JP2001299326A - Incubator - Google Patents

Abstract

(57) [Problem] To provide an incubator that is convenient for transportation and handling, and easy to take out a cell sheet, and can be used for mass culture of the cell sheet. SOLUTION: A lid part 130 is attached to a bottom part 110 so as to be rotatable around a support hole 140.
Is rotated to open the upper surface of the culture surface 112, and the film 10
2 can be easily placed on the culture surface 112. The liquid medium is injected from the inlet / outlet guide 122. A holding pin 142 is disposed on the lid 130, and holds the film 102 at a position below the liquid level of the liquid culture medium 104. When taking out the cell sheet formed in the culture cassette 100,
The top of the culture surface 112 is fully opened by rotating the lid 130, and the culture is taken out in a sheet form.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an incubator, and more particularly to a flat incubator for culturing cells in a liquid medium in the presence of a sheet-like support.

[0002]

2. Description of the Related Art Cultured skin has been used for the purpose of promptly responding to the need for a large amount of skin for transplantation due to burns or the like, and for providing a wound dressing capable of repairing the wound at an early stage. Many studies have been done.

[0003] For example, a cell sheet of epidermal cells and mucosal cells having a plurality of layers can be mentioned, and this cell sheet has an advantage that it can be prepared from own cells. The cell sheet is prepared by isolating epidermal cells or mucosal cells and then culturing them on a plastic plate. The cell sheet thus produced is peeled off from the plate while maintaining the layer structure at the time of use, and is used as a sheet-like graft. In addition, a cell sheet prepared from own cells has an advantage that rejection is low and a survival rate is high.

However, when using this cell sheet,
Since the cells are growing on the plate, the cells need to be subjected to an enzyme treatment and peeled off from the plate. If the cell sheet itself is thin and the concentration of the enzyme is too high, the cell sheet itself is digested by the enzyme. For this reason, a certain degree of skill was required to peel off the cell sheet with an appropriate concentration of enzyme at the time of use. Further, the strength of the cell sheet itself was low, and the cell sheet had a property of being rolled up when peeled off, making it difficult to handle.

[0005] For this reason, a film composed of collagen or the like has been developed for the purpose of enhancing the handleability of the cell sheet. Since such a film can be handled together with a cell sheet formed thereon, it is not necessary to peel off the cell sheet from the plate, and it can also provide a moderate strength required when handling the cell sheet. it can.

On the other hand, such a cell sheet is required to be prepared in a state where it can be used as early as a burn. For this reason, a large number of cell sheets are produced in a dedicated incubator, or stored in a predetermined container and frozen and stored. Usually, in order to culture such a sheet-shaped cell sheet in a laboratory, a plastic bottle-shaped culture flask (for example,
T-25 flask; manufactured by Falcon). The culture flask has a lid attached to an entrance protruding from the main body. For this reason, gas exchange can be easily performed by loosening the lid, and if the lid is tightly closed, there is no liquid leakage, so that it can be easily transported in the laboratory.

[0007] Japanese Patent Application Laid-Open No. 10-43213 discloses a support or container for preparing a cultured skin which prevents contraction of collagen at the time of preparing the cultured skin and supports the prepared cultured skin as it is so that it can be applied to a wound surface. It has been disclosed. Here, the cells are seeded on collagen in sheet, gel or sponge form. In this case, a cultured skin is prepared while preventing the collagen substrate from shrinking, and the cultured skin is taken out of the container together with the support and used. Further, a protrusion is provided on the support or on the bottom of the container to suppress shrinkage of the cultured skin and to make the cultured skin adhere to the support or the bottom of the container.

Further, J. Chem. Eng. Japan 1998, 31 (5),
The 856-859 is capable of culturing the cell sheet in a specific Petri dish with a hydrophilic polyfluoroethylene polymer membrane placed on the culture surface, and equipped with a multi-channel pump to automatically control the exchange of culture medium. An automated culture system has been disclosed. The use of such a system shows the possibility of producing a large amount of cultured skin.

However, usually, a cell sheet is cultured and
Since the place where the cell sheet is stored is often far from the place where such a cell sheet is needed, it is transported in a cultured or preserved state, and will be handled by a different person than the person who handled it up to then . In order to use the cell sheet as an implant in a short time after transporting while maintaining a good state of the cell sheet, ease of transport and convenience for anyone to handle are required.

On the other hand, an incubator such as a petri dish with a lid is convenient for culturing, but inconvenient to transport as it is. A culture flask is convenient for carrying and culturing, but it is difficult to remove sheet-shaped cells from their narrow entrance while maintaining their shape. And take out the cell sheet. Such an operation is not easy for everyone.

[0011]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an incubator which is easy to transport and handle itself. Another object of the present invention is to provide an incubator from which a cell sheet can be easily taken out. Furthermore, an object of the present invention is to provide an incubator that can be used for mass culture of cell sheets and can be stored as it is.

[0012]

The incubator according to claim 1 is a plate-shaped incubator for culturing cells in a liquid medium in the presence of a sheet-shaped support, wherein the support is Is mounted, a bottom portion having a culture surface filled with the liquid medium, a cover portion disposed on the bottom portion to cover the culture surface, and moved to expose the culture surface to the outside, and the support Lifting support means for supporting a part of the liquid medium to prevent the support from floating on the liquid surface of the liquid medium, and liquid exchange means for replacing the liquid medium inside the incubator. It is characterized by:

[0013] According to the incubator of the present invention, the cell surface is formed by culturing cells on the culture surface, because the incubator covers the culture surface and moves to expose the culture surface to the outside world. After that, the culture surface can be exposed to the outside world by moving the cover. Thereby, it is possible to easily take out the cell sheet only by moving the cover portion, and it is possible to easily take out the cell sheet from the exposed culture surface in a sheet shape.

Here, the covering part in the present invention may be of any form as long as it is arranged on the upper surface of the culture surface and can cover the culture surface, and is placed on the bottom in a specific shape. The lid member that covers the culture surface and has flexibility,
In some cases, a film member that wraps around the culture surface according to the shape of the bottom may be used.

Further, since the incubator of the present invention is provided with means for preventing lifting, the support placed on the culture surface can be held in the liquid medium so as not to float from the liquid surface. For this reason, in the liquid medium, when seeding the cells on the support and after starting the culture with the support,
The cells can be reliably cultured in the liquid medium. In addition, since the support and the cell sheet can be held in the liquid medium during transportation, it is possible to prevent the cell sheet from jumping out of the liquid medium during transportation. Thereby, a uniform cell sheet can be reliably produced on the support without coming out of the liquid medium and drying, and handleability during transportation can be improved.

The lifting preventing means may be any one that can support a part of the support and hold the support in the culture solution. Examples of such lifting prevention means include, for example, holding means for holding the support so as not to float, holding means for holding the position by holding the end of the support, and preventing the support from separating from the culture surface. Separation preventing means.

Further, since the incubator of the present invention has liquid exchange means, the liquid medium in the incubator can be easily exchanged. Accordingly, a fresh liquid medium can be supplied to the inside of the incubator and the excess medium can be discharged without exposing the culture surface to the outside by moving the cover portion. Also, by connecting this liquid exchange means to an automatic control system such as a medium supply system capable of automatically controlling the exchange of the liquid medium, a large number of cell sheets can be automatically produced.

The liquid exchange means can be injected into the incubator as long as it is in a liquid state other than the liquid medium. For example, the seeding of cells can also be sent into the incubator via the liquid exchange means. Can be done by Also,
By supplying the cryopreservation liquid from the liquid exchange means,
It is also possible to cryopreserve the whole incubator in a state where the sheet shape is maintained without removing the cell sheet. During cryopreservation, the incubator can be stored in a direction that allows it to be vertically and horizontally, so that a storage form that does not take up space can be selected. The cell sheet thus cryopreserved can be thawed together with the incubator.

One or more tubular members capable of guiding the liquid to the culture surface correspond to such a liquid exchange means. The tubular member may be made of a hard resin such as a hard plastic or metal, or a soft resin, and in any case, is preferably connected to an opening / closing means for enabling or stopping the movement of the liquid.

Therefore, the incubator of the present invention is easy to transport and handle itself, and can easily take out the cell sheet prepared in the incubator, and furthermore, cultivates and preserves the cell sheet in a large amount. Also suitable for.

In the incubator of the present invention, the cover and the bottom may be one continuous member. As in the incubator according to claim 2, the bottom and the cover are connected to each other. It may be separable. In this case, since the bottom and the cover are separable, the cover and the bottom can be handled separately.

According to a third aspect of the present invention, in the incubator according to the first or second aspect, the incubator further comprises a fixing means for fixing the cover in a state of being disposed on the culture surface.

According to the incubator of the third aspect, since the cover is fixed to the state of being placed on the culture surface by the fixing means, it is possible to reliably maintain the state in which the cover covers the culture surface. Can be. As a result, the cover does not come off during transportation, and the handling during transportation can be further improved.

According to a fourth aspect of the present invention, there is provided the incubator according to any one of the first to third aspects, wherein the lifting preventing means is provided on the cover. Claim 4
According to the incubator described in (1), the lifting prevention means is provided on the cover, so when the cover is placed on the culture surface, the lifting prevention means is also placed on the culture surface to support the support. When the cover moves from the culture surface, the lifting prevention means also moves from the culture surface, and the support of the support can be released. Thus, the position of the lifting prevention means can be changed by operating the cover. Further, the support can be easily taken out only by moving the cover from the culture surface.

The incubator according to a fifth aspect is characterized in that, in any one of the first to third aspects, the lifting prevention means is provided at the bottom. According to the incubator according to the fifth aspect, since the lifting prevention means is provided at the bottom, the support can be reliably held in the liquid medium regardless of the presence or absence of the covering portion and the operation. Thus, for example, even if the cover is moved during culture to expose the culture surface, the support does not float on the liquid surface of the liquid medium.

The lifting prevention means in the present invention may press the upper surface of the support, as in the incubator according to the sixth aspect. This makes it possible to reliably prevent the support from being lifted by pressing the support from above. At this time, the shape of the part that contacts the support is
The shape may be a dot such as a circle or a square, or a linear shape such as a plate or a string.

[0027] Further, the portion of the support held by the lifting prevention means may be any part as long as the lifting can be prevented. However, as in the incubator according to claim 7, the support is held at the end of the support. It may be held. By holding the support at its ends, it is possible to reliably prevent the support from lifting without contacting the central portion of the cell sheet on the support. Note that the end held here includes any of the end side, the region along the end side, the corner, and the periphery of the corner.

The position on the support and the number thereof which are pressed by the lifting means may be any positions and numbers which do not significantly hinder the formation of the cell sheet and do not impair the tightness of the produced cell sheet. For example, when pressing around the corners of the cell sheet, a total of four at the four corners,
In the case of pressing down along the end side, a total of two can be provided on two opposing sides.

[0029]

[First Embodiment] FIGS. 1 and 2
A first embodiment of the present invention will be described below with reference to FIG. 1 and 2 show a culture cassette 100 according to a first embodiment of the present invention.

The culture cassette 100 has a rectangular culture surface 1.
12 and a lid 130 that covers the upper surface of the culture surface 112. Such a culture cassette 100 includes a film 102 on which a cell sheet is formed.
Is placed on the culture surface 112 and the culture surface 112 is filled with the liquid medium 104.

The culture surface 112 has three side surfaces 114 (11
4A, 114B, 114C). The side surface 114A and the side surface 114C are disposed to face each other, and the side surface 114B is
C are disposed at opposite ends. The other ends of the side surfaces 114A and 114C protrude from the culture surface 112, respectively.
It is arranged at one end of the culture surface 112 which is the middle part in the longitudinal direction of 4C.

The side surfaces 114A, 114B, 114 surrounding the culture surface 112 and making contact with a covering surface 132 described later.
C and the upper end of the middle plate 116, a lid 130 and a bottom 110
And a sealing material (not shown) for bringing the culture cassette 100 into a sealed state by bringing the culture cassette 100 into a sealed state. In addition, cover fixing recesses 115 are provided near both ends in the length direction of the side surface 114B which is the outer peripheral side of the culture cassette 100, respectively.

At the ends of the side surfaces 114A and 114C protruding from the culture surface, projections 118 for rotatably supporting the lid 130 are provided facing the center of the lid 130, respectively. Also, the side surface 114A and the side surface 1
14C, an exchange hole 120 is provided at a position substantially at the center of the culture surface 112, and an input / output guide 122 is attached to the outside of the culture cassette 100 continuously with the exchange hole 120. ing.

A liquid medium injecting means such as a tube (not shown) and a waste liquid discharging means are connected to each of the input / output guides 122. The liquid medium injecting means and the waste liquid discharging means are further connected to a means for supplying the liquid medium 104 to the culture cassette 100. Such means include, for example, an automatic control medium circulation system for automatically supplying the liquid medium 104 to the culture cassette 100 and discharging the waste liquid. In addition, a clip or a three-way cock that can close the tube may be attached to prohibit the supply and discharge of the liquid medium 104 to and from the culture cassette 100. When the medium is exchanged in a circulating manner, the pH may be adjusted using a HEPES buffer or the like, or the liquid medium 104, which has been previously equilibrated under 5% to 10% CO ₂ to adjust the pH, may be used. May be used.

The cover 130 includes a cover surface 132 covering the upper surface of the culture surface 112, a connecting portion 134 for connecting the cover 130 to the bottom 110, and a fixed support 133 for fixing the cover 130 so as to cover the upper surface of the bottom 110. It is configured.

The connecting portion 134 includes an L-shaped support plate 136 arranged along the width direction of the culture cassette 100 and support surfaces 138 attached to both ends of the support plate 136. On the support surface 138, circular support holes 140 formed in the width direction of the culture cassette 100 are arranged. The fixed support portion 133 is provided with substantially conical fixing projections 135 protruding toward the coupling portion 134 near both ends in the longitudinal direction.

At four corners of the cover surface 132 of the lid 130, columnar pressing pins 142 are respectively attached. The holding pin 142 projects from the covering surface 132 at a substantially right angle. The length of the pressing pin 142 is shorter than the width of the side surface 114, but is lower than the liquid level when the liquid medium is injected into the culture surface 112. If the length of the presser pin 142 is higher than the liquid level when the liquid medium 104 is injected, the film 102 stored in the culture cassette 100 is undesirably floated on the liquid level. It is sufficient if the film 102 pressed by the holding pin 142 is below the liquid level.
0 covers the upper surface of the culture surface 112,
2 is preferably a dimension that allows the tip of the culture surface 112 to be located slightly away from the culture surface 112, but may be a size that allows the film 102 to be in close contact with the culture surface 112.

The culture cassette 100 is made of a transparent or translucent material such as polycarbonate, polypropylene, polyethylene, and polystyrene.
The thickness of the culture cassette 100 is a thickness necessary for culturing cells, and may be a size that is convenient for carrying the culture cassette. For example, a culture space on the culture surface 112 is 1 mm to 20 mm, preferably The height can be set to an extent that a height of 5 mm can be secured. In this case, the liquid medium 104 can be filled from the amount that the film 102 and the cell sheet placed on the culture surface 112 can exist in the culture solution to the maximum amount that can be filled in the culture cassette 100.

The area of the culture surface 112 depends on the cells to be cultured.
Growth rate and desired size of cell sheet
Can be determined, e.g.
cm ^TwoFrom 250cm^TwoNormal cell culture up to
Can be sized. Preferably a portable view
About 100cm from the point^TwoIt is.

On the culture surface 112, a film 102 prepared in a size that can be arranged on the culture surface 112 is placed.
The film 102 serves as a support when cells to be cultured form a cell sheet, and is made of a material to which cells can adhere to the extent necessary for the cells to grow and form a cell sheet. Have been. Examples of such a material include collagen, chitin, polyurethane, gelatin, polylactic acid, calcium alginate, and the like, and any material commonly used for this purpose can be used.

Such a film 102 is used for the liquid medium 1
The cells may be placed on the culture surface 112 filled with 04, or may be immersed in the liquid medium 104 after being placed on the culture surface 112 before the cells are seeded.

The cells that can be cultured in the main culture cassette 100 include any of the cells that can grow alone or form a cell sheet on the film 102. Such cells include epidermal cells, epithelial cells, and fibroblasts. These cells can be multi-layered by growing on the film 102 and used together with the film 102 or alone as a cell sheet for applications such as wound dressing.

Note that the cells seeded in the presence of the film 102
02 may be seeded on the culture surface 112, or after seeding on the film 102 in advance, the film 102 may be placed on the culture surface 112. Further, depending on the selected cells, a cell sheet may be formed on the film 102, or cells may be arranged inside the film 102.

Next, the operation of the culture cassette 100 will be described. The following operations are performed under sterile conditions necessary for cell culture and cell sheet preparation. Side surface 114 of bottom 110
A and the protrusion 118 provided on the side surface 114 </ b> C
By passing through a pair of support holes 140 provided in the coupling portion 134 of the 30, the lid 130 is integrated with the bottom 110 so as to be rotatable about the pair of support holes 140. The pair of input / output guides 122 is connected to a culture medium exchange unit such as an automatic control medium exchange system, and the liquid culture medium 104 is injected into the culture cassette 100.

The cover 130 is rotated about a pair of support holes 140 to expose the entire surface of the culture surface 112. With the lid 130 opened, the film 102
Is placed, a cell suspension medium containing the cells constituting the cell sheet is injected onto the film 102 from above the culture surface 112.

When the seeding of the cells is completed, the lid 130 is
The upper surface of the bottom portion 110 is turned around the support hole 140 so that the cover surface 132 faces the culture surface 112.
Cover with 0. When the fixing projections 135 of the lid 130 are respectively disposed inside the fixing recesses 115 of the bottom 110, the sealing material on the side surface 114 of the bottom 110 and the middle plate 116 is in close contact with the covering surface 132, and the culture cassette 100 is moved. Sealed.

At this time, with the movement of the lid 130, the holding pins 142 arranged at the four corners of the lid 130 move the culture surface 1.
12 toward the film 102 placed on
At the same time, the cover 130 completely covers the upper surface of the culture surface 112, and at the same time, the pressing pin 142 faces the corner of the film 102.

As shown in FIG. 2, when the lid 130 covers the upper surface of the culture surface 112, the presser pins 142 disposed on the cover surface 132 of the lid 130 face the culture surface 112,
The tip is disposed below the liquid surface of the liquid medium 104. On the other hand, the film 102 placed on the culture surface 112
Is lifted by its buoyancy in the liquid medium 104 and comes into contact with the tip of the holding pin 142. As a result, the film 102 hits the holding pin 142 and is prevented from rising further, and is held at a position below the liquid level of the liquid medium 104.

After the cells have been seeded as described above, the culture cassette 100 is placed in an appropriate incubator, and the circulation of the culture solution is started by operating the connected medium circulation system. The cell culture is performed. When cell culture is performed by this circulation type medium exchange, supply of a fresh culture solution and discharge of a waste solution are performed simultaneously with the culture cassette 100 kept in a sealed state, and the culture solution is exchanged. In addition, since the culture cassette 100 is maintained in a closed state, the circulation of the culture medium is stopped and the tube attached to the input / output guide is closed, so that the culture cassette 100 can be carried as it is. The culture in the culture cassette 100 is continued, and after a predetermined culture time, a cell sheet having a plurality of layers formed on the film 102 is formed.

When the cell sheet formed in the culture cassette 100 is taken out, the hermetically sealed state is released by pulling out the fixing projection 135 from the fixing recess 115, and the cover 130 of the culture cassette 100 is placed in the support hole 140. The culture surface 112 is opened by pivoting around the center to expose the entire surface of the culture surface 112 to the outside world.

At this time, at the same time, the holding pin 142 of the lid 130 is
Leave 2 As a result, the film 102 is
The film 102 is released from the load held in the liquid medium 04 and floats on the liquid surface of the liquid medium 104. For this reason,
For example, the film 102 can be easily taken out by simply lifting the corner using tweezers or the like. At this time, since the cell sheet is taken out of the culture cassette 100 together with the film 102, the cell sheet does not get caught or torn at the time of taking out, and does not get caught or torn even after being taken out.

The cell sheet thus prepared was used as a film 102
And used as an implant. When performing a transplant,
In many cases, since the cell sheet is formed on one surface of the film 102, the cell sheet is transplanted with the surface on which the cell sheet is formed being applied to the transplant surface. Thereby, the cell sheet can be transplanted with a simple and convenient operation even when the cell sheet is used as a transplant.

The culture cassette 100 is also used for cryopreserving the prepared cell sheet together with the film 102. In this case, from the input / output guide 122,
A cryopreservation solution is injected instead of the medium. This allows
The cell sheet and the film 102 can be cryopreserved together with the culture cassette 100. Thus, the culture cassette 1
After being frozen and stored at each time, the culture cassette 100 can be stacked or arranged in a narrow place because the culture cassette 100 is thin and flat, so that not only can the limited space be effectively used, but also the space can be transported without taking up space. It also has the advantage of being able to. In addition, the boundary between the lid 130 and the bottom 110 may be fixed with a tape or the like in order to further increase the hermeticity of the culture cassette 100 during transportation. In this case, the cell sheet can be easily taken out as described above by cutting or peeling off the tape at the time of use.

As described above, in the culture cassette 100, the entire surface of the culture surface 112 can be easily exposed by rotating the lid 130, so that the cell sheet produced in the culture cassette 100 can be transferred together with the film 102 to the sheet. It can be easily taken out as it is. In addition, since it is connected to a medium exchange system that can be automatically controlled, a large amount of cell sheets individually housed in the culture cassette 100 can be automatically obtained.

Since the holding pin 142 is disposed above the film 102 and the cell sheet with the cover 130 covering the culture surface 112, the culture cassette 1
Even if the liquid medium 104 is injected at 00, the film 102 and the cell sheet do not float on the liquid surface. For this reason,
During culturing and transportation, a uniform cell sheet can be produced without floating or jumping out of the liquid surface of the liquid medium 104. Further, the holding pin 142
The film 102 and the cell sheet are removed from above the film 102 and the cell sheet with the movement of the cover 130, so that the film 102 and the cell sheet can be easily removed from the culture cassette 100 immediately after the movement of the cover 130.

The culture cassette 100 has a thin flat plate shape that is easy to carry, so that it is easy to hold itself, and because it does not take up space, it can be easily handled during storage or transportation.

In the culture cassette 100 of the present embodiment,
Although the cells are seeded directly on the culture surface 112 by moving the lid 130, the cells may be injected into the culture cassette 100 as a cell suspension from the input / output guide 122.

In the main culture cassette 100, the holding pin 14
Although the upper surface of the film 102 is supported in a dot shape as columnar protrusions 2 disposed at four corners of the lid 130, the present invention is not limited to this. For example, a plate-shaped member extending along the width direction or the longitudinal direction of the lid 130 may be used. In this case, when the upper surface of the culture surface 112 is covered with the lid portion 130, both ends in the width direction or both ends in the longitudinal direction of the film 102 press the film 102 linearly,
It can be more reliably supported. In addition, film 10
A plate-like or thread-like member may be attached to a portion facing the edge of the film 2, and in this case, the vicinity of the edge of the film 102 can be pressed. Further, a thread may be passed to each corner and held. This makes it possible to prevent the cell sheet from rising without contacting the central portion of the cell sheet.

In the culture cassette 100 of the present embodiment,
It is composed of two members, the lid 130 and the bottom 110, but is not limited to this. Except for the matters described below, the description is the same as above. FIG. 3 shows a schematic side view of a modification of the present embodiment. This culture cassette 2
00 has a bottom part 210 and a lid part 230 as a part of one continuous member, and a bent part 202 is disposed between the bottom part 210 and the lid part 230.

The culture cassette 200 includes the culture cassette 1
There is no middle plate 116 as in
2 is wider. Further, the cover 230 is provided with a holding pin 142 protruding from each of the cover surfaces 132.

The bent portion 202 is formed by a groove provided along the side of the lid portion 230. Therefore, the thickness of the bent portion 202 is smaller than the thickness of the lid portion 230 and the bottom portion 210. ing.

The bottom part 210, the lid part 230, and the bent part 202 are made of a flexible resin which can be easily bent, for example, a material such as polypropylene, polyethylene, or polystyrene.

In the culture cassette 200, the culture surface 112
After mounting a film (not shown), the lid 230 is
It is bent toward the bottom 210 around the bent portion 202. Since the bent portion 202 is provided along the side of the lid 230, the lid 230 bends toward the bottom 210 and covers the culture surface 112. When the lid 230 comes into complete contact with the bottom 210, the pressing pin 142 provided on the lid 230 faces a film (not shown) on the culture surface 112. Therefore, even if the culture surface 112 is filled with a liquid medium (not shown), a film (not shown) does not rise to the liquid surface.

When taking out the film and the cell sheet from the culture cassette 200, the lid 230 is bent around the bent portion 202 in a direction away from the bottom 210. As a result, the lid 230 moves and the culture surface 1
12 is exposed, and the pressing of the film and the cell sheet by the pressing pin 142 is released at the same time. As a result, the film and the cell sheet can be easily taken out from the culture surface 112 that is entirely exposed, while maintaining the sheet shape.

According to the culturing cassette 200, the film and the cell sheet can be reliably held in the liquid medium, and the film and the cell sheet can be easily taken out. It can be used as a simple incubator.

[Second Embodiment] A second embodiment of the present invention will be described below with reference to FIG. Except for matters described below, the third embodiment is the same as the first embodiment. FIG. 4 shows a culture cassette 300 according to the present embodiment. The culture cassette 300 includes a lid 330 having holding pins 142 at four corners, a culture surface 112 and side surfaces 314A, 314B, 314C, 314 surrounding the culture surface 112 from four sides.
And a bottom 310 made of D. Side 314
A and 314C are provided with exchange holes 120, and are respectively provided with input / output guides 122 that allow a culture solution or the like to be injected into the culture cassette 300. In addition, a sealing material (not shown) for sealing the culture cassette 300 is disposed at the upper end of the side surface 314A, 314B, 314C, 314D that surrounds the culture surface 112 and is in contact with the lid 330.

The culture cassette 300 is provided with a fixing member 340 capable of closely fixing the lid 330 to the bottom 310.
The fixing member 340 includes a pair of fixing clamps 342 provided on the bottom 310 and a fixing protrusion 346 provided on the lid 330 corresponding to the fixing clamp 342.

The fixing projection 346 is formed so that the lid 330 is
It is arranged on the surface 330A of the lid 330 which is the upper surface of the culture cassette 300 when the culture cassette 300 is closed by covering the culture cassette 300. For this reason, the fixing projection 346 is
0 is placed on the bottom 310, the culture cassette 300
Projecting upward. The fixing projection 346 is a thin rectangular plate-like member, and may be formed integrally with the lid 330 and may be provided with an adhesive or the like.

The fixed clamp 342 is connected to the opposing side surface 31.
4B and the center of the side surface 314D. The fixed clamp 342 is a wide U-shaped member with one side extended, and the end of the extended long side is rotatably fixed to each of the side surface 314B and the side surface 314D by a fastener 344. . The short side, which is the other end side, is approximately the same size as the height of the fixing projection 346 of the lid 330 and is bent in parallel with the long side. The length of the central portion of the fixing clamp 342 is longer than the width of the fixing protrusion 346 of the lid 330, so that the fixing clamp 342 can be locked to the fixing protrusion 346 of the lid 330.

Next, the operation of the culture cassette 300 will be described. In the culture cassette 300, the fixed clamp 342 on the bottom 310 is rotated around the fastener 343,
The locking of the lid 330 with the fixing protrusion 346 is released toward the outside of the culture cassette 300. Cover part 330 at bottom part 3
10 to expose the culture surface 112. afterwards,
The film 102 is placed on the culture surface 112, on which cells are further seeded.

After seeding the cells, the lid 330 is placed on the bottom 310 with the positions of the fixing projections 346 of the fixing member 340 and the fixing clamps 342 facing each other. Lid 330
When the is mounted, the fixed clamp 342 directed outward is
It rotates again around the fastener 344 and locks on the fixing projection 346 of the lid 330. Thereby, the lid 330
Is fixed to the bottom 310, and the culture cassette 300 is sealed by a sealing material (not shown). At this time,
The holding pin 142 of the lid 330 is disposed on the film and the cell sheet placed on the culture surface 112 so as to face the culture surface 112 to prevent them from floating. When removing the film and the cell sheet, the locking of the fixing clamp 342 is released, the culture surface 112 is exposed, and the film and the cell sheet are removed as described above.

As described above, the culture cassette 300 is excellent in the handling of the film and the cell sheet, and the lid 330 covering the culture surface 112 is more stably integrated with the bottom 310, as described above. It is possible to further facilitate transportation in a state in which the container is inserted.

In the culture cassette 300, the fixing projections 34
6 are arranged at one portion on one end side of the lid 330, respectively, but the invention is not limited to this. For example, a fixing protrusion 346 may be provided along one end of the lid 330 along the end. In this case, two or more fixing clamps 342 may be provided.

Further, in the main culture cassette 300, the fixing member 340 constituted by the fixing clamp 342 and the fixing projection 346 is used, but the present invention is not limited to this. FIG. 5 shows a modification of the fixing member applicable to the main culture cassette 300. Except for the matters described below, the description is the same as above.

The culture cassette 400 shown in FIG.
The fixing member 440 has a pair of fixing grooves 442 and claws 444.
The fixing groove 442 is provided on the side surface 414 of the bottom 410 and opens in the lateral direction toward the outside of the culture cassette 400. The claw 444 is provided at an end of the lid 430 and is bent toward the inside of the culture cassette 400.

In the culture cassette 400, after the corresponding claw 444 is engaged with one of the fixing grooves 442, the lid 430 is pressed downward from above. Lid 43
When the “0” is pressed, the other claw 444 of the lid 430 slides from the upper end after contacting the upper end of the corresponding bottom 410, and is disposed inside the corresponding other fixing groove 442. As a result, the lid 430 and the bottom 410 are fixed.

The culture cassette 500 shown in FIG.
The fixing member 540 includes a bottom portion 510 and a cover portion 53 facing each other.
0 and a fixing groove 544. The protrusion 542 on the bottom 510 is
0, and is fixed to the fixing groove 54 of the lid 530.
4 is open toward the bottom 530. In the culture cassette 500, the lid 530 and the bottom 510 are fixed by pushing the protrusion 542 into the fixing groove 544.

The culture cassette 600 shown in FIG.
Has a lid 630 arranged inside the bottom 610, and a pair of side surfaces 6 opposed to each other at the bottom 610.
14A and the side surface 614B are provided with fixing grooves 640, respectively. The fixing groove 640 is provided for the culture cassette 6.
00, and both ends of the lid 630 are held by side surfaces 614A and 614B. Lid 6
A handle 642 is provided on the upper surface of 30.

In this culture cassette 600, after inserting one side of the corresponding lid 630 into one fixing groove 640, one side of the corresponding lid 630 is inserted into the other fixing groove 640 at the upper end of the side surface 614 B of the bottom 610. To place. The lid 630 is pressed from above to push the side surface 614B slightly outward, and the one side of the lid 630 is inserted into the fixing groove 640. As described above, both ends of the lid 630 are fixed to the fixing grooves 6 respectively.
40, the lid 630 and the bottom 610
And fix. When removing the cover 630, the cover 63
0 handle 642, and the side 614A or the side 61
4B side while pulling it upward while slightly pressing it.
One end of 30 is pulled out of the fixing groove 640, and then the other end is pulled out of the fixing groove 640 in the same manner. This allows
Lid 630 is removed from bottom 610.

In the culture cassettes 400, 500, and 600, as described above, the entire surface of the culture surface 112 can be easily exposed.
02 can be easily taken out. In each case, the lids 430, 530, and 630 are
Since it is securely fixed to 0 and 610, the handling of the cell sheet and the film 102 as well as the handling during transportation can be improved.

[Third Embodiment] A third embodiment of the present invention will be described below with reference to FIG. Except for items described below, the configuration is the same as that of the first embodiment. FIG. 6A shows a culture cassette 700 according to the present embodiment. The culture cassette 700 includes a lid 730 having a guide 740 for opening and closing, and a bottom 710 having a corresponding guide groove 744.

The two opposing sides of the cover 730 are bent downward to form guides 742A and 724B. The guide 740 is arranged along the length direction on the inner side surfaces of the guide portions 742A and 742B, and protrudes toward the center of the lid portion 730.

The opposite side surfaces 714 A, 71 of the bottom 710
A guide groove 744 is formed on the outer surface of 4B along the length direction. The size of the guide groove 744 depends on the size of the lid 73.
0 guide 740 can be accommodated.

A holding plate 780 is attached inside the side surfaces 714A and 714B along the length direction of the side surfaces 714A and 714B. The holding plates 780 are provided to project toward the culture surface 112 so as to face each other.
When the degree of adhesion between the side surface 714 and the lid 730 is high, a sealing material for keeping the culture cassette 700 in a sealed state may not be disposed at the upper end of the side surface 714.

The holding plate 780 is located slightly above the culture surface 112 (see FIG. 6B). For this reason,
A space in which the end of the film 102 is arranged is formed between the holding plate 780 and the culture surface 112. Presser plate 780
Is determined depending on the thickness of the film 102 disposed on the culture surface 112.

The width dimension 780 A of the holding plate 780 corresponds to the width of a portion supporting the end of the film 102 disposed on the culture surface 112 and is determined depending on the size of the film 102. 0.1 mm to 10 mm, preferably 1 mm to 5 mm. This size is not particularly limited to the above, since it may depend on the material and thickness of the film 102 to be placed and the amount of the liquid medium 104 filling the culture surface 112.

The side walls 714A and 714B are provided with the exchange holes 12 at positions above the holding plate 780, respectively.
The exchange hole 120 is provided with an inlet / outlet guide 122 for allowing the medium to be injected and discharged.
It is attached to the outside of the culture cassette 700.

Next, the operation of the present embodiment will be described. The guide 740 is guided into the guide groove 744 by arranging the end of the guide 740 of the cover 730 at the end of the guide groove 744 of the bottom 710, and is moved along the guide 740 groove as it is to thereby cover the cover 730. And the bottom 710 are integrated.

The film 10 is placed on the culture surface 112 of the bottom 710.
In the case of disposing 2, the cover 730 slides on the culture surface 112 by moving the cover 730 along the direction in which the guide 740 is formed. The movement of the lid 730 exposes the culture surface 112 little by little from its end.

After the lid 730 has been moved to an extent necessary for accommodating the film 102, one end of the film 102 is placed below the holding plate 780 of the bottom 710, and the other end is placed on the corresponding bottom. 710 is disposed under the other holding plate 780. Thus, both ends of the film 102 are held by the holding plates 780.

When the film 102 is held on the holding plate 780 and placed on the culture surface 112, the culture cassette 700
Of the liquid medium 104 into the inside of the
Seed the cells on 2 above. After seeding the cells, the lid 730
Is moved on the bottom 710 in the direction opposite to the direction of opening to cover the upper surface of the culture surface 112. In the culture cassette 700, the film 102 is supported at its end by the holding plate 780, and does not rise to the liquid level in the liquid medium 104.

[0092] The seeded cells spread on the film 102 and form layers with growth to form a cell sheet. After the cell sheet is formed, the upper surface of the culture surface 112 is moved to a width sufficient for taking out the film 102 in the same manner as described above, and the film 102 and the cell sheet are transferred to the culture cassette 7.
Remove from 00. The film 102 is a film 102
Is moved from under the holding plate 780 by pinching it with tweezers or the like, and is removed from the holding by the holding plate 780.

Thus, the entire surface of the culture surface 112 is exposed to the outside world, and the film 102 and the cell sheet can be easily taken out. In addition, since the lid portion 730 and the bottom portion 710 are stably integrated, transportation is possible. Handling is easy even at times.

In the culture cassette 700, the film 1
02 is prevented from rising to the liquid surface of the liquid culture medium 104 by the holding plate 780 provided on the bottom portion 710 side, so that the lid 730 is not restricted by the movement mode.
The lifting of the film 102 can be prevented.

In this embodiment, the lid 730 is attached to the bottom 71
Sliding on 0 exposes or covers culture surface 112, but is not so limited. Culture cassette 7
00 is circular and the bottom 710 and the lid 73
A groove may be formed in the contact portion with the zero, and the circular cover 730 may be rotated to be detached from the bottom 710.
In this way, by using the rotary type, in addition to the above-described effects, the degree of adhesion between the bottom 710 and the lid 730 can be changed.

In this embodiment, the lid 730 is made of a material having an appropriate hardness such as plastic.
It may be a flexible sheet. In this case,
The culture surface 112 is exposed by rolling up or peeling off the sheet. This also prevents the film 102 and the cell sheet placed on the culture surface 112 from being lifted, and at the same time allows the film 102 and the cell sheet to be easily taken out.

[0097] In the embodiment of the present invention, the one having a floating preventing means in the lid portion 130, such as the culture cassette 100, becomes the front of the exchange hole 120 when the culture surface 112 is closed. A baffle may be provided at an appropriate position. By providing such a baffle plate, the flow of the culture medium injected from the exchange hole 120 can be made uniform and supplied to the inside of the culture cassette 100, or the liquid inside the culture cassette 100 can be discharged with a uniform flow. it can.

In the embodiment of the present invention, the pair of input / output guides 122 are installed so as to cross the culture cassette 100, but the present invention is not limited to this. For example, two may be provided on the same side surface 114. In this case, the medium is injected so that the liquid is sufficiently spread on the culture surface 112 when the medium is charged and discharged, and the medium is discharged without bias. Also,
Although the exchange hole 120 is provided so as to cross the culture cassette 100 in a direction perpendicular to the opening and closing direction of the lid 130, the present invention is not limited to this. A pair of exchange holes 120 may be provided in parallel with the opening and closing direction of the lid 130.

Alternatively, only one inlet / outlet guide 122 may be provided by attaching means for switching the direction of liquid flow, for example, a three-way cock or a switching pump, to the inlet / outlet guide 122. In addition, the number of the input / output guides 122 is not limited to one or two, and can be appropriately selected according to the usage mode, and may be three or more.

In the embodiment of the present invention, CO ₂ required for liquid culture of cells is taken into the liquid medium and supplied to the cells, but the present invention is not limited to this. For example, the culture cassette 10
At 0, a hole for gas exchange may be provided in addition to the exchange hole 120 for medium exchange. Further, for example, in the culture cassette 100, the lid 130 and the bottom 110 are brought into close contact with each other, and the culture solution is circulated to culture the cells. However, the present invention is not limited to this. If desired, the cells may be cultured without contact. Instead of circulating the culture solution, the supply and the waste solution may be cultured separately in a so-called batch system.

The inlet / outlet guide 122, the liquid medium injecting means and the waste liquid discharging means (not shown) in the embodiment of the present invention may be made of a material having an appropriate hardness such as plastic and can be easily cut. It may be a flexible material such as a tube. Furthermore, thermoplastic resin,
For example, polyethylene, polypropylene, polystyrene,
Polyethylene terephthalate may be used. In this case, it can be taken out by carrying out thermocompression bonding at the time of transportation, and detaching it from the culture medium supply system, so that the handleability can be further improved.

[0102]

According to the present invention, it is possible to provide an incubator which is easy to transport and handle itself, and to facilitate taking out of the cell sheet. Further, according to the present invention, it is possible to provide an incubator that can be used for mass culture of a cell sheet and can be stored as it is.

[Brief description of the drawings]

FIG. 1 is a perspective view of a culture cassette according to a first embodiment of the present invention.

FIG. 2 is a cross-sectional view of the culture cassette according to the first embodiment.

FIG. 3 is a schematic sectional view of a culture cassette according to a modification of the first embodiment.

FIG. 4 is a perspective view of a culture cassette according to a second embodiment of the present invention.

FIG. 5A is a schematic sectional view of a culture cassette according to a modification of the second embodiment, and FIG. 5B is a schematic sectional view of a culture cassette according to another modification of the second embodiment; (C)
FIG. 10 is a schematic sectional view of a culture cassette according to still another modification of the second embodiment.

FIG. 6A is a perspective view of a culture cassette according to a third embodiment of the present invention, and FIG. 6B is a cross-sectional view of the culture cassette according to the third embodiment.

[Explanation of symbols]

 REFERENCE SIGNS LIST 100 Culture cassette 102 Film (support) 110 Bottom 115 Fixing dent (fixing means) 112 Culture surface 122 Inlet / eject guide (liquid exchange means) 130 Lid (cover) 133 Fixing projection (fixing means) 132 Covering surface 142 Holding pin (lifting prevention means) 340 Fixing member (fixing means) 740 Guide (fixing means) 744 Guide groove (fixing means) 780 Holding plate (lifting prevention means)

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Toyoichi Kururushima 6-209 Mitani Kitadori, Gamagori City, Aichi Prefecture Japan Tissue Engineering Co., Ltd. (72) Inventor Takeyuki Yamamoto 6-209 Mitani Kitadori, Gamagori City, Aichi Prefecture No. 1 Japan Tissue Engineering Co., Ltd. (72) Inventor Riki Shinohara 6-209 Mitani Kitadori, Gamagori-shi, Aichi F-term in Japan Tissue Engineering Co., Ltd. 4B029 AA08 AA21 BB11 CC02 CC11 GA08 GB01 GB09 GB10

Claims (7)

[Claims] 1. A plate-shaped incubator for culturing cells in a liquid medium in the presence of a sheet-shaped support, wherein the support is placed and a culture surface filled with the liquid medium is provided. A bottom portion, a cover portion that is disposed on the bottom portion to cover the culture surface, and that moves to expose the culture surface to the outside world; and a portion of the support that supports the liquid; An incubator comprising: a lift preventing means for preventing the medium from floating on the liquid surface; and a liquid exchange means for exchanging the liquid medium inside the incubator. 2. The incubator according to claim 1, wherein the bottom and the cover are separable. 3. The incubator according to claim 1, further comprising a fixing means for fixing the covering portion in a state of being arranged on the culture surface. 4. The incubator according to claim 1, wherein the lifting preventing means is provided on the cover. 5. The apparatus according to claim 1, wherein said lifting prevention means is provided on said bottom portion.
The incubator according to any one of the above. 6. The incubator according to claim 4, wherein said lifting prevention means presses an upper surface of said support. 7. The incubator according to claim 6, wherein the lifting prevention means holds the support at an end of the support.