JP4459579B2 - Incubator - Google Patents

Description

  The present invention relates to a culture apparatus and a culture kit that can easily and easily maintain aseptic conditions and can easily perform tissue culture (Explant Culture).

Conventionally, many artificial materials such as artificial bones and wound dressings have been studied, and treatments have been performed by replacing these artificial materials with affected biological parts or covering the affected parts. In recent years, bioabsorbable materials that are absorbed into the living body over time have been studied, and treatment by tissue regeneration in vivo has also been studied. For example, there is a guided tissue regeneration (GTR) method in which the periphery of a defect site of periodontal tissue lost due to periodontal disease is covered with a bioabsorbable membrane and bone tissue is regenerated until gingival invasion is suppressed. It is known (see Patent Document 1).
On the other hand, since the culture technology has been remarkably developed, organ culture, tissue culture (Explant Culture), cell culture (Cell Culture), etc. are selectively used according to the purpose. Culture is mainstream. In particular, recently, a cultured tissue produced by cell culture has been used for research or medicine. As the cultured tissue produced by such a cell culture technique, those composed of specific single cells or those in which cells are held in an artificial material are well known. A cultured tissue in a form in which cells are held in an artificial material is expected to promote tissue regeneration in vivo as compared to the artificial material alone. For example, an osteoinductive material in which periosteal cells are seeded on a biopolymer material having high biocompatibility is disclosed (Patent Document 2).
In addition, as a product that supports such culture technology, many culture vessels and culture media are supplied from each manufacturer, and using these products, cultured tissues are individually produced at each research institution and each medical facility. ing. For example, a hermetically sealed culture vessel capable of easily performing cell culture is disclosed, and a sheet-shaped culture product can be easily produced from dispersed cells (Patent Document 3).

However, culturing operations using general-purpose culturing containers such as ordinary culturing flasks and petri dishes require clean equipment and skilled techniques, and are not easily performed in all facilities. Especially in the medical field in the dental field, there are many privately managed clinics, and it is very burdensome to have enough space and culture facilities necessary for the culture operation. Was difficult. In addition, cell cultures cause contamination (contamination) unless sufficient measures are taken against bacteria, and therefore higher culture techniques and clean facilities are required. Therefore, usually, an external organization equipped with sufficient facilities and techniques has to be requested to produce a cultured tissue.
In addition, in cell culture, a temporary solution has been devised by using a culture vessel that can easily perform cell culture while suppressing contamination as in Patent Document 3, but tissue fragment culture has been devised. However, sufficient culture vessels have not yet been devised. For example, in Patent Document 4, a filter piece that is easy to adhere to a tissue piece is provided on the culture surface. However, it takes a certain amount of time for the tissue piece to adhere to the filter piece. Can not be filled. Therefore, there is a considerable amount of time to be exposed to the air until the tissue piece adheres, that is, until the culture medium is filled, and requires a skillful judgment and time burden of the operator.
JP 07-236688 Special table 2002-522157 Special Table 2002-53983 JP 2003-180337 A

  An object of the present invention is to provide a culture apparatus and a culture kit that can form a preferred culture state early and easily in tissue piece culture in view of the above-mentioned problems of the prior art. Another object is to provide a culture apparatus and a culture kit that can facilitate the culture operation. Furthermore, it aims at providing the culture apparatus and culture kit which can suppress contamination.

The culture apparatus of the present invention comprises:
A culture apparatus for culturing a tissue piece,
A chamber forming a culture cavity containing the tissue piece in a culturable manner;
A cavity opening / closing section provided in the chamber, which opens and closes for loading and unloading of a tissue fragment into and from the culture cavity;
A tissue piece clamping part for clamping the tissue piece at a predetermined position in the culture cavity;
With
The tissue piece sandwiching section is a culture device that at least one forms a culture surface and the other presses and sandwiches the tissue piece so as to be in close contact with the culture surface.

  According to this culture apparatus, since the tissue piece holding part for holding the tissue piece in a predetermined position in the culture cavity is provided in the culture cavity, the tissue piece can be held in the culture cavity in a predetermined position easily and quickly. Can do. Furthermore, since one of the tissue piece sandwiching portions forms a culture surface, the tissue piece can be brought into close contact with the culture surface, and can be adhered at an early stage. Furthermore, since the tissue piece is sandwiched between the tissue sandwiching portions, even if the culture medium is supplied before adhesion to the culture surface, the tissue piece does not float away from the culture surface, and the tissue piece is surely placed on the culture surface. Can be glued. Therefore, a preferable culture state can be formed early by supplying a medium early to the tissue pieces in the culture cavity.

  The tissue piece clamping unit can release and start a tissue piece clamping state by the tissue piece clamping unit as the cavity opening / closing unit opens and closes. By carrying out like this, a tissue piece can be fixed early and easily with carrying in / out of the tissue piece to the culture cavity. The culture surface of the tissue piece sandwiching portion can constitute a part of the chamber.

  In the present culture apparatus, it is preferable that the tissue piece sandwiching section includes a first sheet-like body and a second sheet-like body, and the tissue piece is sandwiched between these sheet-like bodies. In addition, at least a part of the opposing peripheral edge portions of the first sheet-like body and the second sheet-like body are held together, and at least a part of the remaining peripheral edge portion can be opened to each other. It is a more preferable form to form a tissue piece taking in / out part. Further, the tissue piece sandwiching portion including the first sheet-like body and the second sheet-like body can be formed into a bag-like body having a tissue piece taking-in / out portion as an opening.

The tissue piece insertion / extraction part formed in the tissue piece clamping part can be fixed so as to be located at or near the culture cavity opening / closing part. In addition, the tissue piece insertion / extraction part can be opened / closed along with the opening / closing of the culture cavity opening / closing part. Furthermore, the tissue piece insertion / extraction part may constitute at least a part of the culture cavity opening / closing part. A plurality of communication holes can be provided in at least a part of such a tissue piece sandwiching portion, and at least a part of the tissue piece sandwiching portion can be a mesh-like body. Furthermore, it is preferable that at least a part of the tissue piece sandwiching portion, preferably the surface facing the culture surface, is provided with dot-shaped or line-shaped convex portions. According to these forms, it is possible to provide a simple configuration particularly suitable for holding a tissue piece so that it can be cultured.
The culture cavity is preferably formed so as to be able to be sealed at the cavity opening / closing part. By doing so, the culture cavity can be sealed, and the possibility of contamination can be reduced.

  Moreover, in this culture apparatus, it is also preferable that at least a part of the chamber has flexibility. In particular, a flexible bag-like body is preferable. Further, in the present culture apparatus, the tissue piece sandwiching portion may have a rigidity that does not follow the deformation of the chamber, and the tissue piece sandwiching portion may have flexibility. Furthermore, the tissue piece sandwiching portion can be formed so as to float in a certain range in the culture cavity during culture.

  In the main culture apparatus, the chamber may be provided with a liquid take-in / out section. Moreover, in this culture apparatus, the chamber can be provided with a seal portion that can be punctured and can maintain a sealing property. Furthermore, in the present culture apparatus, the chamber may be provided with a gas discharge unit. According to these embodiments, liquid can be easily filled and discharged, so that the possibility of contamination can be reduced. The seal part is useful as a fluid injection / discharge part.

  In the present culture apparatus, the chamber can have oxygen and carbon dioxide permeability. According to this embodiment, even when the chamber is sealed in an incubator having a predetermined gas atmosphere such as a carbon dioxide incubator, the gas can be supplied into the culture cavity to form a predetermined gas atmosphere in the culture cavity.

  The main culture apparatus can be a periosteal tissue piece culture apparatus. According to this embodiment, the cultured periosteum can be easily produced, and can be applied to the GTR method in the oral dental field. In addition, according to the present apparatus, a good cultured periosteum having a uniform thickness can be obtained, so that it can be easily produced by a beginner without requiring a skilled technique.

The culture apparatus of the present invention includes a chamber composed of a lid and a tray that form a culture cavity that encloses the tissue piece so that it can be cultured,
A cavity opening / closing section provided in the chamber, which opens and closes for loading and unloading the tissue piece into and from the culture cavity;
A tissue piece clamping part for clamping the tissue piece at a predetermined position in the culture cavity;
With
The tissue piece sandwiching section can be a culture apparatus in which at least one forms a culture surface and the other presses and sandwiches the tissue piece so as to be in close contact with the culture surface.

  According to this culture apparatus, a preferable culture state can be formed quickly and easily with respect to the tissue piece with a simple configuration. In this culture apparatus, the tissue piece sandwiching section may be configured such that the culture surface is configured by the bottom surface of the tray and the other is provided on the lid. According to this aspect, the tissue piece can be fixed to the culture surface by attaching the lid to the tray. Further, in this culture apparatus, the culture cavity is formed so as to be able to be sealed at the cavity opening / closing portion, and the fluid in the chamber can be poured and discharged into the chamber while maintaining the sealing property of the culture cavity. A fluid injection / discharge section may be provided. Specifically, it can be configured by a mechanism that opens and closes the liquid injection / discharge part with a valve or a cock, or a sealing material that can be punctured and that can maintain sealing performance. According to this, the culture cavity can be formed in a sealed manner, and liquid and gas can be easily filled and discharged, so that the possibility of contamination can be reduced.

The culture kit of the present invention comprises
A culture kit for culturing a tissue piece,
A chamber that forms a culture cavity that encloses a tissue piece in a culturable manner;
A cavity opening / closing part provided in the chamber for opening and closing the culture cavity so that the tissue piece can be carried in and out;
A fluid pouring / discharging unit provided in the chamber and capable of pouring / discharging the fluid in the chamber in a state in which the sealing of the culture cavity is maintained;
A tissue piece clamping part for clamping the tissue piece at a predetermined position in the culture cavity;
At least one of the tissue piece clamping portions forms a culture surface, and the other presses and clamps the tissue piece so as to be in close contact with the culture surface.
A culture device;
A syringe capable of pouring and discharging fluid into the culture cavity via the fluid pouring and discharging section;
A culture kit comprising:

  According to this culture kit, tissue pieces can be fixed easily at an early stage, and a syringe that can be filled and discharged with liquids and gases such as a medium is made into a kit. It is.

  As described above, according to the present invention, tissue piece culture can be easily and stably performed without skilled techniques and large-scale clean facilities.

  Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings illustrating an embodiment of the present culture apparatus. The present invention is not limited to the embodiments illustrated in the drawings, and within the scope of the present invention, the following embodiments have been modified or added by those skilled in the art based on common general technical knowledge. Are also encompassed by the present invention.

FIG. 1 shows a tissue piece culture bag, which is an embodiment of the main culture apparatus 2.
The main culture apparatus 2 is an apparatus for culturing a tissue piece. Examples of the tissue piece include a tissue piece composed of adhesion-dependent cells such as a periosteal tissue piece, a dermis tissue piece, an epithelial tissue piece, and a bone tissue piece, and includes human cells or non-human animal cells.

(Culture cavity)
The main culture apparatus 2 forms a culture cavity 4 for culturing tissue pieces. The culture cavity 4 is a portion that can hold a culture system including a medium that enables culture of tissue pieces, that is, a culture space. In the present invention, the culture cavity 4 is preferably formed to be sealable. By being sealed, the main culture device 2 can keep the components of the culture system inside even if the culture device is held in any state. Moreover, since the culture system can be sealed by sealing, contamination can be prevented. The sealing means is not particularly limited, but when the chamber 10 is a bag-like body, the outer skin part (which is a constituent part of the bag-like body) is overlapped, folded, fitted, bound, and fused in the vicinity of the opening. It can be brought into close contact with a known sealing means such as adhesion, filling with a sealing material or the like. Preferably, a sealing means that can be opened and closed is employed.

(Chamber)
The culture cavity 4 is formed by a chamber 10 in which the culture cavity 4 can be formed. In the culture apparatus 2 illustrated in FIG. 1, the chamber 10 is formed of a liquid-impermeable material that can form the culture cavity 4. As the constituent material of the chamber 10, various known materials that have been conventionally used as this type of material can be used. However, the chamber 10 has at least one of flexibility, transparency, and gas permeability. It is preferable to have all of them. Preferably, it is a plastic material, and examples thereof include polyolefins such as polyethylene and polypropylene, polyvinyl chloride, and copolymers containing these monomers. When the chamber 10 is flexible and can be deformed, the chamber 10 can be deformed to easily fill and discharge a liquid such as a culture medium and gas. At the same time, since the chamber is deformed, it is possible to suppress a large pressure change due to exchange of these media with respect to the tissue pieces in the culture cavity. In addition, if the chamber 10 is flexible and deformable, it can be deformed so as to relieve the pressure difference between the inside and outside of the chamber 10, thereby protecting the tissue piece and avoiding damage to the chamber 10 itself. You can also The flexibility of the chamber 10 may be a material flexibility or a structure flexibility, that is, a deformable structure. In such a case, the deformable part may be the entire chamber or a part thereof.

  Further, at least a part of the constituent material of the chamber 10 is preferably gas permeable. In particular, it is preferable to have permeability to carbon dioxide gas and oxygen. If the permeability of such a gas component is ensured, it can be adapted to many culture systems. In particular, in the present culture apparatus, the inside of the culture cavity 4 can be easily maintained in a favorable environment by being permeable to carbon dioxide and oxygen. Such a gas permeable material can be appropriately selected from known materials.

  The form of the chamber 10 can take various forms as long as the culture cavity 4 can be formed inside. In the form illustrated in FIG. 1, a bag in which two sheet-like bodies are sealed by fusion or the like so that one opening can be formed, and a town having a predetermined width is formed on the other end of the opening. A chamber 10 that is a body (see FIG. 2) is shown. The main culture apparatus 2 preferably has a configuration including a hollow portion that can substantially surround the culture cavity 4 formed inside. Accordingly, it is possible to adopt a flask shape such as a T-shaped flask or a dish shape such as a petri dish composed of a tray and a lid, or a bag shape having at least one opening as illustrated in FIG. . When the chamber 10 is a bag-like body, the chamber 10 may be a rigid body that maintains a certain shape, but is preferably a flexible bag-like body. A flexible bag-like body is advantageous because it can improve the deformability of the chamber 10. Furthermore, it is preferable in that a cavity opening / closing part and / or a tissue piece taking-in / out part described later can be constructed so as to be openable / closable using the flexibility of the cavity.

(Cavity opening / closing part)
The chamber 10 includes a cavity opening / closing part 20 that opens and closes the culture cavity 4. In the form illustrated in FIG. 1, one opening of the bag-shaped chamber 10 constitutes a cavity opening / closing part 20 and includes a sealing part 6 of the culture cavity 4. The cavity opening / closing part 20 is not particularly limited in size, shape, etc., but can be set to such an extent that a tissue piece can be inserted and a cultured tissue can be taken out. Preferably, the size and shape can be inserted into and removed from the culture cavity 4 without deforming the tissue piece or the cultured tissue. The opening / closing mechanism in the cavity opening / closing unit 20 is not particularly limited, but can be configured according to the form of the chamber 10. For example, when the chamber 10 is a bag-like body, the cavity opening / closing portion 20 can be configured by opening and closing the opening edge of the bag-like body constituting the chamber or the vicinity thereof. If it carries out like this, the cavity opening-and-closing part 20 can be comprised with a simple structure. In the case where the chamber 10 has a dish configuration including a tray and a lid, the opening portion of the tray to which the lid is attached or detached or opened and closed constitutes the cavity opening / closing portion 20. In addition, the sealing performance of the culture cavity 4 can be easily ensured by introducing a known sealing structure in the cavity opening / closing unit 20.

  In the form illustrated in FIGS. 1 to 4, the cavity opening / closing part 20 is configured to open by elastic deformation and close by its restoring force. That is, in this embodiment, both ends of the cavity opening / closing part 20 are gripped by a thumb that can face the thumb of one hand and the thumb, and when a load is applied toward the center side of the opening width, the cavity opening / closing part 20 is elastically deformed. Each of the outer skin portions constituting the wings is bent outward and spaced apart. The opening state can be maintained by maintaining the load, and when the load is removed, the cavity opening / closing part 20 is closed by the restoring force. Such a configuration is preferable in that the cavity opening / closing portion 20 can be easily opened and closed, and can be configured to be opened and closed with one hand, for example. Such an opening / closing mechanism can be easily constructed by forming the cavity opening / closing portion 20 with an elastically deformable material and / or structure.

  It is preferable to form the culture cavity 4 in the cavity opening / closing part 20 so that it can be sealed. By doing so, the culture cavity 4 can be unsealed and sealed as the culture cavity 4 is opened and closed. For example, in the embodiment illustrated in FIG. 1, a convex portion is provided on one side and a concave portion is provided on the other side of the opening portion of the bag-like body constituting portion (outer skin portion) in the vicinity of the opening. The sealing property is ensured by providing the sealing part 6 with a zipper that forms the fitting state of the part over the opening width. Such a sealing structure is well known to those skilled in the art, and a person skilled in the art can appropriately select from well-known techniques and apply it to the cavity opening / closing part 20. Therefore, it is not limited to a zipper or a fastener, and may be sealed by thermocompression bonding. In the case of sealing by thermocompression bonding, the cultured tissue T may be taken out by excising a part of the chamber 10 after completion of the culture process. In order to achieve both opening and closing properties and a sealing property, a holding member such as a clip that holds the cavity opening / closing portion 20 from the outside and closely contacts the opposite portion may be employed.

  The chamber 10 can include a liquid inlet / outlet part separately from the cavity opening / closing part 20. By providing the liquid take-in / out part, filling and exchange of the liquid medium and filling and exchange of the cleaning liquid can be performed without opening and closing the cavity opening / closing part 20. It should be noted that the liquid outlet / outlet part is preferably opened and closed in a sealable manner. Moreover, the chamber 10 can be equipped with the seal | sticker part 30 which can puncture and can maintain a sealing performance, as shown in FIG. Puncturable and capable of maintaining sealability means that puncture is possible with a puncture body such as a needle, and that the sealability at that portion can be maintained even when the puncture body is entered and after it is pulled out. is doing. The term “sealability” as used herein includes at least a seal property against a liquid, and more preferably includes a seal property against a liquid and a gas (preferably oxygen and carbon dioxide). Various sealing materials having such characteristics are known, and examples thereof include those containing rubber, silicone, silicone rubber, or an elastomer material.

  By providing such a seal portion 30, it is possible to easily inject and discharge liquid and gas to and from the culture cavity 4 using the syringe having a liquid or gas injection and discharge portion such as a needle through the seal portion 30. become. That is, it can function as a fluid pouring and discharging unit. As a result, gas injection and discharge operations such as medium filling, replacement, and washing can be easily performed aseptically, and the possibility of contamination can be reduced. Moreover, since culture | cultivation replacement | exchange operation etc. with a syringe can be performed without requiring a skillful technique, even a beginner can perform it easily. For example, as shown in FIG. 1, such a seal portion 30 can be provided for an opening communicating between the inside and the outside of the chamber 10. In addition, in order to make the said seal part 30 function as an injection | pouring / discharge part of the fluid which maintained the sealing performance, it is not limited to this seal structure, It can also carry out by well-known valve mechanisms, such as a valve and a cock.

  Although not shown in FIG. 1, the chamber 10 preferably includes a gas discharge unit. By providing the gas discharge part, when supplying the liquid to the culture cavity, the gas in the chamber 10 can be easily discharged with the introduction of the liquid, and the liquid can be easily introduced in a short time. become.

(Tissue piece clamping part)
The main culture apparatus 2 includes a tissue piece clamping unit 40. In FIG. 1, one surface (upper surface in the figure) is mesh-shaped to form a tissue piece pressing portion 52, and the opposite surface (lower surface in the figure) is a bag-shaped body that forms a culture surface 54. The formed tissue piece clamping part 40 is shown. The tissue piece sandwiching section 40 is disposed in the culture cavity 4 and encloses and holds the tissue piece in a predetermined position in the culture cavity 4 so that it can be cultured. Here, “being culturable” means a state in which a group of cells constituting a tissue piece can be proliferated by supplying medium components. Therefore, the tissue piece clamping part 40 is formed so that a culture medium component can be supplied to the tissue piece to be clamped. For example, it is preferable to have a plurality of communication holes in at least a part of the tissue piece sandwiching section 40. When a plurality of communication holes are provided, liquids such as a culture medium and a washing solution are sufficiently supplied to the tissue pieces in the tissue piece holding unit 40, and the circulation of these liquids is improved. Therefore, culture and washing can be efficiently performed. In addition, the tissue piece pressing portion 52 facing the culture surface 54 of the tissue piece sandwiching portion 40 may include a dot-shaped or linear convex portion. In this way, the area of contact with the tissue piece can be reduced for holding, and the tissue piece can be brought into close contact with the culture surface 54 and stimulation to the tissue piece can be suppressed. In addition, a dot-shaped convex part means the convex part which is disperse | distributed in the area | region and is provided independently, and has planar shape of various shapes, such as circular shape and a square shape. Moreover, a line-shaped convex part means the convex part which has a planar shape formed by a single line, a some line | wire parallel or crossing. Furthermore, it is preferable that at least a part of the tissue piece sandwiching portion 40 is a mesh-like body. When it is a mesh-like body, there are merits of both the supply of liquids such as a liquid medium and a cleaning liquid, the improvement of circulation, and the reduction of the contact area. Note that the hole diameter of the material constituting the tissue piece sandwiching section 40 and the opening diameter such as the mesh size are those when the culture cavity 4 is filled with a liquid before the tissue piece adheres to the culture surface 54. However, the size may be any size as long as the tissue piece can be kept in close contact with the culture surface 54, and can be changed depending on the size of the target tissue piece. For example, when the tissue piece is a 3 mm square periosteal tissue piece, it is preferably 1 mm or less.

  The tissue piece may be held in any form as long as it is included in the tissue piece holding part 40, that is, the tissue piece is held inside the tissue piece holding part 40 by holding it. The holding form for holding the tissue piece includes a form for holding the whole or a part of the tissue piece. In the form illustrated in FIG. 1, the tissue piece clamping unit 40 is configured to clamp the entire tissue piece. When a part of the tissue piece is sandwiched, for example, as shown in FIG. 4, it is preferable that the pressing portion 52 sandwich at least a part of the peripheral edge such as both end parts and one end part of the tissue piece. It should be noted that when sandwiching tissue pieces, in particular, when contacting and sandwiching the tissue piece itself, in a natural state where no force is applied, the tissue pieces are configured to contact each other with an appropriate pressure. For example, as shown in FIG. 1, when the tissue piece T to be cultured is sandwiched between the pressing portion 52 and the culture surface 54, the appropriate pressure referred to here is destroyed and adhered to the culture surface. This means that the pressure is not so strong that the state cannot be maintained or all the cells constituting the tissue piece T are killed.

  Although the material which comprises the tissue piece clamping part 40 is not specifically limited, Preferably a transparent material is used. According to this, it is possible to easily observe the state of tissue piece retention and the state of cell proliferation. In addition, regardless of the structure, that is, regardless of whether the material is porous or mesh-like, it is possible to adopt a material having liquid permeability. Moreover, it is preferable that at least the tissue piece contact portion of the pressing portion facing the culture surface 54 has a surface layer to which cells are difficult to adhere. Examples of such a surface layer include those made of polyethylene, polypropylene, fluororesin, polytetrafluoroethylene (PTFE), polycarbonate, polyester and the like, and those coated on the surface with agarose, hyaluronic acid, and the like. However, even these materials can be easily adhered by surface treatment (for example, rough surface treatment).

  On the other hand, the culture surface 54 preferably has a surface layer that imparts or improves adhesion so that cells adhere. Examples of such a surface layer include those made of polystyrene or the like, and those coated on the surface with collagen, fibrin or the like. However, even these materials can be made difficult to adhere by surface treatment (for example, mirror treatment).

  Moreover, it is preferable that the material which comprises the tissue piece clamping part 40 has flexibility itself. By forming in this way, the tissue piece clamping part 40 provided with the elastic deformation property can be formed. Thereby, the tissue piece holding property of the tissue piece clamping unit 40 and the opening / closing property of the tissue piece inserting / removing unit 60 can be easily secured. In addition, the elastic deformability of the tissue piece clamping part 40 can also be provided with the structure, and can also be provided with a material and a structure. In addition, by constituting a part or all of the tissue piece sandwiching portion 40 with a magnetic material, it is possible to sandwich the tissue piece by attracting the upper surface and the lower surface with a magnetic force.

(Tissue piece taking in / out part)
The tissue piece clamping unit 40 includes a tissue piece insertion / removal unit 60. The tissue piece take-out / insertion unit 60 is an opening for inserting a tissue piece inside the tissue piece holding unit 40 and taking out the cultured tissue from the site. The tissue piece insertion / extraction unit 60 may be always open, but is preferably formed to be openable and closable. The opening form and the closed form of the tissue piece taking-in / out part 60 are not particularly limited, and various settings can be made according to the form of the tissue piece or the tissue piece holding part 40. In the embodiment illustrated in FIG. 1, the tissue piece insertion / extraction unit 60 is integrated with the cavity opening / closing unit 20, and the opening / closing of the cavity opening / closing unit 20 and the opening / closing of the tissue piece insertion / extraction unit 60 are realized at the same time. . When the tissue piece insertion / extraction part 60 is opened, the holding state by the tissue piece holding part 40 is released, and when the tissue piece insertion / removal part 60 is closed, the holding state by the tissue piece holding part 40 is started. In this exemplary embodiment, the cavity opening / closing part 20, that is, the tissue piece taking-in / out part 60 is opened by elastic deformation and is closed by restoring the deformation.

  In the main culture apparatus 2, it is preferable that the tissue piece insertion / extraction unit 60 is released and started to be held by the tissue piece clamping unit 40 in accordance with opening and closing thereof. According to this embodiment, it is possible to easily hold and release the tissue piece accompanying the insertion and removal of the tissue piece from the tissue piece clamping unit 40. Furthermore, in this culturing apparatus 2, the tissue piece sandwiching section 40 can be connected or fixed so that the tissue piece inserting / removing section 60 is positioned at or near the cavity opening / closing section 20. According to this embodiment, it is possible to more easily insert / remove the tissue piece into / from the tissue piece holding part 40 in the culture cavity 4. In addition, the tissue piece insertion / extraction unit 60 can be opened / closed with the opening / closing of the cavity opening / closing unit 20 of the culture cavity 4. By doing so, the tissue piece can be taken in and out and the tissue piece can be released and started as the culture cavity 20 is opened and closed. Further, in these forms, the tissue piece taking-out / inserting part 60 can constitute a part or the whole of the cavity opening / closing part 20. This further facilitates the insertion and removal of the tissue piece from the culture cavity 4 and the tissue piece holding part 40.

  Furthermore, it is preferable that when the tissue piece taking-in / out part 60 is opened, almost the entire culture surface 54 of the tissue piece holding part 40 on the tissue piece taking-in / out part 60 side can be opened. If formed in this manner, there is no barrier to the work of carrying in / out the tissue piece, so that the tissue piece can be easily grasped and the carry-in / out itself can be carried out easily and without damaging the tissue piece. Further, in this embodiment, it is preferable that the culture cavity opening / closing part 20 is also configured so that substantially the whole of the culture surface 54 on the tissue piece taking-in / out part side can be opened. By doing so, it becomes possible to carry in and out the tissue piece more easily and safely. In the culturing apparatus 2 illustrated in FIG. 1 and the like, the tissue piece is taken in and out with the culturing apparatus 2 in a substantially horizontal state. In the culturing apparatus 2, the tissue piece taking in / out portion 60 and the cavity of the culturing apparatus 2 are in this state. When the opening / closing part 20 is formed on the side of the culture surface 54 and the tissue piece insertion / removal part 60 and the cavity opening / closing part 20 are opened, these openings are opened so as to open substantially the entire side of the culture surface 54. Is formed.

  Furthermore, it is preferable that the tissue piece insertion / extraction part 60 is at least a part of the cavity opening / closing part 20, and the cavity opening / closing part 20 includes the sealing part 6. In this case, the insertion and removal of the tissue piece, the opening and closing of the culture cavity, and the sealing of the culture cavity can be performed simultaneously or incidentally, and these operations can be performed promptly.

  The form of the tissue piece clamping part 40 including the tissue piece taking-in / out part 60 can take various forms depending on the various holding forms and the types of tissue pieces. For example, as shown in FIG. 1, the tissue piece sandwiching portion 40 includes a first sheet-like body 52 and a second sheet-like body 54, and the tissue piece is sandwiched and held between these sheet-like bodies. Can be. By making the tissue piece clamping part 40 into such a form, it is convenient to hold the tissue piece with a simple configuration. The tissue piece sandwiching section 40 can be configured by allowing the sheet-like bodies 52 and 54 to face each other close to each other or to overlap each other, and to form a tissue piece insertion / extraction section 60 having an appropriate opening shape. For example, a bag is formed by forming a part of the opposed sheet-like body as an opening and making the remaining peripheral edges closely contact each other, and forming the opening as a tissue piece insertion / removal part 60 and connecting it in the vicinity of the cavity opening / closing part 20. A tissue piece sandwiching section 40 is formed. According to this embodiment, the tissue piece can be reliably put in and out of the tissue piece holding portion 40 and can be held. On the other hand, each side of the opposing sheet-like body is held mutually, and each other side can be opened from each other, so that the one side can be opened and closed with the one side as a rotation fulcrum. It can function as the tissue piece taking-in / out part 60. In this case, since these side edges are not connected, not the bag-shaped tissue piece sandwiching portion 40 but the foldable or openable tissue piece sandwiching portion 40 is formed (not shown).

  Also, in the case of partial clamping shown in FIG. 4, the tissue piece insertion / extraction part 60 is opened and closed with the back side of the chamber 10 of the tissue piece clamping part 40 as the center of rotation, and this opening / closing is interlocked with the cavity opening / closing part 20. Can be opened and closed.

  When the chamber 10 is flexible, the tissue piece sandwiching portion 40 is configured to maintain the shape regardless of the deformation of the chamber 10, and even if the chamber 10 is deformed indefinitely, the tissue piece Can be securely sandwiched and the adhesion to the culture surface 54 can be maintained. Moreover, the tissue piece clamping part 40 can also be formed so that it can float within a certain range in the culture cavity 4 while maintaining its overall shape. For example, in each of the forms illustrated in FIGS. 1 to 4, since the tissue piece sandwiching portion 40 is only partially connected to the chamber 10, depending on the connected state, Can float in the presence of a liquid such as a medium. By doing so, it is possible to avoid the influence of deformation such as a change in the shape of the culture cavity and a pressure change in the culture cavity. In addition, it is not restricted to the connection form of FIGS. 1-4, The tissue piece clamping part 40 can be easily floated with the connection state with respect to the chamber 10 in the culture cavity 4 of the tissue piece clamping part 40. FIG.

  Further, when the chamber 10 has flexibility, the tissue piece sandwiching portion 40 can be formed to be deformable following the deformation form of the chamber 10. The tissue piece can be reliably held by changing the form of the tissue piece holding portion 40 by applying an external force to the chamber 10 and deforming it. For example, as shown in FIG. 5, the tissue piece sandwiching portion 40 is connected to the chamber 10 so as to be deformed following the deformation of the chamber 10. In this example, by bending the chamber 10, the tissue piece sandwiching portion 40 is also curved, and as a result, a curved shape applied to the tissue piece T and the cultured tissue T ′ sandwiched between the tissue piece sandwiching portions 40 can be provided. ing.

  Next, a process of culturing a sheet-like tissue piece T as an example of the tissue piece using the culture apparatus 2 will be described. First, as shown in FIG. 2 (a), the culture apparatus 2 is kept almost horizontal, and the cavity opening / closing part 20 is opened by applying a load from both ends of the cavity opening / closing part 20 to the center of the culture apparatus 2, At the same time, the tissue piece insertion / extraction part 60 is opened. With the release of the tissue piece insertion / removal unit 60, the tissue piece clamping unit 40 is released from the holding state, and as a result, the tissue piece T can be inserted and placed therein. After placing the tissue piece T on a predetermined portion of the exposed surface of the sheet-like body 54 (culture surface) of the tissue piece sandwiching section 40, the load applied to the cavity opening / closing section 20 is removed, and the cavity opening / closing section 20 is closed. As a result, as shown in FIG. 2 (b), the tissue piece insertion / removal part 60 and the tissue piece holding part 40 are also closed together, and the whole tissue piece T is held between the upper and lower sheet-like bodies 52, 54, A state of being in close contact with the culture surface 54 with an appropriate pressing force and being held in the tissue piece sandwiching section 40 is formed. Further, the culture cavity 4 is sealed by the sealing part 6 provided in the cavity opening / closing part 20.

  Next, the culture medium is injected into the culture apparatus 2. In the injection, a syringe 80 including a needle 82 and a piston 84 and a cylinder 86 is used as a pouring and discharging unit for a fluid such as a liquid. One form of the present invention is a culture kit in which the culture apparatus 2 and the syringe 80 are combined. As shown in FIG. 3 (a), after inserting and holding the tissue piece T in the culture apparatus 2 and sealing the chamber 10, the needle 82 of the syringe 80 containing the liquid medium M is entered into the seal part 30, The syringe 80 is temporarily fixed to the seal part 30. After the syringe 80 is fixed, the piston 84 is slid to inject the medium M in the cylinder 8 into the chamber 10 (culture cavity 4). After the injection of the medium M, the volume of the chamber 10 increases due to the injection of the medium M, but the pressure in the chamber 10 is relaxed and uniformed by the deformation of the chamber 10, and the The pressure is not applied more than necessary.

  Next, as shown in FIG. 3 (b), the seal portion 30 is disposed so that the gas in the chamber 10 gathers in the vicinity of the seal portion 30, and the piston 84 is slid upward to thereby move the chamber. The gas (air) in 10 is sucked into the cylinder 86. As a result, as shown in FIG. 3C, all the gas in the chamber 10 is sucked and the chamber 10 is filled with the medium M. At this time, the volume in the chamber 10 decreases as the gas is sucked. However, since the internal pressure is stabilized with the external pressure by the deformation of the chamber 10, the tissue piece T is not pressurized more than necessary. .

  When the injection of the medium M and the discharge of the gas are completed, the syringe 80 is removed from the seal portion 30. Even when the needle 82 of the syringe 80 is entered and removed, the sealing performance of the seal portion 30 is maintained, the sealing performance of the chamber 10 is maintained, and leakage of the liquid medium M can be prevented. The culture apparatus 2 into which the culture medium M has been injected is placed in a carbon dioxide incubator (not shown), and tissue piece culture is performed. The adjusted gas component in the carbon dioxide incubator is dissolved in the medium M in the chamber 10 via the gas permeable material constituting at least a part of the chamber 10 and supplied to the tissue piece T. The medium M can be supplied to the tissue piece T through the mesh-shaped pressing portion 52 and sufficient nutrient components are supplied. The method of standing in the carbon dioxide incubator is not particularly limited, but a hook may be provided in a part of the culture apparatus 2 and may be arranged so as to be suspended in the incubator. In this way, many culture apparatuses 2 can be cultured at once, and can be cultured in a small space.

  Further, in the above-described embodiment, it has been described that all the gas in the chamber 10 is exhausted. However, depending on the state of the culture apparatus 2 at the time of culture, the state where the tissue piece T is immersed in the medium is maintained. For example, the gas may remain in the chamber 10.

  It should be noted that the medium exchange can be easily performed if it is performed in accordance with the above-mentioned medium injection. Specifically, after injecting gas (air) into the chamber 10 via the syringe 80, the chamber 10 is arranged so that the seal portion 30 is located below, and the used medium M is sucked into the syringe 80. After removing the syringe 80 that has aspirated the spent medium M, the needle 82 of the syringe 80 containing the fresh liquid medium M is newly entered into the seal portion 30 and the medium M is injected into the chamber 10 as described above. Medium exchange is complete.

  After completion of the culture, the spent medium M is discharged as described above, and then the needle 82 of the syringe 80 containing the cleaning liquid is entered into the seal portion 30 to fill the chamber 10 with the cleaning liquid. As described above, the cleaning process can be easily performed in the chamber 10 by repeatedly injecting and discharging the cleaning liquid. After the washing step, with the washing liquid in the chamber 10 discharged, the sealing portion 6 is opened, and the cultured tissue T ′ is taken out from the tissue piece taking-in / out portion 60. At this time, the tissue piece insertion / removal unit 60 is opened in accordance with the opening of the cavity opening / closing unit 20, and the holding state of the cultured tissue T ′ by the tissue piece clamping unit 40 is released. The extracted cultured tissue T ′ is used for various purposes according to the purpose.

  In the above-described embodiment, the gas is also discharged using the seal portion 30, but the gas can be discharged separately in the gas discharge portion. If the gas discharge port is configured to automatically discharge the gas in the chamber 10 according to the pressure in the chamber 10, the gas in the chamber 10 is automatically discharged as the medium is injected. It is possible to shorten the working time in the process.

  In addition, the culture apparatus of this invention is not restricted to the form illustrated to FIGS. 1-5, The following forms are also included. That is, the culture surface of the tissue piece sandwiching portion is a form that constitutes a part of the chamber, and in particular, the chamber that forms a culture cavity that encloses the tissue piece in a culturable manner is constituted by a lid and a tray, A cavity opening / closing portion that opens and closes for loading / unloading the tissue piece into / from the culture cavity, and a tissue piece holding portion that holds the tissue piece at a predetermined position in the culture cavity, the tissue piece holding portion comprising: At least one may form a culture surface, and the other may be configured to press and hold the tissue piece in close contact with the culture surface. In this culturing apparatus, it is preferable that the tissue piece sandwiching portion is configured such that the culture surface is configured by the bottom surface of the tray and the other is provided on the lid. The culture apparatus having such a configuration will be briefly described with reference to FIG.

  In the culture device 100, a culture cavity 110 is formed by a lid 102 and a tray 104. The tissue piece sandwiching portion in this configuration includes a tissue piece pressing portion 106 provided on the lid 102 and a culture surface 108 on the bottom surface of the tray, and the tissue piece T is sandwiched so as to be in close contact with the culture surface 108. Thereby, a tissue piece can be easily fixed to a culture surface early and reliably, and a suitable culture environment can be formed by supplying a medium early. In addition, a plurality of protrusions can be provided on the culture surface 108 side of the tissue piece pressing portion 106, and the tissue piece T can be pierced with these protrusions to be securely held. The culture cavity opening / closing unit is configured by opening / closing the lid 102 and the tray 104, and the release and start of the clamping of the tissue piece T are performed in conjunction with the opening / closing of the culture cavity opening / closing unit.

  The culture apparatus is preferably formed with a culture cavity sealed by a lid and a tray. According to this, since the culture cavity is sealed, the possibility of contamination can be reduced. As a sealing means for obtaining a sealed structure, a known method such as screwing or adhesion can be used. In addition, when the culture cavity has a sealed structure, it is preferable to include the fluid injection / discharge section as described above. In particular, a structure that includes a seal portion that can be punctured and that can maintain a sealing property and that allows liquid and fluid to be taken in and out via a syringe having a puncture body is preferable. As a result, liquid and gas can be easily poured into and discharged from the culture cavity, and gas filling and discharging operations such as medium filling, replacement, and washing can be easily performed aseptically. The possibility can be reduced. Moreover, since culture | cultivation replacement | exchange operation etc. with a syringe can be performed without requiring a skillful technique, even a beginner can perform it easily.

  When the tissue piece pressing portion is provided on a chamber constituent member such as a lid, the tissue piece pressing portion is not interlocked with a chamber locking operation (for example, a lid rotating operation when the lid is fixed to the tray by screwing or fitting). It is preferable to configure. For example, when the locking operation is performed by rotating the lid, the tissue piece pressing portion is provided so as to be rotatable with respect to the lid, thereby preventing the tissue piece pressing portion from rotating with respect to the tray as the lid rotates. it can. By doing so, it is possible to prevent the tissue piece from being damaged by rotating the tissue piece as the lid rotates.

  FIG. 7 shows a modified example of the configuration of the tissue piece pressing portion in the culture apparatus 100 configured to be able to rotate and fix the lid 102 with respect to the tray 104. As shown in FIG. 7, the tissue piece pressing portion 106 includes a tissue piece contact portion 106a, and can include a shaft body portion 112 and a locking body 114 above the tissue piece contact portion 106a. The tissue piece pressing portion 106 is inserted through the shaft body portion 112 through a through-hole formed in the center of the lid 102 and is mounted so as to be movable up and down and rotatable within a predetermined range. The locking body 114 is locked and fixed to a step of a shallow concave portion formed on the outer periphery of the through hole. Thus, the tissue piece pressing portion 106 attached to the lid 102 is formed so that the upper end thereof does not protrude from the surface of the lid 102 even when the culture cavity is formed. According to such a configuration, the tissue piece pressing portion 106 is rotatably fixed to a through hole formed in the lid 102 within a predetermined range. Therefore, in order to lock the lid 102 with respect to the tray 104, Even when it rotates by a predetermined range, it does not rotate with the rotation of the lid 102. As a result, the tissue piece T can be fixed without rotating during the locking operation for sealing the culture cavity, and it is avoided that the tissue piece is damaged. Further, since the locking body 114 does not protrude upward from the surface of the lid 102, the culture apparatus 100 can be stacked. In this configuration, since the tissue piece pressing portion 106 is formed to be movable up and down within a predetermined range, a predetermined pressing force is exerted on the tissue piece T by the weight of the tissue piece pressing portion 106. In addition, as described later, the position of the tissue piece pressing portion 106 can be adjusted.

  Moreover, the tissue piece pressing part should just be what can press-fix to such an extent that a tissue piece can be closely_contact | adhered to a culture surface in the appropriate state, and a tissue piece can be cultured. Therefore, for example, in the case of a tissue piece composed of adhesion-dependent cells, these are brought into close contact with the culture surface and pressed to such an extent that the culture is not hindered. Such a pressing state can appropriately secure the distance between the tissue piece contact portion of the tissue piece pressing portion and the culture surface so that the fixed state can be realized. For this purpose, for example, an elastic body is attached to the end of the tissue piece contact portion of the tissue piece pressing portion toward the culture surface, and an appropriate distance between the tissue piece contact portion and the culture surface is set by the elasticity of the elastic body. It can also be secured. It is also possible to adjust the distance between the culture surface and the fixing member by forming the tissue piece pressing portion so as to be movable up and down within a predetermined range.

  In addition, although the tissue piece clamping part of the culture apparatus of this invention can also be provided in a chamber integrally like the form mentioned above, it can also be provided separately from a chamber. In this way, the tissue piece can be inserted into the chamber after the tissue piece is held outside the chamber at the tissue piece holding portion. At this time, the cavity opening / closing part of the culture cavity needs to be provided so that the tissue piece holding part holding the tissue piece can be carried in and out. As such a tissue piece clamping part, it can also be comprised from a sheet-like body as illustrated to FIGS. 1-5, and a tissue piece is pressed with respect to the plate which comprises a culture surface, and the said culture surface. For example, a pressing member such as a tissue pressing unit 106 illustrated in FIG. 6 can be used.

  In the culture apparatus as described above, when cultured using canine periosteum tissue pieces, cultured periosteum having tough strength could be produced. In addition, when this cultured periosteum was applied to a periodontal disease model dog as a GTR membrane, it was confirmed that periodontal tissue was regenerated after 16 weeks and periodontal disease symptoms were improved. did it.

1 is a schematic external view of a culture apparatus according to an embodiment. It is explanatory drawing explaining opening and closing of the tissue piece taking-in / out part and the tissue piece clamping part in the culture apparatus of embodiment. It is explanatory drawing regarding the culture medium injection | pouring method with respect to the culture apparatus of embodiment. It is a figure which shows another example of the tissue piece clamping part. It is explanatory drawing regarding the form which a tissue piece clamping part deform | transforms following the deformation | transformation of a chamber. It is the schematic which shows the modification example which concerns on the culture apparatus of this invention. It is a figure which shows the modification of the tissue piece press part which is an example of the tissue piece clamping part.

Explanation of symbols

  2,100 culture apparatus, 4,110 culture cavity, 10 chamber, 20 cavity opening / closing part, 30 seal part, 40 tissue piece clamping part, 52 pressing part (sheet-like body), 54 culture surface (sheet-like body), 60 tissue Single insertion / removal part, 80 syringe, 82 needle, 84 piston, 86 cylinder, 102 lid, 104 tray, 106 tissue piece pressing part, 108 culture surface, 106a tissue piece contact part, 112 shaft body part, 114 locking body.

Claims (12)

A culture apparatus for culturing a tissue piece,
A chamber which is bag-like body of flexible enclosing a culturable cultured cavity the tissue piece,
A cavity opening / closing section provided in the chamber, which opens and closes for loading and unloading of a tissue fragment into and from the culture cavity;
A tissue piece sandwiching portion disposed in the chamber and sandwiching the tissue piece at a predetermined position in the culture cavity;
With
The tissue piece sandwiching portion is a bag-like body separate from the chamber provided with the first sheet-like body and the second sheet-like body, and the first sheet-like body and the second sheet-like body One of the sheet-like bodies forms a culture surface, and the other presses and clamps the tissue piece so as to be in close contact with the culture surface. The opening of the part opens and closes, and the sandwich of the tissue piece between both sheet-like bodies is released or the tissue piece is sandwiched,
Culture device. The culture apparatus according to claim 1, wherein a plurality of communication holes are provided in at least a part of the tissue piece sandwiching portion. The culture apparatus according to claim 1 or 2 , wherein at least a part of the tissue piece sandwiching portion is a mesh-like body. The culture apparatus according to any one of claims 1 to 3 , wherein a surface of the tissue piece sandwiching portion facing the culture surface includes a dot-shaped or line-shaped convex portion. The cultures cavity, said being sealably formed in the cavity opening and closing unit, culture apparatus according to any one of claims 1-4. The culture apparatus according to any one of claims 1 to 5, wherein the tissue piece sandwiching portion has a rigidity that does not follow the deformation of the chamber. The culture apparatus according to any one of claims 1 to 6 , wherein the tissue piece sandwiching section is formed so as to float in a certain range in the culture cavity during culture. The culture apparatus according to any one of claims 1 to 7 , wherein the chamber includes a liquid take-in / out part. The culture apparatus according to any one of claims 1 to 8 , wherein the chamber includes a fluid injection / discharge section having a sealing material that can be punctured and maintain sealing performance. Wherein the chamber comprises a gas discharge portion, the culture device according to any one of claims 1-9. The chamber is provided with an oxygen and carbon dioxide permeability, culture device of any of claims 1-10. The culture apparatus according to any one of claims 1 to 11 , which is a periosteal tissue piece culture apparatus.

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