JP7330044B2 - Vessel for treatment of sheet-like cell culture with protrusions - Google Patents

Description

The present invention relates to a container for processing sheet-like cell cultures having projections.

In recent years, new regenerative medicine has been developed as a solution for treating severe heart failure. As an example, a method of applying a sheet-like cell culture prepared using a tissue-engineered temperature-responsive culture dish to the surface of the heart has been attempted for severe myocardial infarction and the like. Techniques using sheet-like cell cultures can safely transplant a large amount of cells over a wide area. , particularly useful in the treatment of heart disease (eg, heart failure, especially chronic heart failure) associated with systolic dysfunction (eg, left ventricular systolic dysfunction).

For clinical application of such a sheet-like cell culture, for example, the prepared sheet-like cell culture must be subjected to a treatment such as washing to bring it into a state where it can be applied to a subject. Such processing includes removing only the liquid from a vessel containing the sheet-shaped cell culture and the liquid. Methods for removing liquid include using a device such as a micropipette to aspirate the container, tilting the container to release the liquid from the rim of the container, or tilting the container to collect the liquid in the container. For example, a device is used to suck out the collected liquid (Patent Document 1). However, it is difficult to remove only the liquid from the container containing the sheet-shaped cell culture and the liquid, and the liquid often remains in the container. Moreover, since the absolute physical strength of the sheet-like cell culture is low, liquid remaining in the container often causes wrinkling, tearing, breakage, and the like.

JP-A-2006-149268

Under such circumstances, the present inventors found that when removing only the liquid from a container containing the sheet-shaped cell culture and the liquid, the operation of removing the liquid damages the sheet-shaped cell culture. After the removal operation, the sheet-like cell culture slides on the bottom surface of the container due to the liquid remaining in the container, causing wrinkles. Accordingly, an object of the present invention is to solve such problems and to efficiently remove only the liquid from a container in which the sheet-shaped cell culture and the liquid are accommodated, thereby providing a step of processing the sheet-shaped cell culture. , to provide a container for reducing the risk of wrinkling, tearing, and breakage.

In order to solve the above problems, the present inventors have conducted intensive research, and have found that the container has a convex portion that protrudes upward from the bottom surface, and the sheet-like cell culture is placed at a high position on the convex portion. Therefore, the container for use in treating the sheet-like cell culture, which allows excess liquid attached to the sheet-like cell culture to flow in a lower direction, efficiently attached to the sheet-like cell culture. The inventors have found that the excess liquid can be removed, and completed the present invention.

That is, the present invention relates to the following.
[1] A container for use in processing a sheet-like cell culture,
Having a convex portion protruding upward from the bottom surface of the container,
The container, wherein the sheet-shaped cell culture is placed at a high position on the convex portion, thereby allowing excess liquid adhering to the sheet-shaped cell culture to flow in a lower direction.
[2] The container according to [1], wherein at least the portion of the projection on which the sheet-like cell culture is placed has a dome shape.
[3] The container according to [1] or [2], wherein an annular space is formed between the projection and the inner surface of the container.

[4] The container according to any one of [1] to [3], wherein the protrusion has one or more pillars outside the portion on which the sheet-like cell culture is placed.
[5] The container according to any one of [1] to [4], wherein at least a portion of the protrusions and/or the pillars has elasticity.
[6] The container according to any one of [1] to [5], including a cutting die whose size can be adjusted.

[7] A container for use in processing a sheet-like cell culture,
Having a convex portion protruding upward from the bottom surface of the container,
a mounting portion for mounting the sheet-like cell culture on the convex portion;
The container having a liquid-retaining space surrounding the mounting portion.
[8] A mounting portion is provided at a high position of the convex portion, and by mounting the sheet-shaped cell culture on the mounting portion, excess liquid adhering to the sheet-shaped cell culture flows in a lower direction. The container of [7], which is capable of
[9] A kit comprising the container according to any one of [1] to [8] and an outer container for housing the container.

[10] A method for processing a sheet-like cell culture, comprising:
(a) placing the sheet-like cell culture on a high position of the convex portion of a container having a convex portion that protrudes upward from the bottom;
(d) maintaining the sheet-like cell culture at a high position on the convex part, and causing excess liquid adhering to the sheet-like cell culture to flow in a lower direction;
The above method, comprising
[11] After step (a) and before step (d),
(b) washing the sheet-like cell culture by adding a wash solution until the sheet-like cell culture is submerged;
(c) removing the washing solution;
The method of [10], further comprising

[12] The method of [11], wherein the washing solution is added and/or removed from the outside of the portion on which the sheet-like cell culture is placed.
[13] After step (d),
(e) laminating a polymer-gelable compound on the sheet-shaped cell culture to produce a laminate;
The method according to any one of [10] to [12], further comprising
[14] After step (e),
(f) cutting out a portion of the laminate containing the sheet-like cell culture;
The method of [13], further comprising
[15] A sheet-like cell culture treated according to the method of any one of [10] to [14].
[16] A method of treating a disease ameliorated by applying a sheet-like cell culture, comprising applying an effective amount of the sheet-like cell culture according to [15] to a subject in need thereof. The method above.

According to the container of the present invention, excess liquid adhering to the sheet-like cell culture can be easily removed from the sheet-like cell culture. Therefore, it is possible to reduce the risk of wrinkles, breakage, and tearing without moving a pipette or the like close to the sheet-like cell culture in order to remove the liquid adhering to the surface of the sheet-like cell culture. In addition, by using the container of the present invention, the sheet-like cell culture can be in a stretched state without wrinkles. It can be applied in a wrinkle-free state even when applied to. Furthermore, since the liquid adhering to the sheet-like cell culture of the present invention and the liquid used for washing can be easily removed from one place, operations such as the washing step of the sheet-like cell culture are facilitated. be able to.

In addition, according to the container of the present invention, the position of the sheet-like cell culture or laminate can be fixed. Therefore, it is possible to reduce the risk of wrinkling, tearing, and damage due to displacement of the sheet-like cell culture or laminate, and also facilitates cutting out of the laminate in the step of cutting out the laminate.

FIG. 1 shows a container according to a first embodiment of the invention. FIG. 1A shows a top view of the container and FIG. 1B shows a cross-sectional view of the container. Figure 2 shows a container according to a second embodiment of the invention. FIG. 2A shows a top view of the container and FIG. 2B shows a cross-sectional view of the container.

According to one aspect of the present invention, the container has a convex portion protruding upward from the bottom surface of the container, and the sheet-like cell culture is placed on a high position of the convex portion, so that the sheet-like cell culture is attached to the sheet-like cell culture. It relates to a container for use in processing sheet-like cell cultures, which allows a liquid to flow downward.

In the present invention, the term "sheet-like cell culture" refers to a sheet-like cell culture formed by connecting cells to each other. Cells may be connected to each other directly (including through cellular elements such as adhesion molecules) and/or through intermediaries. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include extracellular matrix. The mediator is preferably of cell origin, in particular of cells that make up the cell culture. The cells are at least physically (mechanically) linked, but may also be functionally linked, eg chemically, electrically. The sheet-like cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminated (multilayered) body, e.g., two layers, three layers, 4 layers, 5 layers, 6 layers, etc.). Moreover, the sheet-like cell culture may have a three-dimensional structure in which the cells do not exhibit a clear layered structure and have a thickness exceeding the thickness of a single cell. For example, in a vertical cross section of a sheet-like cell culture, the cells may be non-uniformly arranged (for example, in a mosaic pattern) without being evenly aligned in the horizontal direction.

The sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells onto and/or into their surfaces and maintain the physical integrity of sheet-like cell cultures, such as polyvinylidene difluoride ( PVDF) membranes and the like are known, but the sheet cell culture of the present disclosure can maintain its physical integrity without such a scaffold. Moreover, the sheet-like cell culture of the present disclosure preferably consists only of substances derived from the cells that constitute the sheet-like cell culture, and does not contain other substances.

Cells constituting the sheet-like cell culture are not particularly limited as long as they can form the sheet-like cell culture, and include, for example, adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (e.g., myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) and so on. Somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells). Non-limiting examples of cells that make up the sheet-like cell culture include myoblasts (e.g., skeletal myoblasts), mesenchymal stem cells (e.g., bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, umbilical cord blood-derived, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synoviocytes, chondrocytes, epithelial cells (e.g., oral mucosal epithelial cells) , retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., liver parenchymal cells, etc.), pancreatic cells (e.g., pancreatic islet cells, etc.), renal cells, adrenal cells , periodontal ligament cells, gingival cells, periosteal cells, skin cells, and the like. Non-limiting examples of iPS cell-derived adhesive cells include iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, and periosteal cells. , skin cells, synovial cells, chondrocytes, and the like.

The cells that make up the sheet-like cell culture can be derived from any organism that can be treated with the sheet-like cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (such as mice, rats, hamsters, guinea pigs, etc.), rabbits, and the like. is included. In addition, the number of types of cells constituting the sheet-like cell culture is not particularly limited, and it may be composed of only one type of cell, or may be composed of two or more types of cells. When there are two or more types of cells that form a sheet-like cell culture, the content ratio (purity) of the largest number of cells is 50% or more, preferably 60% or more, more preferably 60% or more at the end of formation of the sheet-like cell culture. is 70% or more, more preferably 75% or more.

Cells may be heterologous or allogeneic. As used herein, "heterologous cells" refer to cells derived from organisms of a species different from that of the recipient when sheet-like cell cultures are used for transplantation. For example, when the recipient is a human, heterologous cells include cells derived from monkeys or pigs. Also, "allogeneic cells" refer to cells derived from the same species of organism as the recipient. For example, if the recipient is human, human cells are allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous or autologous cells), ie cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not result in rejection upon transplantation. However, it is also possible to use xenogenic or allogeneic non-autologous cells. Immunosuppressive treatment may be required to prevent rejection when xenogeneic or allogeneic non-autologous cells are used. In this specification, cells other than autologous cells, that is, heterologous cells and allogeneic non-autologous cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cells are autologous or allogeneic. In one aspect of the disclosure, the cells are autologous cells. In another aspect of the disclosure, the cell is an allogeneic cell.

The sheet-like cell culture can be produced by any known method (see, for example, JP-A-2007-528755, International Publication No. 2017/13802, JP-A-2010-081829, JP-A-2011-110368, etc.). A method for producing a sheet-like cell culture typically includes the steps of seeding cells on a culture substrate, forming a sheet from the seeded cells, and peeling the formed sheet-like cell culture from the culture substrate. Including but not limited to steps. The step of seeding the cells onto the culture substrate may be preceded by steps of freezing the cells and thawing the cells. Additionally, a step of washing the cells may be performed after the step of thawing the cells. Each of these steps can be performed by any known method suitable for producing a sheet-like cell culture.

In the present invention, the term “treatment of sheet-like cell culture” refers to an operation for changing the state of the sheet-like cell culture from the time the sheet-like cell culture is formed until the sheet-like cell culture is provided to a subject. point to Such manipulations include any manipulation that requires the removal of excess liquid attached to the sheet-like cell culture, such as, but not limited to, washing of the sheet-like cell culture, lamination including sheet-like cell cultures. The production of the body and the cutting of the laminate are included.
In the present invention, "removing excess liquid adhering to the sheet-like cell culture" refers to removing liquid that is unnecessary for the formation of the sheet-like cell culture, but is not limited to , removing the liquid soaking the sheet-shaped cell culture in a washing process or the like, and removing the liquid adhering to the sheet-shaped cell culture after removing the soaking liquid. In the present invention, removing excess liquid adhering to the sheet-like cell culture does not include removing water contained in the composition of the sheet-like cell culture.
In the present invention, the liquid includes all liquids used for treatment of sheet-like cell cultures, but is not limited to these, for example, medium, washing solution, buffer solution, thrombin solution, fibrinogen solution, and other compounds capable of polymer gelation. and their effluents. These are sometimes collectively called liquid.

In the present invention, the "container" is not particularly limited as long as it can contain a sheet-like cell culture, liquid, etc. and does not leak liquid, and any container including commercially available containers can be used. . Materials for the container include, for example, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethylacrylamide, metal ( Examples include, but are not limited to, iron, stainless steel, aluminum, copper, and brass. In addition, the container preferably has a bottom surface large enough to process the sheet-like cell culture inside. Examples thereof include, but are not limited to petri dishes, cell culture dishes, and cell culture bottles. The area of the bottom of the container is typically 1.13-78.5 cm ² , preferably 12.6-78.5 cm ² , more preferably 9.1-60.8 cm ² .

In the present invention, the term "protrusion" refers to a portion that protrudes upward from the bottom surface of the container and creates a height difference with respect to the bottom surface of the container. In the present invention, the portion of the projection on which the sheet-like cell culture is placed is referred to as the top portion (placement portion). The top has sufficient area for placing a sheet-like cell culture. The top part may have any shape, and may be flat, as long as the sheet-like cell culture can be placed thereon. In one aspect, the top is gently sloping. Here, the term "gently inclined" means that when the sheet-shaped cell culture is placed on the inclined top, the self weight of the sheet-shaped cell culture causes frictional force between the sheet-shaped cell culture and the top, etc. It means that the sheet-like cell culture is slanted to the extent that the sheet-like cell culture does not slide down overcoming the force of stopping at the top of the plate. Since the top portion is gently slanted, the liquid adhering to the sheet-like cell culture gently flows downward along the slope. Therefore, the adhering liquid can easily flow along the slope, and the liquid adhering to the sheet-like cell culture portion can be removed more efficiently. In particular, the gentle slope of the top also allows removal of liquid present between the sheet-like cell culture and the surface of the vessel. In one aspect, the top is gently rounded. Here, "gently rounded" means that the placed sheet-like cell culture is rounded to the extent that the shape of the apex does not damage it. The top part can be gently rounded to provide a gentle slope so as not to damage the sheet-like cell culture.

In one aspect of the invention, the shape of the apex is at least partially domed. Here, the dome-like shape means that the container has a curved surface that has a vertex at the highest position and gently slopes from the vertex toward the outside of the container. In other words, it indicates that the height gradually decreases from the top toward the outside of the container. The position of the sheet-shaped cell culture placed at this time is determined by being caught by the vertex. Furthermore, the sheet-like cell culture caught on the vertex is pulled by its own weight and stretched along the gently sloping curved surface, leaving no wrinkles. In this embodiment, when the sheet-like cell culture is at least partially placed on the dome-shaped top, only excess liquid adhering to the sheet-like cell culture is low along the inclined curved surface of the top. It flows down into position.

In the present invention, the material for the protrusion is not particularly limited as long as the sheet-like cell culture can be placed on the top of the protrusion, and for example, the same material as the container can be used. In one aspect of the present invention, the convex portion may be integrally molded with the container.
In one aspect of the present invention, the convex portion is made of an elastic material. Materials having elasticity include, but are not limited to, natural rubber, synthetic rubber (e.g., isoprene rubber, butadiene rubber, styrene-butadiene rubber, chloroprene rubber, nitrile rubber, butyl rubber, ethylene propylene rubber, chlorosulfone rubbers (especially vulcanized ones) such as polyethylene, fluororubber, silicone rubber such as polydimethylsiloxane, etc., thermoplastic elastomers, and the like. In this aspect, the convex portion distorts its shape by being pushed. Therefore, for example, when recovering a sheet-shaped cell culture placed on a convex portion, the shape of the convex portion is distorted by pushing a spatula or tweezers, and the sheet-shaped cell culture and the convex portion are penetrated. Therefore, the sheet-like cell culture can be easily collected with a spatula or tweezers.

In one aspect of the present invention, the container forms an annular space between the projection and the inner surface of the container. Here, the annular space may have any shape as long as it is a continuous space surrounding the top. In other words, any liquid that adheres to the sheet-like cell culture flows down into one annular space. Here, the liquid that has flowed down is retained in the annular space. Therefore, the surrounding environment is not polluted by the falling liquid. Similarly, when liquid is added to the container during operations such as washing, the added liquid flows down into one annular space. Since the liquid that has flowed down is retained in the annular space, it is possible to easily recover the liquid therefrom.

In one aspect of the present invention, the protrusion has one or more pillars outside the top. In one aspect, the posts are spaced a width apart to allow liquid addition and withdrawal operations between the posts and the inner surface of the container. In one aspect, the pillars are at a distance from the top that allows the laminate to be cut out, leaving a margin in the gel portion.
In the present invention, the pillar refers to a portion protruding from the convex portion to the outside of the top portion. The pillar slows down the liquid flow when the liquid is filled into the container or when the liquid is removed from the container with a pipette, etc., and the risk of wrinkling, breaking, or tearing of the sheet-like cell culture due to the liquid flow is further reduced. make it possible to reduce In addition, when the laminate is produced, the columns are involved and the polymer compound is gelled, thereby fixing the gel layer in the laminate and making it easier to cut out the laminate. and

In the present invention, the material of the column is not particularly limited as long as it does not adversely affect the processing of the sheet-like cell culture, and for example, the same material as the container can be used. In one aspect of the present invention, the column may be integrally molded with the projection.
In one aspect of the invention, the post is constructed from a resilient material. Materials having elasticity include, but are not limited to, natural rubber, synthetic rubber (e.g., isoprene rubber, butadiene rubber, styrene-butadiene rubber, chloroprene rubber, nitrile rubber, butyl rubber, ethylene propylene rubber, chlorosulfone rubbers (especially vulcanized ones) such as polyethylene, fluororubber, silicone rubber such as polydimethylsiloxane, etc., thermoplastic elastomers, and the like. In this aspect, the post can be distorted and bent in shape by pushing. Therefore, for example, when collecting a sheet-shaped cell culture placed on a convex portion, pushing in a spatula or tweezers bends the pillar, and does not prevent the collection of the sheet-shaped cell culture by the spatula or tweezers. A sheet-like cell culture can be easily recovered.

Another aspect of the present invention is a sheet-like container having a protrusion on the bottom surface of the container that can remove excess liquid adhering to the sheet-like cell culture by placing the sheet-like cell culture. The present invention relates to a kit comprising a container for use in treating cell cultures and an outer container for housing the container.
In one aspect, the outer container may have a structure that can be sealed. In one aspect, the outer container may have a structure capable of maintaining sterility inside. In one aspect, the outer container is sealed and the container containing the sheet-like cell culture is contaminated in the common area by aseptically maintaining the container containing the sheet-like cell culture contained therein in the common area. It is possible to bring it into the highly clean area of the operating room without any effort.

The kit in the present invention may further include, but is not limited to, for example, culture medium, washing solution, buffer solution, equipment used for cell culture, transport container, instructions on usage, and the like.
Instruments used for cell culture include, for example, pipettes, cell strainers, and cell scrapers.
Instructions on usage include, for example, instruction manuals, media recording information on manufacturing and usage, such as flexible discs, CDs, DVDs, Blu-ray discs, memory cards, and USB memories.

Still another aspect of the present invention includes (a) placing a sheet-like cell culture on a high position of the convex portion of a container having a convex portion protruding upward from the bottom surface, (d) the sheet-like cell culture at a raised position and allowing excess liquid adhering to the sheet-like cell culture to flow downward.
In the present invention, the sheet-shaped cell culture is placed at a high position on the convex portion of the container, and by maintaining that state, excess liquid adhering to the surface of the sheet-shaped cell culture is eliminated by the difference in height of the convex portion. , it flows down to the lower position without any other operation. In one aspect of the invention, the container has, in addition to the protrusion, one or more pillars outside the top of the protrusion. In one aspect of the invention, the protrusion and/or the one or more posts are elastic.

In one aspect of the present invention, the method comprises, after (a) and before step (d), (b) adding a wash solution until the sheet-like cell culture is submerged; (c) removing the washing solution. In the present invention, the washing solution is not particularly limited as long as it does not adversely affect the sheet-like cell culture, and examples thereof include Hank's Balanced Salt Buffer, HEPES, DMEM, RPMI and Ham's F-12. .
In one aspect of the invention, the addition and/or removal of the cleaning liquid is performed from outside the top of the protrusion. Preferably, the addition and/or removal of the cleaning liquid is done from the outside of any one of the pillars of the projection. More preferably, the washing solution is added gradually from the outside of any one of the columns until the sheet-like cell culture is submerged, and the washing solution is removed from the outside of any one of the columns until the sheet-like cell culture is submerged. This is done gradually until there is no liquid left around the culture.

In one aspect of the present invention, the method further comprises, after step (d), the step of (e) layering a polymeric gellable compound on the sheet-like cell culture to create a laminate. In the present invention, any compound capable of polymer gelation can be used as long as it does not adversely affect the living body. Gels formed by such compounds include, but are not limited to, fibrin gels, gelatin, collagen and sodium alginate. In one embodiment, gels formed by such compounds are bioerodible gels, and are degraded in vivo, absorbed into the body, metabolized, and excreted.
In one embodiment, gels formed by such compounds are gelled by mixing two liquids. In one aspect, the gel is in a solidified state around room temperature. In one aspect, the gel does not exhibit reversibility with temperature. Examples of such gel include, but are not limited to, fibrin gel obtained by mixing fibrinogen solution and thrombin solution, anti-adhesion agent obtained by mixing NHS-modified CM dextrin and trehalose aqueous solution with sodium carbonate or sodium bicarbonate aqueous solution, and the like. is mentioned. These are commercially available, for example, as Volheal (registered trademark) for tissue adhesion (manufactured by Teijin Pharma Limited), Beliplast (registered trademark) for tissue adhesion (manufactured by CSL Behring), and Adspray (registered trademark) (manufactured by Terumo Corporation). It is possible and can be used in the present invention. In the present invention, the gel is preferably fibrin gel.

In one aspect of the present invention, the method further comprises, after step (e), the step of (f) cutting out a portion of the laminate containing the sheet-like cell culture. In one embodiment, cutting out from the laminate is performed by fixing a portion of the laminate with tweezers and using a scalpel or scissors. In one aspect, cutting out from the laminate is performed by pressing against the laminate using a standard cutting die. In one embodiment, the size of the cutting die can be adjusted as appropriate, and the cutting range can be determined according to the size of the sheet-like cell culture. Examples of the cutting die whose size can be adjusted as appropriate include a ring-shaped cutting die that includes an adjuster for adjusting the diameter of the ring.

Another aspect of the invention relates to sheet-like cell cultures treated according to the method of the invention.

Another aspect of the present invention is a method of treating a disease ameliorated by the application of a sheet-like cell culture, comprising an effective amount of the sheet-like cell culture treated by the method of the present invention. (hereinafter sometimes referred to as "the treatment method of the present invention").

The treatment method of the present invention may further comprise treating the sheet-like cell culture according to the treatment method of the present invention. The treatment method of the present invention may further comprise, before the step of producing the sheet-like cell culture, the step of collecting cells for producing the sheet-like cell culture or a tissue serving as a source of the cells from the subject. . In one embodiment, the subject from which cells or tissue as a source of cells is collected is the same individual as the subject to which the sheet-like cell culture or composition or the like is administered. In another embodiment, the subject from whom the cells or tissue from which the cells are sourced is a separate individual of the same species as the subject to whom the sheet-like cell culture or composition or the like is administered. In another embodiment, the subject from whom cells or tissue as a source of cells is collected is an individual of a different species than the subject to whom the sheet-like cell culture or composition or the like is administered.

In the present invention, the term "subject" means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present invention, the subject may be healthy or suffer from some disease, but the disease ameliorated by the application of the sheet-like cell culture (e.g., disease related to tissue abnormality, etc.) When treatment of is contemplated, it typically refers to a subject having or at risk of having the disease.

The term "treatment" is also intended to encompass all types of medically acceptable prophylactic and/or therapeutic interventions aimed at curing, temporarily relieving or preventing disease. For example, the term "treatment" includes delay or cessation of progression of diseases (e.g., diseases associated with tissue abnormalities) ameliorated by application of sheet-like cell cultures, regression or disappearance of lesions, and prevention of disease onset. It encompasses medically acceptable interventions for various purposes, including prophylaxis or prevention of recurrence.

In the present invention, an effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, alleviating symptoms, or delaying or stopping progression (e.g., size, weight, number of sheet-like cell cultures, etc.). and is preferably an amount that prevents the onset and recurrence of the disease or cures the disease. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such an amount can be appropriately determined by, for example, testing in experimental animals such as mice, rats, dogs or pigs, or disease model animals, and such testing methods are well known to those skilled in the art. Also, the size of tissue lesions to be treated can be an important indicator for determining the effective dose.

Application methods typically include direct application to tissue. The application frequency is typically once per treatment, but multiple sheets may be applied, such as when the desired effect is not achieved. For direct application to tissue, for example, a method of applying the sheet-like cell culture or the like of the present invention to a diseased site of tissue is used.

Tissues to be treated (applicable tissues) include, but are not limited to, heart (myocardium), cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gums, kidneys, thyroid, skeletal muscle, middle ear and the like. In addition, the diseases to be treated include, but are not limited to, heart diseases (e.g., myocardial injury (myocardial infarction, cardiac trauma, etc.), cardiomyopathy (ischemic cardiomyopathy, dilated cardiomyopathy, diastolic cardiomyopathy, diastolic cardiomyopathy, large cardiomyopathy, etc.), corneal diseases (e.g., corneal epithelial stem cell exhaustion, corneal damage (thermal/chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scarring, Stevens-Johnson syndrome, ocular pemphigoid) etc.), retinal diseases (e.g., retinitis pigmentosa, age-related macular degeneration, etc.), esophageal diseases (e.g., prevention of esophageal inflammation/stenosis after esophageal surgery (esophageal cancer removal), etc.), skin diseases (e.g., Skin injuries (trauma, burns, etc.), joint diseases (e.g., osteoarthritis, etc.), cartilage diseases (e.g., cartilage damage, etc.), liver diseases (e.g., chronic liver diseases, etc.), pancreatic diseases (e.g., diabetes, etc.) ), dental disease (e.g., periodontal disease), kidney disease (e.g., renal failure, renal anemia, renal osteodystrophy, etc.), thyroid disease (e.g., hypothyroidism, etc.), muscle disease (e.g., , muscle injury, myositis, etc.), middle ear diseases (eg, otitis media, etc.).

When the applied tissue is an organ such as the heart, and when the applied site moves frequently due to pulsation, peristalsis, etc., the sheet-like cell culture, etc. of the present disclosure may fall off from the applied site simply by pasting. be. Therefore, in a preferred embodiment of the treatment method of the present invention, after applying the sheet-like cell culture or the like of the present invention to a disease site, the sheet-like cell culture or the like of the present disclosure is sutured and fixed by passing a suture needle or the like through the penetration site. is included. This can prevent the sheet-like cell culture or the like of the present disclosure applied to the diseased site from falling off from the applied site.

Preferred embodiments of the present invention will be described in detail below with reference to the drawings.
[First embodiment]
First, a first embodiment of the present invention will be described.
FIG. 1 is a top view and cross-sectional view of a container according to a first embodiment of the present invention. In addition, in each drawing in the present disclosure, the size of each member is appropriately emphasized for ease of explanation, and each member shown does not indicate the actual size.

As shown in FIG. 1, the container 1 according to the first embodiment of the present invention has a convex portion 2 protruding upward from the bottom surface of the container, and a sheet-like cell culture 3 is placed on a high position of the convex portion 2. By placing it, excess liquid adhering to the sheet-shaped cell culture can be made to flow downward. The container 1 is a general-purpose petri dish-like container having a convex portion at the center of the bottom surface, and constitutes an accommodation space capable of accommodating a sheet-like cell culture, a liquid, and the like. The convex portion 2 has a top portion 21 on which the sheet-like cell culture is placed. Here, the top portion 21 has a dome-like shape, the surface of which is gently rounded, and gently slopes from the highest portion of the top portion 22 toward the outside of the container. are doing. In this embodiment, the convex portion 2 is positioned at the center of the bottom surface of the container, and an annular space is formed between the top portion 21 and the container. Therefore, the liquid flowing down from the sheet-shaped cell culture 3 can be communicated and collected from one place.

Next, a usage example of the container of this embodiment will be shown.
(1) Removal of excess liquid adhering to sheet-shaped cell culture In this embodiment, first, the sheet-shaped cell culture 3 prepared in a separate container is placed on the top of the convex portion 2 of the container according to this embodiment. 21. Although the sheet-like cell culture 3 is placed in the center of the protrusion in FIG. 1, it is not limited to this. In order to hook the sheet-shaped cell culture to the vertex 22, it is preferably placed so that a part of the sheet-shaped cell culture is positioned on the vertex 22. At this time, excess liquid attached to the sheet-like cell culture in another container flows down from the top 21 to a lower position of the container along the curved surface inclined toward the outside of the container, and thus the sheet-like Excess liquid adhering to the cell culture can be removed. Furthermore, by placing the sheet-shaped cell culture 3 so that a portion of the sheet-shaped cell culture 3 is positioned at the vertex 22 , the sheet-shaped cell culture 3 stretches under its own weight while being caught by the vertex 22 . Wrinkles are stretched along the inclined curved surface.

(2) Washing sheet-like cell culture Sheet-like cell culture can be washed using the container according to the present embodiment. , The user places the sheet-like cell culture 3 on the top 21 of the projection in the same manner as in (1) above, and then gradually adds the washing liquid into the container. When the liquid level rises to the extent that the sheet-like cell culture 3 is immersed, the components adhering to the sheet-like cell culture 3 are washed away with the washing liquid. Then, the washing liquid is gradually drawn out from the container. At this time, the washing liquid may be added and/or removed from the outside of the top to minimize the influence of the liquid flow generated when adding and/or removing the washing liquid to the sheet-like cell culture. Here, since the outer side of the top portion 21 is formed with an annular space through which the liquid can communicate, the liquid can be collected from one place and removed from the container. At this time, the sheet-like cell culture 3 is stopped by being caught by the vertices 22 of the projections 2, and the washing liquid containing the components adhering to the sheet-like cell culture 3 is removed. Furthermore, since the cleaning liquid adhering to the sheet-like cell culture 3 also flows down along the inclined curved surface toward the outside of the container from the vertex 21, it is possible to prevent the sheet-like cell culture 3 from being brought close to instruments such as pipettes. Cleaning liquid can be removed from the outside of the top 21 . The sheet-like cell culture is washed by performing these operations one or more times. Even if the sheet-like cell culture 3 is slightly wrinkled when immersed in the washing solution, the wrinkles are stretched out along the inclined curved surface of the top 21 by its own weight when it is caught on the top 21 of the protrusion 2. go.

(3) Production of a laminate of a sheet-like cell culture and a polymer-gelable compound In addition, using the container according to the present embodiment, a polymer-gelable compound is placed on the sheet-like cell culture 3. can be laminated to produce a laminate. The user places the sheet-like cell culture 3 on the top portion 21 of the convex portion, and then adds a polymer-gelable compound onto the sheet-like cell culture 3 . At this time, as described above, excess liquid adhering to the sheet-shaped cell culture 3 flows down to a lower position of the container, so that such liquid does not react with the compound to which it is added. The concentration of the compound is not diluted by the liquid. In addition, since the top part 21 on which the sheet-like cell culture 3 is placed is gently inclined with the vertex 22 as the highest position, by adding the viscous compound from the vertex 22, the tilted It gently spreads over the entire sheet-like cell culture 3 along the curved surface.

(4) Cut-out of Laminate Further, the container according to the present embodiment can be used to cut out a laminate produced from the sheet-like cell culture 3 and a compound capable of polymer gelation. After preparing the laminate of the sheet-like cell culture 3 and the compound capable of polymer gelation as described above, excess liquid adhering to the sheet-like cell culture 3 flows down, especially the top 21 Since the excess liquid between the sheet-shaped cell culture and the container flows down due to the inclination, the sheet-shaped cell culture 3 slips on the liquid during the cutting operation, and is difficult to be pushed out. becomes easy to cut out. Furthermore, by hooking the sheet-like cell culture 3 on the vertex 22, the position of the sheet-like cell culture 3 is fixed more firmly, and the cutting operation can be performed more easily. In addition, even if a washing operation is further performed to wash away the ungelled compound after preparing the laminate, the washing solution contains the sheet-like cell culture of the laminate as described above in (2). Since it does not remain in the part, it does not affect the cutting work.

[Second embodiment]
Next, a second embodiment of the invention will be described.
FIG. 2 is a top view and cross-sectional view of a container according to a second embodiment of the present invention. In addition, in each drawing in the present disclosure, the size of each member is appropriately emphasized for ease of explanation, and each member shown does not indicate the actual size.
In addition, in the figure, the same reference numerals are given to the same configurations as the configuration of the container 1 according to the first embodiment, and the differences from the first embodiment will be described in detail below. , the description is omitted.

As shown in FIG. 2, the container 1 according to the second embodiment of the present invention further includes columns 4 outside the portion on which the sheet-like cell culture 3 is placed.

Next, a usage example of the container of this embodiment will be shown.
(1) Washing sheet-like cell culture Sheet-like cell culture can be washed using the container according to the present embodiment. As described in (1) of the first embodiment, the user places the sheet-like cell culture 3 on the top 21 of the convex portion, and then gradually adds the washing liquid from the outside of the column 4. . Here, the impetus due to the flow when adding the liquid is loosened by the column 4 . When the liquid level rises to the extent that the sheet-like cell culture 3 is immersed, the washing liquid is gradually removed from the outside of the columns 4 . By these operations, the sheet-like cell culture 3 is washed as described in (2) of the first embodiment.

(2) Preparation of a laminate of a sheet-like cell culture and a polymer-gelable compound In addition, using the container according to the present embodiment, the sheet-like cell culture 3 and a polymer-gelable compound can be A laminate can be produced by The method of producing the laminate is as described in the first embodiment (3), but in this embodiment, the compound capable of polymer gelation gels in such a manner that the pillars 4 are involved.
(3) Cutout of Laminate Furthermore, the container according to the present embodiment can be used to cut out a laminate produced from the sheet-like cell culture 3 and the gel. Since the gel portion of the laminate is gelled in such a manner as to involve the columns, the position of the sheet-like cell culture 3 is further fixed by the gel portion than at the time of cutting out in the first embodiment, and it is not pushed out by the cutting operation. , the clipping operation can be performed more easily. In addition, since the sheet-like cell culture 3 does not move due to the cutting operation, it is also possible to cut out the laminate by pressing a cutting die. For example, the size of the cutting die can be freely set, and cutting can be performed according to the size of the sheet-like cell culture.

Description of Reference Symbols 1 Vessel 2 Convex part 21 Top part 22 Vertex 3 Sheet-like cell culture 4 Column

Claims (13)

A container for use in processing a sheet-like cell culture,
Having a convex portion protruding upward from the bottom surface of the container,
Here, at least the portion of the convex portion on which the sheet-like cell culture is placed has a dome shape, and
By placing the sheet- shaped cell culture so that part of it is located at the top of the dome-shaped shape , excess liquid adhering to the sheet-shaped cell culture can flow in a lower direction. Said container. 2. A container according to claim 1 , configured to form an annular space between the protrusion and the inner surface of the container. 3. The container according to claim 1 or 2 , wherein the protrusion has one or more pillars outside the portion on which the sheet-like cell culture is placed. The container according to any one of claims 1 to 3 , wherein at least part of the protrusions and/or pillars has elasticity. A container according to any one of claims 1 to 4 , comprising a cutting die whose size is adjustable. A container for use in processing a sheet-like cell culture,
a convex portion protruding upward from the bottom surface of the container;
a dome-shaped mounting portion on which the sheet-shaped cell culture is mounted in the convex portion;
and a liquid-retaining space surrounding the mounting portion. By placing the sheet -shaped cell culture partly on the apex of the dome shape of the placing part, excess liquid adhering to the sheet-shaped cell culture is pushed downward. 7. A container according to claim 6 , which is flowable. A kit comprising the container according to any one of claims 1 to 7 and an outer container for housing the container. A method for processing a sheet cell culture, comprising:
(a) placing the sheet-like cell culture on the protrusions of a container having protrusions projecting upward from the bottom surface , wherein at least the sheet-like cell culture is placed on the protrusions; the part has a dome shape, and the sheet-like cell culture is placed so that a part thereof is located at the apex of the dome shape;
(d) maintaining the sheet-like cell culture on the convex portion and allowing excess liquid attached to the sheet-like cell culture to flow downward;
The above method, comprising after step (a) and before step (d),
(b) washing the sheet-like cell culture by adding a wash solution until the sheet-like cell culture is submerged;
(c) removing the washing solution;
10. The method of claim 9 , further comprising: 11. The method according to claim 10 , wherein the washing solution is added and/or removed from the outside of the part on which the sheet-like cell culture is placed. after step (d),
(e) laminating a polymer-gelable compound on the sheet-shaped cell culture to produce a laminate;
A method according to any one of claims 9 to 11 , further comprising after step (e),
(f) cutting out a portion of the laminate containing the sheet-like cell culture;
13. The method of claim 12 , further comprising: