thick to be in focus or too large to be placed on a moving stage that can move in the XY direction on the microscope. It was not preferable.

  In addition, depending on the type of gas concentration regulator, the system may be depressurized or pressurized when used in an airtight state. As a result, the ceiling and the bottom plate are warped and deformed, becoming unsmooth, unsuitable for microscopic observation, and eventually the container itself is damaged.

JP 2003-93041 A JP 2005-326495 A JP 2004-329099 A Utility model registration No. 3034364 JP 09-252766 A

  The present invention relates to a method for observing a culture process under a microscope, and to provide an airtight container for microscope observation used in a simple cell culture observation system that can be observed while culturing a sample on a microscope stage. It is in.

The inventors of the present invention have achieved the present invention as a result of intensive studies on an airtight container that can be easily adjusted in gas concentration and can be used as it is in culture and microscopic observation.
That is, the present invention has a culture part provided with a transparent part on which a culture vessel is placed and which can be observed with a microscope, and a storage part for a gas concentration regulator that controls the culture part to a predetermined gas atmosphere, and , An airtight container for microscopic observation that can be mounted on a microscopic observation stage, and has a rectangular main body (1) whose ceiling surface is optically transparent and smooth and joined at the lower end of the main body upper part 1 A sealing material (between the bottom frame (2), the transparent sheet (3) placed on the inner surface of the bottom frame 2, and the periphery of the transparent sheet (3) on the upper body (1) and the bottom frame (2) 4) and an airtight container for microscopic observation in which a gas concentration adjusting agent storage portion (5) is provided on one side wall portion of the main body upper portion 1 and a pressure adjusting portion (6) is provided on the side wall portion.

  In a preferred embodiment of the present invention, the sealing material (4) is a soft resin, and the transparent sheet (3) has a thickness of 0.1 to 1.5 mm. In addition, the transparent sheet (3) has a concave shape in the mounting portion of the culture vessel so that the bottom surface of the bottom frame (2) and the lower surface of the transparent sheet (3) are in the same plane, or The transparent sheet (3) is formed by forming irregularities corresponding to the outer shape of the bottom shape of the culture vessel. This is an airtight container for microscopic observation that satisfies the requirement that the height of the predetermined portion of the bottom frame (2) is 0.5 to 1 cm.

By using this airtight container, cultured cells can be observed in a specific gas environment using various general small culture containers. Moreover, also in culture, a necessary gas environment can be individually set for each airtight container in a thermostat, and cross contamination between the airtight containers can be prevented. Furthermore, it has the portability which can be moved between a microscope and an incubator, maintaining each culture gas environment.
By adopting a structure in which only the parts of the bottom surface portion 2 that are scratched and easily interfere with observation due to repeated use of the airtight container are replaced, the other portions can be used repeatedly, so that the economy is high.

First, the present invention will be specifically described with reference to the accompanying drawings.
FIG. 1 is a perspective view of an airtight container for microscopic observation of the present invention. 2 is a side view of FIG. 1, and FIG. 3 is a plan view of FIG. FIG. 4 is an enlarged view of a typical example of the seal portion.
In FIG. 1, an airtight container for microscopic observation includes a rectangular main body upper portion (1) whose ceiling surface is optically transparent and smooth, a rectangular bottom frame (2) having a substantially L-shaped cross section, and a transparent sheet (3). , A sealant (4) between the upper part (1) of the main body and the periphery of the transparent sheet (3) on the bottom frame (2); (5) and a pressure adjusting part (6) on the side wall.

As shown in FIGS. 2 and 3, the main body upper part 1 is openable and closable as a lid, and the main body upper part 1 is formed by a bottom frame 2 and a sealing material 4 on the upper surface of the periphery of the transparent sheet 3 supported by the bottom frame 2. In a state where the lid is covered, a sealed structure is formed. In addition, a cushioning material (4 ′) is appropriately disposed between the transparent sheet 3 and the bottom frame 2 to improve the face-to-face alignment between the bottom surface of the bottom frame 2 and the bottom surface of the transparent sheet 3, and improve the alignment. It shall assume a role such as fixing to the frame 2.
And the main body upper part 1 has the accommodating part (5) of a gas concentration regulator and the pressure regulator (6) in the one side wall part. The gas concentration adjusting agent storage unit 5 is detachably attached to the upper part 1 of the main body at the upper part thereof, and the side part is appropriately formed with an opening, and between the culture vessel stored on the transparent sheet. Gas exchange is facilitated.
Further, a pressure adjusting unit 6 is installed on the side wall of the main body upper portion 1 provided with the storage unit 5.

FIG. 4 is an enlarged view of the seal portion, and shows a seal portion composed of the main body upper portion 1, the bottom frame 2, the transparent sheet 3, the seal material 4 and the buffer material 4 ′. This airtight container is kept airtight by the upper body 1, the transparent sheet 3 and the sealing material 4. The bottom frame 2 and the cushioning material 4 ′ are auxiliary members for stably holding the seal portion composed of 1, 4, and 3. Also, b and c in FIG. 4 are provided with a bent portion in the film to make it difficult to shift even if there is a pull in the inner direction.
In addition, although not described in this drawing, the main body upper part 1 and the bottom frame 2 are separately fixed using a fastener.

In this airtight container, the ceiling surface of the upper part 1 of the main body and the transparent sheet 3 are made of an optically transparent material, and other parts, the side wall of the upper part 1 of the main body, the storage part 5 and the bottom frame are Since it is a shape retaining material and a structure retaining material, a material having sufficient strength is used.
Glass, polycarbonate, cycloolefin polymer, polymethylpentene, polystyrene, polymethyl methacrylate, cycloaliphatic acrylate resin, polyethylene terephthalate, styrene / methyl are those that have high total light transmittance and can be used as optically uniform materials. Examples thereof include a methacrylate resin and a styrene / acrylonitrile resin.
Of course, the transparent materials mentioned above can be used for the side wall and bottom frame, but in addition to the above, glass fiber and other reinforcing materials are blended with the resins such as polyvinyl chloride, polyethylene, polypropylene, and fluoroethylene resin. Examples thereof include fiber reinforced plastics, aluminum and alloys thereof.

Various optical techniques are applied to the microscopic observation, and it is necessary to use an optical material suitable for each technique.
In the phase-contrast microscope, a material with low birefringence is essential, and examples thereof include glass, polymethyl methacrylate, cycloolefin polymer, polymethylpentene, alicyclic acrylate resin, and polystyrene / polycarbonate block copolymer resin.
Observation with a fluorescence microscope usually uses light having a wavelength of 350 to 750 nm as the excitation light, and observes fluorescence having a wavelength longer than that of the excitation light. Therefore, for observation with a fluorescent microscope, a material having a high transmittance over a wide wavelength region is essential, and glass or a cycloolefin polymer (ZENOX, ZEONOR made by Nippon Zeon Co., Ltd.) having a wavelength of 350 nm or more and a total light transmittance of 80% or more. ) Is exemplified. On the other hand, polyethylene naphthalate is exemplified as a container material for shielding ultraviolet rays.
Examples of the container material used for observation with visible light include glass and polycarbonate having excellent strength.

Microscopic observation is usually performed from the lower side in a state where the culture container is placed on the transparent sheet 3 of the airtight container and mounted on the microscope observation stage.
Therefore, the transparent sheet 3 must have optical characteristics that can be observed with a microscope. In this respect, the thickness is preferably 0.05 to 3 mm, particularly preferably 0.1 to 1.5 mm.
In addition, since the transparent sheet 3 is in direct contact with the bottom surface of the culture vessel, the microscope stage surface, the incubation thermostat bath surface, and the like, a protective film is suitably temporarily attached to the outer bottom surface as appropriate. It is preferable to peel off and use only during microscopic observation. Moreover, the culture container mounting surface of the transparent sheet 3 is appropriately subjected to an antifogging process or attached with an antifogging film. In addition, as a countermeasure against scratches on the part in contact with the culture container, a hard coat is appropriately applied. Furthermore, when it is not directly bonded to the bottom frame 2, it is replaced with a new one when inconvenience due to scratches or the like occurs.

  In addition, from the viewpoint of microscopic observation, it is preferable that at least a part corresponding to the culture part is in direct contact with the microscopic observation stage. From this requirement, as shown in FIG. 2, the periphery of the transparent sheet 3 is formed into a structure that protrudes from the bottom frame 2 and a portion corresponding to the thickness of the buffer material used as appropriate. In this molding, if wrinkles or the like occur at the four corners, the sealing state is likely to deteriorate, so that this occurrence does not occur and no optical distortion remains inside. In addition, the transparent sheet 3 is naturally sized to fit the bottom frame, but in accordance with the culture container, in order to fix the culture container, a recess (convex portion around the size) suitable for the size may be formed. As illustrated in FIG. 4, the shape of the edge of the transparent sheet may be a flat shape, a shape rising along the outer frame, or a shape in which the container inside of the seal portion rises along the shape of the outer frame. .

As the material of the sealing material 4, an elastomer such as natural rubber or synthetic rubber is applicable. Although these sealing materials do not come into direct contact with the culture solution, it is preferable that there are few volatile components in order to reduce cytotoxicity.
In the present invention, the pressure difference between the inside and outside of the container is adjusted to a range in which the ceiling and bottom surfaces (transparent sheet 3) of the container are not substantially deformed by the pressure adjusting unit 6, so that the pressure difference applied to the sealing material is small. . Therefore, since a softer material can withstand deformation due to a pressure difference, a soft material that can be sealed without excessive tightening can be suitably used for hermetic sealing. Specifically, ultra-soft urethane resin or oil-impregnated polyethylene is preferable.
Furthermore, it can be selected appropriately in consideration of the relationship with the main body material using a curable liquid sealing material. For example, in the case of polycarbonate, a one-component solventless silicone sealing agent can be used.

Since it is not exposed to high temperatures during the culture period, it is not particularly necessary to consider heat resistance. However, if it is used after heat sterilization (autoclave sterilization), or if it is used for long-term culture, the use conditions The material of the sealing material is selected in consideration.
Examples of the high heat resistance include silicon rubber, fluorine rubber, ethylene propylene rubber, and acrylic rubber. Examples of the sealing material having a lower gas permeability include butyl rubber, polysulfide rubber, epichlorohydrin rubber, high nitrile rubber, and fluorine rubber.
Moreover, as a cross-sectional shape of the sealing material, a sealing material such as a rectangle, a round shape, a U shape, a V shape, an L shape, and a U shape can be applied.

  Further, as the cushioning material 4 ′, the same sealing material as that described above can be used for general cushioning applications. However, in the present invention, as shown in FIGS. 4A, 4B, and 4C, the transparent sheet 3 can be used for the function of holding and fixing the shape. For example, a metal, fiber reinforced resin, or the like is used, and the transparent sheet 3 can be appropriately bonded and fixed thereto.

Naturally, the airtight container is sized to accommodate the culture container and to be placed on the microscope stage. In observation from the lower surface side, for example, in an inverted phase contrast microscope, the outer height is usually 6 cm or less. In addition, in the state where the culture vessel, the gas concentration adjusting agent storage unit 5 and the pressure adjusting unit 6 are provided, in particular, from the condition that automatic observation is easy with a movable stage that can move in the XY direction, the outer size is 20 cm or less in width. The length is preferably 12 cm or less.
The bottom frame 2 of the airtight container has a height of 2 cm or less so that it is easy to take out a short culture container after taking the upper part 1 of the main body. In order to facilitate, the portion corresponding to the arm position is preferably 1 cm or less. From the viewpoint of maintaining strength, the height is preferably 0.5 cm or more and 1 cm or less. The main body upper part 1 is 5 cm or less in height, and usually selected from 1 to 5 cm.

The above airtight container is required to have airtightness capable of maintaining a predetermined gas environment for at least 5 days, preferably 2 weeks. When a gas concentration adjusting agent is used, it is difficult to maintain a predetermined gas environment if the outside air intrusion amount is large. Therefore, it is usually preferable that the allowable amount of outside air intrusion is 1% or less, preferably 0.5% or less, of the volume in the container per day.
Since this airtightness can be achieved if there is no leakage from the seal portion, a structure and a seal material that do not cause air leakage at the seal portion are selected.

In FIG. 1 of the present application, the bottom transparent sheet 3 is sandwiched between the cushioning material 4 ′ and the seal 4 between the bottom frame 2, the seal 4 is sandwiched between the main body upper portion 1 and the transparent sheet 3, and the main body upper portion 1 and the bottom The frame 2 was detachably crimped using a stopper.
From this shape, the transparent sheet 3 constituting the bottom surface can be easily replaced. Further, as described in the description of the sealing material, the use of a softer sealing material enables sufficient sealing.

This airtight container is provided with a gas concentration adjusting agent storage portion 5 on one side wall portion (usually one side wall on the short side) of the main body upper portion 1 and a pressure adjustment portion 6 on the side wall thereof. With this positional relationship, when the gas is replenished to the airtight container from the outside or by attaching an air bag or the like, changes in the gas composition supplied to the culture unit via the gas concentration regulator are suppressed.
The gas concentration adjusting agent storage unit is usually provided with an auxiliary tool for storing the gas concentration adjusting agent so that the gas concentration adjusting agent is prevented from coming into contact with the culture vessel and the gas concentration adjusting agent can be easily attached and detached. It can be detachably attached to a ceiling surface near one side wall, side walls on both sides, and the like. For example, it is exemplified that an auxiliary tool is attached to the ceiling so that a partition wall can be formed on the ceiling, and in this case, the water vapor generated from the gas concentration regulator in the initial stage of the reaction usually corresponds to the microscope observation part. It is possible to suppress wrapping around the part.

Examples of the culture container include a cell culture container having no airtightness in which air flows even if a commonly used lid is used.
Specific examples include multiwell plates, dishes, chamber slides, culture bags and containers made of gas permeable materials, and flasks with gas filters.

The gas concentration regulator is used to maintain the gas atmosphere in the hermetic container (a sealed container having a predetermined volume) of the present invention within a predetermined concentration range for a certain period of time.
Usually, the gas concentration regulator is taken out from the outer bag of the gas barrier property (usually an aluminum barrier), placed in the gas concentration regulator storage part 5 of the hermetic container, and immediately sealed and used. After sealing, a predetermined gas concentration is reached after a lapse of a certain time, and this state is maintained for a predetermined time.
In the main culture, the gas component whose concentration is adjusted is mainly oxygen and carbon dioxide, which lowers the oxygen concentration and maintains the carbon dioxide concentration at a predetermined concentration of, for example, about 3 to 10%. Is saturated.

When the carbon dioxide gas concentration is constant and the oxygen concentration is 0.1% or less in a deoxygenated state or a reduced atmosphere, the main component is usually an organic compound having both oxygen absorption ability and carbon dioxide generation ability. , Activated carbon or the like as an auxiliary.
As a gas concentration regulator, there is a composition comprising ascorbic acid, metal salt, porous carrier, alkaline earth metal hydroxide, alkali metal carbonate and water (Patent Document 5).
Examples of products embodying these include a cell culture gas concentration regulator Culture Pal (trade name, manufactured by Mitsubishi Gas Chemical Co., Ltd.). Specifically, in a container of a predetermined volume, Culture Pal CO ₂ has an oxygen concentration of 15% and carbon dioxide gas concentration of 5%, Culture Pal Five has an oxygen concentration of 5% and carbon dioxide gas concentration of 5%.・ Zero is a sterilized gas concentration regulator designed to adjust the oxygen concentration to 0% and carbon dioxide concentration to 5%, respectively.

  By selecting a predetermined gas-tight container and a predetermined gas concentration regulator, a certain range of gas concentration is maintained for a certain period of time. Naturally, the gas concentration in the gas-tight container can be measured or monitored. . Specifically, it is excited by installing a small gas monitor in this airtight container, measuring it with a gas concentration measuring device as needed, and applying LED light that enables non-contact continuous gas concentration monitoring. For example, a fluorescent probe or sensor pad may be installed in an airtight container, or an OxoPlate (Presens) or the like having a sensor pad combined with a well plate may be used.

  Depending on the type of the gas concentration regulator, the inside of the system may be depressurized, and this may deform the observation surface of the microscope. A pressure adjusting unit (6) for adjusting the pressure change due to the agent was installed. The pressure adjusting unit 6 includes a movable cylinder, a gas barrier plastic film or a bag or balloon made of an aluminum film installed at the gas inlet for pressure adjustment outside the container, or one installed inside the container. Can be used. These can be used in combination with a check valve, a filter or a valve as required.

In addition, since the pressure adjusting unit 6 is installed on the side wall of the gas concentration adjusting agent storage unit 5, air introduced through a filter or the like from the outside, or when a bag or balloon is installed inside the container, The air leaking from the air flows into the culture vessel through the gas concentration regulator.
Further, not only the volume change caused by the gas adjusting agent described above but also the volume change at the time of heating to a culture temperature of 37 ° C., the pressure adjusting unit works effectively and is deformed due to the pressure fluctuation at the bottom. Can be avoided.
Furthermore, a rapid pressure change due to the gas regulator in a short time can be dealt with by a valve or a check valve, and a volume change due to a temperature change can be dealt with by reversible pressure regulation of a bag or a balloon.

The microscope used for observation in the present invention is preferably an inverted microscope that observes the sample from the lower surface side. As an observation method, an optical microscope, a phase contrast microscope, a fluorescence microscope, a differential interference microscope, a confocal laser microscope, or the like is appropriately selected according to the purpose.
In the case of continuous observation, continuous monitoring of cell culture can be realized by installing a small microscope unit with an electronic imaging device and this culture container kit in a normal thermostat. Examples of the phase contrast microscope with an electronic imaging device include an inverted cell observation microscope cell watcher (manufactured by Corefront Corporation).

The culture is usually maintained at a predetermined temperature (about 37 ° C. for cell culture). For this temperature control, a constant temperature bath is usually simple. The constant temperature bath may be anything that can control the temperature within the required temperature range.
In addition, by using the hermetic container of the present invention in the culture microscope apparatus described above, it is possible to easily create and maintain different gas concentration compositions for each hermetic container containing the culture container. Furthermore, cross contamination between the airtight containers can be prevented.
Using what has been described above, microscopic observation is performed while performing the cell culture of the present invention.

It is a perspective view of an airtight container used for the present invention. It is a side view of FIG. It is a top view of FIG. It is an enlarged view of the seal part in an airtight container.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Main body upper part 2 Bottom frame 3 Bottom transparent sheet 4 Seal material 4 'Buffer material 5 Gas concentration adjustment part 6 Pressure adjustment part

Claims (5)

It has a culture part with a transparent part on which a culture vessel can be placed to enable microscopic observation, and a storage part for a gas concentration regulator that controls the culture part to a predetermined gas atmosphere, and is mounted on a microscope observation stage possible a airtight container for microscopic observation, the ceiling surface is optically transparent smooth rectangular upper main body (1), rectangular bottom frame of the joint case at the lower end of the body upper (1) (2 ), transparent sheet (3 which is搭置on the inner surface of the bottom frame (2)), the sealing material between the periphery of the upper body (1) and bottom frame (2) on the transparent sheet (3) (4) And an airtight container for microscopic observation, in which a gas concentration adjusting agent storage portion (5) and a pressure adjusting portion (6) are provided on one side wall portion of the main body upper portion (1). The hermetic container for microscopic observation according to claim 1, wherein the sealing material (4) is a soft resin. The airtight container for microscopic observation according to claim 1, wherein the transparent sheet (3) has a thickness of 0.1 to 1.5 mm. The transparent sheet (3) according to claim 1, wherein the mounting portion of the culture vessel is concavely shaped so that the bottom surface of the bottom frame (2) and the lower surface of the transparent sheet (3) are coplanar. Airtight container for microscopic observation. The hermetic container for microscopic observation according to claim 1, wherein the transparent sheet (3) is formed with irregularities corresponding to the outer shape of the bottom shape of the culture container.

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