JP2008048654A - Tray-formed vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vessel having little risk of bacterial contamination of culturing cell, enabling easy observation of the culture state, having good culture performance, giving easily understandable culture result and enabling easy handling. <P>SOLUTION: The tray-formed vessel is provided with the first vessel wall 3 and the second vessel wall 4 placed opposite to each other and having sealed circumferential wall edges and a port 5 connected to a space formed between the first vessel wall 3 and the second vessel wall 4. The first vessel wall 3 has a recess 6 and a flange 7 formed on the circumferential edge of the recess 6, and the second vessel wall 4 is made of a soft material. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a container suitable for stimulating, differentiating, inducing, mutating, or proliferating cells (hereinafter collectively referred to as culture), and more particularly, in a form suitable for culturing cells. It relates to a tray-like container.

  Conventionally, petri dishes, flasks and the like (hereinafter referred to as petri dishes and the like) have been used in culture at a laboratory level, for example, culture for research purposes, culture for inspection purposes, and culture for evaluation purposes. Furthermore, in recent years, with the development of cell medicine, culture for therapeutic purposes has also been frequently performed. In most cases, the culture vessel used for this is the same as the laboratory test. In the present situation, petri dishes and the like are used as they are.

These therapeutic cultures include, for example, patient corneal cell culture, skin cell culture, chondrocyte culture, etc., and culturing and proliferating patient cells to regenerate them into membranes or plates. Some of them are transplanted to patients.
In addition, hematopoietic cells collected from the patient, such as T cells, NK cells, dendritic cells, etc. are cultured, that is, the cells are activated, induced or mutated, and further proliferated before the patient's Some return to the body for antiviral treatment and anticancer treatment.

Thus, when using cultured cells and the like for medical purposes, particularly when cells, proteins, and other components obtained by culture are returned to the body, the culture container used in the process is The culture performance should be good, the culture state can be easily observed, the cells in culture are less likely to be contaminated with bacteria, and easy to handle. That is, the aseptic operability must be good.
However, petri dishes and the like that are generally used cannot be said to have good aseptic operability.

For example, in cell culture, an operation of introducing a medium, cultured cells, additives, etc. into the container at the beginning of the culture and an operation of taking out the harvested cells such as cultured cells and proteins synthesized by the cells are always required at the end of the culture. In addition, even during the culture period, it is often necessary to repeat the operation of replenishing the medium and additives, but in a petri dish or the like, this operation is usually performed by opening the lid and opening the opening in the atmosphere. Since it must be repeated with a pipette or the like while it is open, there are frequent opportunities for bacteria to enter from the opening. A container that requires such an operation must be said to be a container with a high risk of bacterial contamination.

Normally, this operation is performed in a sterile room or a sterile booth, so although there is little risk of contamination, the space called the sterile room or sterile booth is controlled by the bacteria per unit volume of the space. The number is kept below a certain number, and it does not mean that there are no bacteria in the space inside the sterile room or the sterile booth. The longer the opening is open, the longer the opening operation becomes. The more complex, the greater the risk of contamination.

In fact, there have been many reports of contamination by bacteria during the culturing operation. In particular, the frequency of occurrence is high for unskilled workers with little culturing experience, workers who are not good at precise work, etc. In many cases, it is necessary to select a person to work on.

Furthermore, in a culture system using a petri dish or the like, it is considered that not only the handling of the petri dish or the like but also the sterility of the entire system using these containers is deteriorated.
That is, when culturing using a petri dish or the like, skill is required not only for handling a culture vessel but also for handling a culture tool such as a pipette.

The skill required of the worker means that even if the work is not problematic from the viewpoint of the skilled worker, the operation is complicated, an operation error is likely to occur, and the operation error leads to a large risk. It is clear that the operability should be improved.

In addition, by connecting a tube or a connecting adapter in advance to a flask equipped with a breathable sterile filter, by adding more cytokines, adding medium, transferring to other containers, etc. There are also attempts to improve aseptic operability.

However, even if the flask is an empty container, the volume at the time of culture does not change, and the volume at the time of culture must be twice or more than the volume of the required medium. There are many cases where it cannot be obtained. Furthermore, in addition to the above reasons, the flask needs to have strength in the container wall in order to maintain the structure, and the thickness of the container wall has to be increased. When connecting these containers, connection operability is poor and it is difficult to construct a total closure system.

As used herein, a closed system refers to an aseptic closed system in which the contents are moved between a plurality of aseptically produced containers without being exposed to contaminated outside air, that is, aseptically moved and required. Say that processing will be done.
Furthermore, if the surface of the air-permeable filter is covered with a medium, the air-permeability is remarkably deteriorated. Therefore, it is necessary to handle the medium with care so that the medium does not touch the filter surface.

In addition, an attempt to improve the above-mentioned problem by changing the culture container to a bag-like container provided with a port and a tube connected to the port for filling and discharging the content liquid into the container instead of the flask. Has been done.
For example, cells are cultured in a bag-like container having a container wall with good gas permeability, and supply of oxygen necessary for cell growth and discharge of carbon dioxide, which is one of the metabolites of cells, Is going to go through.

Since the volume of the bag-like container changes depending on the amount of the content liquid, the content liquid can be filled and discharged from the attached tube without discharging the air in the container or supplying air into the container. In other words, the medium can be filled and drained aseptically from the tube connected to the port without an aseptic filter for exhaust or supply, and the volume occupied by the empty container is significantly smaller than the required amount of medium. As for the volume at the time of culturing, the above-mentioned problem in the flask is improved because the volume is slightly larger than the required amount of medium.
However, in the bag-like container in which the planar sheets are simply overlapped, for example, the following problems are newly generated.

In general, it is known that floating cells fall to the bottom of the container by gravity during culture, where they are stimulated or proliferated. In addition, it is preferable that the cells or clusters in which the cells spontaneously gather are uniformly present in the container in order to manage the metabolism of the cells themselves or the exchange of information between the cells. That is, it is preferable to manage the cells or clusters so that they are uniformly dispersed on the bottom of the culture vessel.

However, in a bag-like container that is simply a stack of flat sheets, the two-dimensional flat sheet is transformed into a three-dimensional curved sheet when the contents liquid, that is, the culture medium is inflated. Inevitably, as it is, that is, in a situation where no external forcing force is given, it is difficult to keep only the bottom surface of the container flat, for example, the bottom surface forms a large curved surface, It causes wrinkles.

Therefore, the cells are forced to move to a low position along a curved surface or a wrinkle regardless of the requirements of the cells themselves, and cannot be uniformly present on the bottom surface. In general, floating cells have little interaction with the bottom surface of the culture vessel even if they proliferate, that is, they do not adhere to the bottom surface, so that migration and accumulation to a low position become remarkable with time.
Further, since the depth from the liquid surface is deep at the low position, the amount of cells that settle and deposit increases, and accumulation is promoted to a lower position.

In cell culture, it is usually performed by observing the growth state of a cell and observing its form, etc. to determine the next treatment, that is, addition of a medium, addition of cytokines, termination of culture, etc. It has been broken.
In general, adherent cells grow by adhering to the bottom surface, and suspended cells often grow by sinking to the bottom surface, so it is common to observe with an inverted microscope through the bottom surface of the container. Sure.

However, if the bottom surface of the container is curved or wrinkled, the position of the bottom surface changes, that is, the distance from the objective lens to the cell changes only by moving the observation point, resulting in a defocus. The image of the cell was blurred, and the focus had to be adjusted each time. Moreover, it has been difficult to maintain a clear visual field when observing while moving.

In addition, in a bag-like container in which flat sheets are simply overlapped, the culture conditions at the time of sealing and the culture conditions in the vicinity of the center of the container are significantly changed. For example, at the time of sealing, the thickness of the culture medium becomes thinner from around the center, and in this part, it becomes difficult to receive the supply of nutrients, and conversely, it becomes easier to receive the supply of oxygen.

In general, for small-volume culture vessels such as petri dishes, the purpose of cell culture is often experimental or evaluation. It is not preferable to be different.

The problem to be solved by the present invention is that, when used for culturing cells, there are few opportunities for the cells being cultured to be contaminated with bacteria, the culture performance is good, the culture results are easy to understand, easy to handle, and more desirable. An object of the present invention is to provide a container that allows easy observation of the culture state.
That is, when used for culturing cells, aseptic operation is easy, it is easy to construct a total closed system, cells or clusters are easily dispersed uniformly on the bottom of the culture vessel, and there are differences in culture conditions within the vessel. It is desirable to provide a container in which cultured cells and the like can be easily observed with an inverted microscope.

More specifically, even if there is no opening in the container, gas exchange with the outside of oxygen and carbon dioxide necessary for cell culture is possible, accompanied by exhausting air from the container or supplying air to the container. In addition, the contents can be filled and discharged from the attached tube, and the volume occupied by the empty container is significantly smaller than the required medium volume. An object of the present invention is to provide a container that can maintain a uniform thickness and can easily observe the state of cells under an inverted microscope.

The invention according to claim 1 is the first container wall and the second container wall facing each other, whose peripheral edges are sealed,
A port communicating with a space formed between the first container wall and the second container wall, wherein the first container wall has a concave portion and a flange shape formed on a peripheral edge of the concave portion. And the second container wall has flexibility.

That is, from the beginning, the first container wall is formed into a three-dimensional structure, that is, a concave shape, so that the content liquid can be filled without deforming the first container wall, and the second container wall can be made flexible. By adopting the material having, it is intended to obtain a container with a port capable of filling the container with the content liquid and discharging the internal solution from the container without air coming in and out.
Here, the fact that the first container wall does not deform does not necessarily mean that the shape immediately before filling the content liquid and the shape after filling do not change, but also the shape that has already been formed immediately before filling is deformed. This includes the case of returning to the originally formed shape by filling the content liquid.

Here, the resin forming the first container wall may be hard or soft, and a general resin is used. For example, styrene resin, polyester resin, polycarbonate resin, polymethyl methacryl resin, cycloolefin resin, polyethylene resin, ethylene vinyl acetate copolymer, polypropylene resin, and a mixture of these resins are used.
Moreover, you may form the 1st container wall with the composite sheet which compounded these resin.
The composite sheet here refers to a sheet made of a resin mixture, a laminate film, a sheet made of a plurality of resins such as resin coating on the resin film, and resin printing on the resin film.

  In consideration of the case of culturing adhesive cells, it is preferable that the surface in contact with the cell is a hydrophilic surface, so that the hydrophilic resin or the surface subjected to the hydrophilic treatment may be formed on the inside. preferable.

Further, the resin forming the second container wall, that is, the flexible resin includes low density polyethylene resin, ethylene-vinyl acetate copolymer, polypropylene resin, ethylene-propylene copolymer resin, polybutadiene resin, styrene- Examples thereof include butadiene copolymer resins and hydrogenated resins, polyurethane resins, and the like, and mixtures of these resins.

Examples of the low density polyethylene include not only general low density polyethylene but also linear low density polyethylene resins and metallocene catalyst-based low density polyethylene resins.
The polypropylene resin also includes a stereo block polypropylene resin and a mixture of the polypropylene resin and the stereo block polypropylene resin.

Here, the flexibility refers to the followability of the container wall that secures the volume by deforming to the extent that the content liquid can flow into or out of the container without air coming in and out. This followability is not simply determined by the physical values of the material forming the container wall, but also varies depending on the container form, the amount of air present in the container, etc. It is not appropriate to restrict with a numerical value.

Considering the use of the present invention, the flexibility of the container of the present invention is such that the inner solution is at a pressure of about a drop, for example, a pressure of about 50 cm water column, and the required amount of content liquid flows in or out. It only has to be flexible.
More specifically, for example, in a container in which the container wall facing when the inside of the container is sucked at 5000 Pa is in close contact, the peripheral seal between the container walls facing each other when the pressure of 5000 Pa is applied. What is necessary is just to have flexibility to such an extent that it becomes a half or more.
Since the first container wall is also formed of the flexible resin, the followability from the first container wall to the second container wall is also added, so that a container with better followability can be obtained. .

The invention described in claim 2 is characterized in that, in addition to the configuration described in claim 1, the second container wall has a convex portion that can be accommodated in the concave portion of the first container wall.
Since the convex part of the second container wall can be stored in the concave part of the first container wall, the volume of the space formed by the first container wall and the second container wall is reduced, and the content liquid is discharged. Since the amount of liquid remaining in the gap formed by the first container wall and the second container wall is also reduced, a container having a container wall with better followability, that is, a flexible container can be obtained.

Usually, even if air of about 20% of the amount of liquid to be filled remains in the container, there is often no problem in handling, so there is a gap between the first container wall and the second container wall. The volume of the space formed by is preferably 20% or less of the amount of liquid to be filled, that is, the volume of the concave portion of the first container wall.
This is because the reference value of the amount of liquid that is filled in the container during normal use is the volume of the concave portion of the first container wall.

The invention according to claim 3 is characterized in that, in addition to the structure according to claim 2, the bottom surface of the recess is planar.
That is, if the bottom surface of the recess is flat, the culture medium can be maintained at a constant thickness, so that the culture environment in the container can be made uniform.

According to a fourth aspect of the present invention, in addition to the configuration described in the third aspect, the bottom surface of the concave portion is transparent.
In cell culture, it is common to observe the shape and color of cells under a microscope, observe the progress of the culture, the state of the cells, and perform the following treatment. The container wall at least on the microscope observation side of the container is preferably flat and transparent so that cells can be observed with a microscope without being curved or wrinkled.

In general, since microscopic observation of cultured cells and the like is often observed with an inverted microscope, in the present invention, the container wall on the microscope observation side has a flat bottom surface of the concave portion because the bottom surface of the concave portion hits the container wall. In addition, it is preferable that it is not curved or wrinkled, and is transparent to the extent that cells can be observed with a microscope, that is, the light transmittance is 80% or more.

In addition to the structure described in any one of claims 1 to 4, the invention described in claim 5 is characterized in that the gas permeability of the second container wall is the gas permeability of the first container wall. It is characterized by being larger than.
That is, by supplying oxygen necessary for cells into the container and discharging carbon dioxide outside the container through the container wall, that is, by not allowing the inside and outside of the container to communicate directly, the sterility inside the container can be reduced. Is to maintain.

For example, in a general culture vessel such as a flask made of styrene resin, a vent called a vent is provided and exchanged with the outside of oxygen and carbon dioxide, but the culture solution spills out from the vent or the vent In the present invention, the container wall itself is made to have gas permeability, particularly oxygen and carbon dioxide permeability. Yes.

Here, gas permeability is mainly oxygen and carbon dioxide permeability, but oxygen permeability and carbon dioxide permeability tend to be similar to various materials, and carbon dioxide permeability. In general, the gas permeability of a container used for cell culture is evaluated by the oxygen permeability.

Since the first container wall needs to maintain its shape with respect to the weight of the medium when the medium enters, it is usually preferable to use a hard material that is difficult to deform, Since the material generally has poor gas permeability, it is preferable to form the second container wall with a material having better gas permeability.

The oxygen permeability required for the container wall of the container of the present invention varies depending on the amount of cells to be cultured and the area of the container wall through which oxygen permeates. That is, the oxygen permeability required for the container wall is proportional to the amount of cells to be cultured and inversely proportional to the area of the container wall through which oxygen permeates.

The container of the present invention is formed of two different oxygen-permeable container walls, and the amount of cells when cultured most efficiently is proportional to the amount of the medium. The value obtained by dividing the sum of the oxygen permeability per effective area of the container wall and the oxygen permeability per effective area of the second container wall by the amount of the liquid content is 0.2 cc / atm · day · ml or more In this way, it is possible to complete a culture vessel in which cells do not fall short of oxygen during culture.

Here, the oxygen permeability is a numerical value representing the amount of oxygen permeating the container wall per 1 m ² when the oxygen pressure of 1 atm differential pressure is continuously applied to the container wall at 25 ° C. for 24 hours.
The oxygen permeability per effective area of the container wall is a value obtained by multiplying the oxygen permeability of the container wall by the area of the container wall, and represents the ability of the container wall to supply oxygen into the container. The sum of oxygen permeability per effective area of all container walls of the container represents the ability of the container to supply oxygen into the container.

Furthermore, by dividing the total oxygen permeability per effective area by the amount of the inner solution, that is, the amount of the medium in which the cells are cultured, the amount of oxygen that the container can supply to the medium per ml can be expressed.

The invention described in claim 6 is used for culturing cells in addition to the structure described in any one of claims 1 to 5, and is further characterized in that the invention described in claim 7 is provided. Is characterized in that, in addition to the structure described in claim 6, cells to be cultured are injected or embedded in the human body or attached to a damaged part of the human body.
That is, since the container of the present invention can be easily closed, the cells, proteins, etc. cultured in the container of the present invention can not only reduce the risk of contamination by general bacteria, but also can cause cross contamination. Virus contamination from body fluids can be reduced.

The container of the present invention is generally used for various cultures performed in a petri dish or the like.
For example, the container of the present invention having a hydrophobic inner surface can be used as it is as a closed system non-trivial petri dish or flask for culturing floating cells as it is, and by coating various proteins, it can be used for culturing adhesive cells or floating cells. It can also be used for systemic cell stimulation.

Furthermore, the container of the present invention having a hydrophilized inner surface can be used as it is as a closed system petri dish or flask for culturing adherent cells as it is, and it is also possible to culture special cells by coating proteins. is there.
In addition, if the container of the present invention is prepared by coating a styrene resin on the inner surface of a flexible resin, for example, a vinyl acetate copolymer container having a vinyl acetate content of 20%, it can be used for normal culture. A closed system culture vessel with the same surface as the styrene petri dish or flask used is made.

Moreover, as a method for hydrophilizing the inner surface of the container of the present invention, for example, after forming a styrene-coated sheet into a tray shape, the inner surface is irradiated with ultraviolet rays including wavelengths of 185 nm and 254 nm, and after hydrophilization, What is necessary is just to finish in a sealed container.

As described above, according to the present invention, when used for culturing cells, the cells in culture are less likely to be contaminated by bacteria, the culture state can be easily observed, the culture performance is good, and the culture results are understood. An easy-to-handle container can be provided.
That is, aseptic operation is easy, it is easy to construct a total closed system, it is easy to observe cultured cells, etc. with an inverted microscope, cells or clusters are easily dispersed uniformly on the bottom of the culture vessel, A container with little difference in culture conditions can be provided.

Embodiments of the present invention will be described based on examples with reference to the drawings.
[Example 1]
As shown in FIG. 1 and FIG. 2, the culture vessel 1 of the present embodiment has two opposite vessel walls whose peripheral edges are sealed and sealed by a peripheral seal 2, that is, a vessel wall 3 (first vessel wall) and A container comprising a container wall 4 (second container wall) and a port 5 for communicating with a space formed between the container wall 3 and the container wall 4 and for filling or discharging the content liquid; The container wall 3 has a recess 6 and a flange-shaped portion 7 formed on the periphery of the recess 6, and the container wall 4 is made of a material having flexibility and good oxygen permeability. The bottom surface of the recess 6 is rectangular in plan view and is a single plane, and is transparent to the extent that cells can be observed with a microscope, that is, the light transmittance is 80% or more. It is configured.

Further, a cap 8 is provided at the open end of the port 5 for sealing the culture vessel 1, and the content liquid is discharged to the peripheral seal portion on the opposite side to the port 5 of the culture vessel 1 of this embodiment. A suspension port 9 is provided for suspending and discharging the culture vessel 1 of the present embodiment.
Here, a 200 μm sheet of polypropylene resin formed by inflation molding was used for the container wall 3, and a 100 μm sheet of polypropylene resin formed by inflation molding was used for the container wall 4.

It should be noted that a thick sheet is provided on the container wall 3 so that the vacuum-formed structure is not easily crushed, and the container wall 4 is provided with a container wall 4 that can follow the contents liquid in and out. In order to improve the permeability, a thin sheet was used.
Moreover, in order to raise the whole softness | flexibility, the stereo block copolymer excellent in a softness | flexibility and oxygen permeability was used for the polypropylene resin.

In the culture container 1 of this example, after forming a recess 6 having a length of 100 mm, a width of 60 mm, and a depth of 9 mm by vacuum forming on the former sheet, it is overlapped with the latter sheet, and the periphery is sealed by heat sealing. It is formed by heat sealing the port 5 after trimming the part.
When 40 ml of the culture medium was filled into the culture container 1 of the present example and discharged, some air was discharged before filling the medium, but thereafter, the medium could be filled and discharged without air coming in and out.

In addition, after coating the anti-CD3 antibody on the culture container 1 of this example, the T cells were cultured. As in the petri dish or flask, the T cells were uniformly dispersed on the floor surface, and further dispersed on the floor surface. The cells were easily observed with an inverted microscope. For example, even if the field of view was moved while observing, it was easily observed without changing the focal point.

[Example 2]
As shown in FIG. 3, in the culture container 1 of the present example, in addition to the configuration of Example 1, a convex part 10 that can be stored in the concave part 6 of the container wall 3 is formed on the container wall 4.
Here, a two-layer sheet of cycloolefin resin layer 30 μm and polypropylene resin layer 170 μm formed by multilayer extrusion molding is used for the container wall 3, and a vinyl acetate copolymer (acetic acid acetate) formed by inflation molding is used for the container wall 4. A 100 μm single-layer sheet having a daily vinyl content of 20% was used.

In the culture container 1 of the present embodiment, the concave portion 6 is formed on the former sheet in the same manner as in the first embodiment, and then the convex portion 10 is formed on the latter sheet by the same method. After arranging so as to overlap, it is formed in the same manner as in the first embodiment.
When the culture container 1 of this example was filled with 40 ml of the medium and discharged, not only the medium could be filled and discharged without entering and leaving the air, but also the air remaining in the bag after filling was small, and at the time of discharge The bag was discharged without negative pressure.

After coating the anti-CD3 antibody on the culture container 1 of this example and then culturing T cells, the same performance as in Example 1 was confirmed.
In Examples 1 and 2 above, the tray-shaped container of the present invention is used as a culture container. However, the use of the tray-shaped container of the present invention is not limited to these, and any It is.

As described above, when the tray-like container according to the present invention is used for culturing cells, the cells in culture are less likely to be contaminated with bacteria, the culture state can be easily observed, and the culture performance is good. It has the effects of easy understanding and handling of culture results, and is extremely suitable as a cell culture container.

It is a perspective view which shows the culture container which concerns on one Example of this invention. FIG. 2 is a schematic cross-sectional view when the culture vessel of FIG. 1 is cut along a vertical plane passing through A-A ′. It is a schematic cross section which shows the culture container which concerns on the other Example of this invention. FIG. 4 is a schematic cross-sectional view of a state in which a medium is contained in the culture container of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture container 2 Perimeter seal 3 Container wall (1st container wall)
4 container wall (second container wall)
5 ports
6 Concave portion 7 Flange-shaped portion 8 Cap 9 Suspension port 10 Convex portion

Claims (7)

A first container wall and a second container wall facing each other with a peripheral edge sealed, and a port communicating with a space formed between the first container wall and the second container wall;
The first container wall has a concave portion and a flange-shaped portion formed on the periphery of the concave portion,
The tray-like container, wherein the second container wall has flexibility. The tray-like container according to claim 1, wherein the second container wall has a convex part that can be accommodated in the concave part of the first container wall. The tray-like container according to claim 1 or 2, wherein a bottom surface of the concave portion is planar. The tray-like container according to claim 3, wherein a bottom surface of the recess is transparent. The tray-like container according to any one of claims 1 to 4, wherein the gas permeability of the second container wall is larger than the gas permeability of the first container wall. The tray-like container according to any one of claims 1 to 5, which is used for cell culture. The tray-like container according to claim 6, wherein the cultured cells are injected or embedded in a human body, or attached to a damaged part of the human body.

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