JP7262443B2 - Sheet-like cell culture with penetrating structures - Google Patents

Description

The present invention includes a sheet-like cell culture having at least one peripheral penetrating structure for passing a needle through, a laminate of the sheet-like cell culture and a biocompatible gel, a method for producing the same, and the laminate. The present invention relates to a pharmaceutical composition for transplantation, a kit for producing the composition for transplantation, a method for treating diseases using the sheet-shaped cell culture, and the like.

In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, attempts have been made to utilize fetal myocardial cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. for the repair of myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. (Non-Patent Document 1).

As part of such attempts, a cell structure formed using a scaffold and a sheet-like cell culture in which cells are formed into a sheet have been developed (Patent Document 1).
Regarding the therapeutic application of sheet-like cell cultures, the use of cultured epidermal sheets for skin damage due to burns, etc., the use of corneal epithelial sheet-like cell cultures for corneal damage, and the oral mucosa sheet-like use for endoscopic resection of esophageal cancer. Studies are underway centering on regenerative medicine, such as the use of cell cultures.

As described above, sheet-like cell cultures are highly useful in regenerative medicine and the like, but they are generally fragile as they are, and tend to wrinkle or tear during isolation from the culture substrate or during subsequent manipulations. Operations such as transfer, storage, and transplantation are extremely difficult. In order to solve this problem, a fibrinogen solution and a thrombin solution are simultaneously sprayed onto the sheet-like cell culture to form a fibrin-containing support layer on the sheet-like cell culture, thereby exposing the sheet-like cell culture and fibrin. A method of manufacturing a laminate with is attempted (Patent Document 2).

Japanese Patent Publication No. 2007-528755 JP 2011-172925 A

Haraguchi et al., Stem Cells Transl Med. 2012 Feb;1(2):136-41

When applying sheet-like cell culture to organs such as the heart, it is done by attaching the sheet-like cell culture to the organ. There has been a problem that the sheet-like cell culture that has been applied is detached and dropped from the application site. To deal with such a problem, the sheet-like cell culture that has been applied is generally prevented from falling off by sewing at least one needle to the organ.

However, sheet-like cell cultures that do not have a reinforcing layer have a high risk of being damaged during sewing, and formation of a reinforcing layer such as the above-mentioned support layer makes handling difficult. It was newly found that the laminated body made easy has a problem that the sewing operation becomes difficult because the presence of such a reinforcing layer makes it difficult for a sewing needle to penetrate the laminated body. Therefore, the present invention provides a sheet-like cell culture that does not have these problems, is excellent in operability and is suitable for transplantation, a laminate of the sheet-like cell culture and a biocompatible gel, a method for producing the same, and the laminate An object of the present invention is to provide a pharmaceutical composition for transplantation containing the body, a kit for producing the composition for transplantation, and a method for treating diseases using the sheet-like cell culture.

The present inventors have studied a sheet-like cell culture that is excellent in operability and facilitates transplantation techniques including sewing. The present inventors have found that forming a sheet-like cell culture can be easily sewn up, and as a result of continuing intensive research, the present invention has been completed.

That is, the present invention relates to:
[1] A sheet-like cell culture for transplantation for suture fixation, which has at least one penetrating structure in its peripheral edge.
[2] The sheet-like cell culture for transplantation of [1], wherein the penetrating structure is present only in the peripheral portion.
[3] The sheet-like cell culture for transplantation of [1] or [2], wherein the penetrating structure is a hole or a notch.
[4] The sheet-like cell culture for transplantation of [1] to [3], which is a monolayer sheet-like cell culture.
[5] The sheet-like cell culture for transplantation of [1] to [4], containing myoblasts or cardiomyocytes.
[6] The sheet-like cell culture for transplantation according to [1] to [5], which is produced by forming a penetrating structure in the sheet-like cell culture.
[7] A medical laminate comprising a sheet-like cell culture layer containing at least one monolayer sheet-like cell culture and a reinforcing layer containing a biocompatible gel, wherein at least one reinforcing layer penetrating structure The medical laminate having in.
[8] The medical laminate of [7], which contains one or at least two sheet-shaped cell cultures.
[9] A pharmaceutical composition for implantation, comprising the medical laminate of [7] or [8] and a needle for suture fixation.
[10] A transplantation kit comprising a sheet-like cell culture for transplantation and a biocompatible gel having at least one penetrating structure in the periphery.
[11] An effective amount of the sheet-like cell culture of [1] to [6], the medical laminate of [7] or [8] and/or the pharmaceutical composition for transplantation of [9] is applied to the diseased site of interest. A method of treating a disease ameliorated by the application of a sheet-like cell culture, characterized in that the penetrating structure is sutured and fixed by passing a needle through the penetrating structure.

The sheet-like cell culture and the medical laminate of the present invention reduce the risk of breakage of the fragile sheet-like cell culture, are excellent in operability, and are easy to apply to the affected area. In addition, since the stitching operation can be simplified, the difference in operation due to the skill level of the operator is reduced, and it is possible to reliably treat the disease, so further spread of regenerative medicine and the like can be expected.

1 is a photographic representation of a medical laminate of the present disclosure made in Example 1. FIG. A portion surrounded by a dotted line has a hole as a penetrating structure. A is a photographic view when trying to pierce a medical laminate having no penetrating structure with a suture needle. Since there is no penetrating structure, it can be seen that the suture needle does not penetrate easily because it is blocked by the reinforcing layer. B is a photographic view when a suture thread is passed through the medical laminate of the present disclosure. By passing the suture needle through the penetrating structure, the medical laminate can be easily penetrated by the suture needle. A is a photographic diagram when sheet-forming cells were seeded on a culture substrate on which obstacles were placed in Example 2. FIG. B is a photograph of the sheet-like cell culture immediately before peeling from the culture substrate in Example 2. FIG. A portion surrounded by a dotted line has a hole as a penetrating structure. C is a photograph of the sheet-like cell culture peeled from the culture substrate in Example 2. FIG. A portion surrounded by a dotted line has a hole as a penetrating structure. 1 is a schematic diagram representing some aspects of a medical laminate of the present disclosure; FIG. A represents an aspect having a hole as a penetrating structure, and B represents an aspect having a notch as a penetrating structure. In both embodiments, the sheet-like cell culture 10 and the reinforcing layer 11 are laminated to form the medical laminate 1, and the hole 101 or the incision is formed so as to penetrate the sheet-like cell culture 10 and the reinforcing layer 11. 102.

The present disclosure will now be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are hereby incorporated by reference in their entirety. In addition, if there is any discrepancy between the publications referred to in this specification and the description in this specification, the description in this specification shall take precedence.

In the present disclosure, a “sheet-like cell culture” refers to a sheet-like cell 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 an 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 a sheet-like cell culture (hereinafter sometimes referred to as "sheet-forming cells") are not particularly limited as long as they can form a sheet-like cell culture. )including. 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, synovial cells, 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., hepatocytes, 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.

In the present disclosure, "myoblasts" are striated muscle cell progenitor cells and include skeletal myoblasts and cardiac myoblasts.
In the present disclosure, "skeletal myoblast" means myoblasts present in skeletal muscle. Skeletal myoblasts are well known in the art, and can be prepared from skeletal muscle by any known method (e.g., the method described in JP-A-2007-89442) or commercially available. available (eg Lonza, Cat# CC-2580). Skeletal myoblasts have markers such as, but not limited to, CD56, α7 integrin, Myosin heavy chain IIa, Myosin heavy chain IIb, Myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, Myogenin, Desmin, PAX3. can be identified by In certain aspects, the skeletal myoblasts are CD56 positive. In a more particular embodiment, the skeletal myoblasts are CD56 positive and desmin positive. Skeletal myoblasts can be derived from any organism with skeletal muscle, including but not limited to humans, non-human primates, rodents (such as mice, rats, hamsters, guinea pigs), rabbits, dogs, cats, pigs, It may be derived from mammals such as horses, cows, goats, and sheep. In one aspect, the skeletal myoblast is a mammalian skeletal myoblast. In certain embodiments, the skeletal myoblasts are human skeletal myoblasts.

In the present disclosure, "cardiac myoblast" means myoblasts present in the myocardium. Cardiac myoblasts are well known in the art and can be identified by markers such as Isl1. Cardiac myoblasts can be derived from any organism that has a heart muscle, including but not limited to humans, non-human primates, rodents (such as mice, rats, hamsters, guinea pigs), rabbits, dogs, cats, pigs, horses, It may be derived from mammals such as cows, goats, and sheep. In one embodiment, the cardiac myoblast is a mammalian cardiac myoblast. In certain embodiments, the cardiac myoblasts are human cardiac myoblasts.
In the present disclosure, "cardiomyocyte" means a cell having cardiomyocyte characteristics, which include, but are not limited to, expression of cardiomyocyte markers, presence of autonomous beating, and the like. . Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (aka SIRPA or SHPS-1), KDR (aka CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM, and the like. . Cardiomyocytes are preferably exemplified by iPS cell-derived cardiomyocytes.

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 forming 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, cells derived from monkeys or pigs are examples of heterologous cells. 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.

A sheet-like cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, 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. The production method of the present disclosure may include the step of producing a sheet-like cell culture, in which case the step of producing the sheet-like cell culture is a substep of the above method for producing a sheet-like cell culture may include one or more of In one aspect, there is no step of growing the cells after the step of thawing the cells and before seeding the cells onto the culture substrate.

The culture substrate is not particularly limited as long as cells can form a cell culture thereon, and includes, for example, vessels made of various materials, solid or semi-solid surfaces in vessels, and the like. The container preferably has a structure and material that does not allow a liquid such as a culture solution to pass through. Examples of such materials include, without limitation, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like. It is also preferred that the container has at least one flat surface. Examples of such vessels include, but are not limited to, culture vessels having a bottom surface composed of a culture substrate capable of forming a cell culture and liquid-impermeable sides. Particular examples of such culture vessels include, without limitation, cell culture dishes, cell culture bottles, and the like. The bottom of the container may be transparent or opaque. When the bottom surface of the container is transparent, cells can be observed and counted from the back side of the container. The container may also have a solid or semi-solid surface inside it. Solid surfaces include plates and containers of various materials as described above, and semi-solid surfaces include gels, soft polymer matrices, and the like. The culture substrate may be produced using the materials described above, or commercially available ones may be used. Preferred culture substrates include, without limitation, substrates having an adherent surface suitable for forming, for example, sheet-like cell cultures. Specifically, substrates having a hydrophilic surface, for example, corona discharge-treated polystyrene, substrates coated with a hydrophilic compound such as collagen gel or hydrophilic polymer on the surface, collagen, fibronectin, and laminin. , vitronectin, proteoglycans, glycosaminoglycans and the like, and substrates coated with cell adhesion factors such as cadherin family, selectin family and integrin family on the surface. Also, such substrates are commercially available (eg, Corning ^(R) TC-Treated Culture Dish, Corning, etc.). The culture substrate may be wholly or partially transparent or opaque.

The culture substrate may be coated with a material whose physical properties change in response to a stimulus such as temperature or light. Examples of such materials include, but are not limited to, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (e.g., N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide amide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), having a cyclic group ( meth)acrylamide derivatives (e.g. 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1 -(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl) - morpholine, etc.), or a temperature-responsive material composed of a homopolymer or copolymer of a vinyl ether derivative (e.g., methyl vinyl ether), a light-absorbing polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydroxide and an acrylamide-based monomer A copolymer with a polymer, and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (for example, JP-A-2-211865, JP-A-2003-33177). ). By applying a predetermined stimulus to these materials, their physical properties, such as hydrophilicity and hydrophobicity, can be changed to promote detachment of cell cultures adhered to the materials. Culture dishes coated with temperature-responsive materials are commercially available (eg, UpCell® from CellSeed Inc. ⁾ and can be used in the manufacturing methods of the present disclosure.

The culture substrate may be of various shapes, but is preferably flat. The area is not particularly limited, but may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² . For example, the culture substrate may be a circular culture dish with a diameter of 10 cm. In this case, the area will be 56.7 cm ² .
The culture substrate may be coated (coated or coated) with serum. A sheet-like cell culture with a higher density can be formed by using a serum-coated culture substrate. “Serum-coated” means a state in which serum components adhere to the surface of the culture substrate. Such a state can be obtained, without limitation, for example, by treating the culture substrate with serum. Treatment with serum includes contacting the serum with the culture substrate and optionally incubating for a period of time.

Heterologous serum and/or allogeneic serum can be used as the serum. Heterologous serum means serum derived from an organism of a different species than the recipient when sheet cell cultures are used for transplantation. For example, when the recipient is human, serum derived from cows or horses, such as fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc., correspond to heterologous serum. Also, "allogeneic serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum is an allogeneic serum. Allogeneic serum includes autologous serum (also referred to as autologous serum), ie, serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient. In the present specification, sera other than self sera, that is, heterologous sera and allogeneic sera may be collectively referred to as non-self sera.
Serum for coating the culture substrate is commercially available, or can be prepared by standard methods from blood collected from the desired organism. Specifically, for example, the collected blood is left at room temperature for about 20 to about 60 minutes to coagulate, centrifuged at about 1000×g to about 1200×g, and the supernatant is collected. etc.

When incubating on the culture substrate, serum may be used neat or diluted. Dilutions can be made in any medium, including, without limitation, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.) can be used. The dilution concentration is not particularly limited as long as the serum components can adhere to the culture substrate. /v), more preferably from about 5% to about 40% (v/v).

The incubation time is also not particularly limited as long as the serum components can adhere to the culture substrate. 24 hours, more preferably about 2 hours to about 12 hours. The incubation temperature is also not particularly limited as long as serum components can adhere to the culture substrate. is.

Serum may be discarded after incubation. As a serum disposal method, a conventional liquid disposal method such as aspiration with a pipette or decantation can be used. In preferred embodiments of the present disclosure, the culture substrate may be washed with a serum-free wash solution after serum disposal. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the culture substrate. Liquid (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15 , SkBM, RITC80-7, etc.). The washing method may be a conventional washing method of the culture substrate, for example, without limitation, a method of adding a serum-free washing solution onto the culture substrate, stirring for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarding it. etc. can be used.

In the present disclosure, culture substrates may be coated with growth factors. Here, "growth factor" means any substance that promotes cell proliferation compared to its absence, e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast Including cell growth factor (FGF) and the like. The method of coating, discarding, and washing the culture substrate with a growth factor is such that the dilution concentration at the time of incubation is, for example, about 0.0001 μg/mL to about 1 μg/mL, preferably about 0.0005 μg/mL to about 0.1 μg/mL. 05 μg/mL, more preferably from about 0.001 μg/mL to about 0.01 μg/mL, but essentially the same as serum.

In the present disclosure, the culture substrate may be coated with a steroidal agent. As used herein, the term “steroidal agent” refers to a compound having a steroidal nucleus, which is capable of exerting adverse effects on living organisms, such as adrenal cortex insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, and the like. In the method of coating, discarding, and washing the culture substrate with a steroid agent, the dilution concentration during incubation is, for example, about 0.1 μg/mL to about 100 μg/mL, preferably about 0.4 μg/mL to about 0.4 μg/mL as dexamethasone. It is essentially the same as serum except that it is about 40 μg/mL, more preferably about 1 μg/mL to about 10 μg/mL.

The culture medium may be coated with any one of serum, a growth factor and a steroid agent or any combination thereof, i.e. serum and growth factor, serum and steroid agent, serum and growth factor and steroid agent, Alternatively, it may be coated with a combination of growth factors and steroid agents. When coating with multiple components, these components may be mixed and coated at the same time or in separate steps.

The culture substrate may be seeded with cells immediately after being coated with serum or the like, or may be stored after being coated and then seeded with cells. The coated substrate can be stored for a long period of time, for example, by keeping it at about 4°C or lower, preferably about -20°C or lower, more preferably about -80°C or lower.
Cells can be seeded onto the culture substrate by any known technique and conditions. Cells may be sown on the culture substrate, for example, by injecting a cell suspension obtained by suspending cells in a culture solution into the culture substrate (culture vessel). For injecting the cell suspension, an instrument suitable for injecting the cell suspension, such as a dropper or a pipette, can be used.

<1> Sheet-shaped cell culture and medical laminate of the present disclosure In one aspect of the present disclosure, there is provided a sheet-shaped cell culture for transplantation for suture fixation, which has at least one penetrating structure in the periphery.
In the present disclosure, the term “periphery” refers to a portion of a structure such as a sheet-like cell culture or a laminate structure, which is located relatively outside the shape of the upper surface or the lower surface. Specifically, for example, on the line segment connecting the outer edge of the shape of the upper or lower surface to the center, 50% from the outer edge, 40% from the outer edge, 30% from the outer edge, 20% from the outer edge, 15% from the outer edge %, preferably up to 10% from the outer edge, more preferably up to 5% from the outer edge.

In the present disclosure, the term “penetrating structure” means a structure penetrating through a structure in a certain direction. Typically, for example, a hole or a notch penetrating from the upper surface to the lower surface of the sheet-like cell culture is exemplified.
The sheet-like cell culture of the present disclosure is a sheet-like cell culture that is fixed by suturing to an application site, for example, an organ such as the heart during transplantation. Therefore, even if the application site frequently moves due to pulsation or peristaltic movement, it will not come off.

The sheet-like cell culture of the present disclosure has at least one penetrating structure in the periphery. Such a penetrating structure is for inserting a needle, and exists so as to penetrate from the upper surface to the lower surface of the sheet-like cell culture. Such a penetrating structure may be any structure as long as it is a structure through which a needle can be passed. It is weaker than other parts and has a structure that makes it easy for a needle to pass through.

A plurality of penetrating structures may be present in the sheet-like cell culture, but at least one is present in the periphery of the sheet-like cell culture. In a preferred embodiment, the penetrating structures are present only at the periphery of the sheet-like cell culture. A plurality of penetrating structures may be present as described above, but from the viewpoint of the strength of the sheet-like cell culture, the number of constituent cells, etc., the smaller the number, the better. Preferable specific examples of penetrating structures include, for example, one, two, three, four, etc., and more preferably one or two.

As described above, the penetrating structure is a structure through which a needle can be passed, and is preferably a structure in which cell connections are disrupted, such as holes or notches. When the penetrating structure is a hole, the shape can be any shape, including but not limited to circular, elliptical, polygonal, and the like. The size of the pore (here, "pore size" means the diameter of the largest circle inscribed in the pore) is not particularly limited as long as a surgical suture needle can pass through it, for example about 200 μm or more, about 500 μm or more. , about 1 mm or more, about 1.5 mm or more, about 2 mm or more, and the like. On the other hand, if the pores are too large, the strength of the entire sheet-like cell culture and the number of cells constituting the sheet-like cell culture are lowered, which is not preferable. Thus, preferably, the pore size is no greater than about 5 mm, no greater than about 4 mm, no greater than about 3 mm, no greater than about 2.5 mm. It may be about 2 mm or less, about 1.5 mm or less, about 1 mm or less, and the like.

Therefore, the possible range of pore size may be any combination of the above lower limit and upper limit, such as 200 μm to 5 mm, 200 μm to 4 mm, 200 μm to 3 mm, 200 μm to 2.5 mm, 200 μm to 2 mm, 200 μm-1.5 mm, 200 μm-1 mm, 500 μm-5 mm, 500 μm-4 mm, 500 μm-3 mm. 1 .5mm-5mm, 1.5mm-4mm, 1.5mm-3mm, 1.5mm-2.5mm, 1.5mm-2mm, 2mm-5mm, 2mm-4mm, 2mm-3mm, 2mm-2.5mm etc. could be.

When the penetrating structure is a notch, its shape is not particularly limited, and may be linear, cross-shaped, or the like. The size of the incision (herein, "the size of the incision" means the length of the largest incision) is not particularly limited as long as the surgical suture needle can pass through, for example, about 200 μm or more, about 500 μm or more, about It may be 1 mm or greater, about 1.5 mm or greater, about 2 mm or greater, and the like. On the other hand, if the incision is too large, the strength of the entire sheet-like cell culture will decrease, which is not preferable. Therefore, preferably, the size of the cut is no more than about 5 mm, no more than about 4 mm, no more than about 3 mm, no more than about 2.5 mm. It may be about 2 mm or less, about 1.5 mm or less, about 1 mm or less, and the like.

Therefore, the possible range of the size of the cut may be any combination of the above lower limit and upper limit, for example, 200 μm to 5 mm, 200 μm to 4 mm, 200 μm to 3 mm, 200 μm to 2.5 mm, 200 μm to 2 mm, 200 μm-1.5 mm, 200 μm-1 mm, 500 μm-5 mm, 500 μm-4 mm, 500 μm-3 mm. 1 .5mm-5mm, 1.5mm-4mm, 1.5mm-3mm, 1.5mm-2.5mm, 1.5mm-2mm, 2mm-5mm, 2mm-4mm, 2mm-3mm, 2mm-2.5mm etc. could be.

The sheet-like cell culture of the present disclosure may be a single-layer sheet-like cell culture or a laminate in which a plurality of sheet-like cell cultures are laminated as described above, but preferably a single-layer It is a sheet-like cell culture.
Cells used in the sheet-like cell culture of the present disclosure include, for example, the cells described in detail in the above-mentioned "cells constituting sheet-like cell culture". A sheet-like cell culture containing myoblasts or cardiomyocytes is preferable in view of the fragility of the sheet, the site of application, and the like.

The sheet-like cell culture of the present disclosure can be produced by the below-described production method. In particular, the penetrating structure possessed by the sheet-like cell culture of the present disclosure may be formed during or after formation of the sheet-like cell culture. From the viewpoint of ease of forming the sheet-like cell culture, in a preferred embodiment of the present disclosure, the penetrating structure is formed after forming the sheet-like cell culture.

Any method known in the art can be used as a method for forming the penetrating structure. A method for forming a penetrating structure during formation of a sheet-like cell culture is not limited to this, but for example, a method of seeding sheet-forming cells on a culture substrate having a cell non-adhesive region to form a sheet. , a method of forming a sheet by seeding sheet-forming cells on a culture substrate with obstacles or the like placed thereon, and the like.

The method for forming a penetrating structure after forming a sheet-like cell culture is not limited to this, but for example, a method of forming a cut or a hole with a knife such as a scalpel or scissors, or a method of penetrating with a projection such as a needle. method, a method of forming holes with a punch or the like, and a method of eliminating cells with a laser beam or the like.

Another aspect of the present disclosure encompasses a medical laminate comprising at least one monolayer sheet-like cell culture and a reinforcing layer comprising a biocompatible gel. Such medical laminates have at least one perforation structure at the periphery. Here, the penetrating structure present in the medical laminate has a structure that penetrates at least the reinforcing layer, and preferably has a structure that penetrates both the sheet-like cell culture and the reinforcing layer.

The biocompatible gel that can be used in the laminate of the present disclosure may be any gel that does not adversely affect the body when introduced into the body, and is not limited to this. Fibrin gel, fibrinogen gel, gelatin gel, collagen gel and the like.

A method known in the art can be used to form a reinforcing layer containing a biocompatible gel. Examples of such methods include, but are not limited to, a method of spraying a biocompatible gel onto a sheet-like cell culture, a method of laminating a sol-like biocompatible substance on a sheet-like cell culture, and forming a gel. In addition to the method of solidifying the gel, the method of solidifying the gel after immersing it in a liquid gel, and the like, the method described in JP 2016-52271 A and the like can be mentioned.

The method of forming a structure that penetrates at least the reinforcing layer is not limited to this, but for example, a method of forming a reinforcing layer with an obstacle placed on the sheet-like cell culture, a method of forming a reinforcing structure After that, a method of forming holes or incisions with a knife, protrusion, punch, etc. so as to penetrate the sheet-like cell culture and the reinforcing layer, and a state in which a suture needle or the like is passed through the formed sheet-like cell culture and the like to form a reinforcing layer.

The medical laminate of the present disclosure contains at least one monolayer sheet-like cell culture. In a preferred embodiment, the medical laminate of the present disclosure contains one monolayer sheet-like cell culture. In another preferred aspect, the medical laminate of the present disclosure includes at least two monolayer sheet-like cell cultures. Such at least two monolayer sheet-like cell cultures may be layered and included as one layered sheet-like cell culture, or at least two independent sheet-like cell cultures (monolayers or layers) It may be included in a form connected by one reinforcing layer.

In another aspect, the present disclosure includes a pharmaceutical composition for implantation, which includes the medical laminate of the present disclosure and a needle for suture fixation. The needle for suture fixation may be packaged separately from the medical laminate of the present disclosure, or may be attached while passing through the penetrating structure of the medical laminate of the present disclosure.
In embodiments in which a needle for suture fixation is attached while penetrating the penetrating structure, such a composition for transplantation may be produced by forming a reinforcing layer on the sheet-like cell culture and then penetrating the reinforcing layer, It may be produced by forming a reinforcing layer after piercing the sheet-like cell culture with a needle.

In another aspect, the present disclosure also includes an implantation kit for manufacturing the medical laminate of the present disclosure. Such a kit may include, for example, a sheet-like cell culture for transplantation having at least one penetrating structure in its periphery and a biocompatible gel. In one aspect of the implant kit of the present disclosure, the implant kit may further include needles for suture fixation.

<2> Method for producing the sheet-shaped cell culture and/or medical laminate of the present disclosure In another aspect, the present disclosure includes a method for producing the sheet-shaped cell culture and/or medical laminate of the present disclosure. do.
The method for producing a sheet-like cell culture of the present disclosure includes the following steps:
(i) seeding a culture substrate with a cell population comprising at least one sheet-forming cell;
(ii) sheeting the cell population seeded in (i) above to form a sheet-like cell culture in a cell culture medium; and (iii) forming a sheet-like cell culture formed in (ii) above. exfoliating from the culture substrate;
and optionally forming a penetrating structure.

In step (i), a cell population comprising at least one sheet-forming cell is seeded onto a culture substrate. Here, the “sheet-forming cells” and the “culture substrate for seeding the cell population containing the sheet-forming cells” are as described above. Furthermore, the culture substrate may have a member for forming a penetrating structure at a position corresponding to a portion where the penetrating structure of the sheet-like cell culture exists. The member for forming the penetrating structure is not limited to this, but for example, a removable obstacle (e.g., a columnar additional member, a protrusion that can be broken off, etc.), a culture surface formed of a cell non-adhesive substance etc.

In (ii), the seeded cell population is incubated in a cell culture medium to form a sheet, forming a sheet-like cell culture.
Sheeting of the seeded cells can be performed by any known technique and conditions. Non-limiting examples of such techniques are described, for example, in Patent Document 1, WO 2014/185517, and the like. Formation of a cell sheet is thought to be achieved by the cells adhering to each other through an intercellular adhesion mechanism such as adhesion molecules or extracellular matrix. Therefore, sheet formation of the seeded cells can be achieved, for example, by culturing the cells under conditions that form intercellular adhesions. Any conditions can be used as long as intercellular adhesion can be formed, but usually, intercellular adhesion can be formed under conditions similar to general cell culture conditions. Such conditions include, for example, culturing at about 37° C. and 5% CO ₂ . In addition, the culture can be performed under normal pressure (atmospheric pressure, non-pressure). Cultivation can be performed in vessels of any size and shape. The size and shape of the sheet-like cell culture can be adjusted by adjusting the size and shape of the cell-attached surface of the culture vessel, or by placing a mold of desired size and shape on the cell-attached surface of the culture vessel. It can be arbitrarily adjusted by culturing cells therein. In this specification, the culture for forming the seeded cells into a sheet is sometimes referred to as "sheet forming culture". The sheet culture reduces the thickness of the sheet-like cell culture on the culture substrate (inside the culture vessel). That is, after the cells are seeded, the thickness of the cell layer on the culture substrate decreases due to subsequent sheet formation, but the sheet-like cell culture shrinks due to detachment from the culture substrate and increases in thickness again. . The reduction in thickness due to sheet formation is about 90% to about 10% when the thickness of the cell layer immediately after seeding is taken as 100%. The sheet-like cell culture shrinks again by detachment from the culture substrate, and the increase rate of the thickness of the sheet-like cell culture at that time is about 120% to 300%, assuming that the thickness of the cell layer immediately after detachment is 100%. is.

The incubation time for sheet formation is not particularly limited as long as the sheet can be formed. The time during which a sheet can be formed may vary depending on the type of cells contained in the seeded cell population (especially the type of sheet-forming cells) and the state of the cells. For example, cells containing skeletal myoblasts as sheet-forming cells When a mass is seeded, sheets can be formed in as little as 2 hours. Thus, in one aspect, the incubation time for sheeting is 2-12 hours, 2-11.5 hours, 2-11 hours, 2-10 hours, 2-9 hours, 2-8 hours, 2-7 hours. , 2-6 hours, 2-5 hours or 2-4 hours, preferably 2-4 hours or 2-6 hours.

The cell culture medium used for culture (sometimes simply referred to as "culture medium" or "medium") is not particularly limited as long as it can maintain the survival of cells, but typically contains amino acids, vitamins, and electrolytes. can be used as the main component. In one aspect of the present disclosure, the culture medium is based on a basal medium for cell culture. Such basal media include, but are not limited to, DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (such as MCDB102, 104, 107, 120, 131, 153, 199), L15, SkBM, RITC80 -7 and so on. Many of these basal media are commercially available and their compositions are also known.
The basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be changed as appropriate according to the cell type and cell conditions. Accordingly, basal media for use in the present disclosure are not limited to those of known composition, but include those in which one or more components have been added, removed, increased or decreased.

Amino acids contained in the basal medium include, but are not limited to, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc. Vitamins include, but are not limited to, calcium D-pantothenate, choline chloride, folic acid, i - inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B12, adenine, thymidine, etc. and electrolytes such as, but not limited to, CaCl ₂ , KCl, MgSO ₄ , NaCl, NaH _{2PO4 , NaHCO3} , Fe _{(NO3)3, FeSO4, CuSO4, MnSO4, Na2SiO3 , (NH4)6Mo7O24 , NaVO3 , NiCl2 , ZnSO4 , etc. , respectively .} be In addition to these components, the basal medium may contain sugars such as D-glucose, sodium pyruvate, pH indicators such as phenol red, putrescine, and the like.

In one aspect of the present disclosure, the concentrations of amino acids contained in the basal medium are L-arginine: about 63.2 mg/L to about 84 mg/L, L-cystine: about 35 mg/L to about 63 mg/L, L-glutamine : about 4.4 mg/L to about 584 mg/L, glycine: about 2.3 mg/L to about 30 mg/L, L-histidine: about 42 mg/L, L-isoleucine: about 66 mg/L to about 105 mg/L, L-leucine: about 105 mg/L to about 131 mg/L, L-lysine: about 146 mg/L to about 182 mg/L, L-methionine: about 15 mg/L to about 30 mg/L, L-phenylalanine: about 33 mg/L ~ about 66 mg/L, L-serine: about 32 mg/L to about 42 mg/L, L-threonine: about 12 mg/L to about 95 mg/L, L-tryptophan: about 4.1 mg/L to about 16 mg/L, L-tyrosine: about 18.1 mg/L to about 104 mg/L, L-valine: about 94 mg/L to about 117 mg/L.
In one aspect of the present disclosure, the concentrations of vitamins contained in the basal medium are calcium D-pantothenate: about 4 mg/L to about 12 mg/L, choline chloride: about 4 mg/L to about 14 mg/L, folic acid : about 0.6 mg/L to about 4 mg/L, i-inositol: about 7.2 mg/L, niacinamide: about 4 mg/L to about 6.1 mg/L, riboflavin: about 0.0038 mg/L to about 0 .4 mg/L, thiamine: about 3.4 mg/L to about 4 mg/L, pyridoxine: about 2.1 mg/L to about 4 mg/L.

In addition to the above, the cell culture medium may contain one or more additives such as serum, growth factors, steroid drug components, and selenium components. However, if these components are not self-derived, they may become impurities derived from the manufacturing process that may cause side effects such as anaphylactic shock to the recipient in clinical practice. It may be desirable to exclude derived components. Accordingly, in preferred embodiments of the present disclosure, the cell culture medium does not contain an effective amount of at least one of these non-autologous additives. Also, in a more preferred aspect of the present disclosure, the cell culture is substantially free of at least one of these non-autologous additives. Furthermore, in particularly preferred embodiments of the present disclosure, the cell culture medium is substantially free of non-autologous additives. In one aspect, the cell culture medium may contain only basal medium.

In one aspect of the present disclosure, the cell culture medium is substantially free of serum. A cell culture medium substantially free of serum is sometimes referred to herein as a "serum-free medium." Here, "substantially free of serum" means that the serum content in the culture medium does not adversely affect the application of the sheet-shaped cell culture to a living body (e.g., the serum content in the sheet-shaped cell culture The serum albumin content is less than about 50 ng), preferably, it means that these substances are not actively added to the culture medium. In the present disclosure, in order to avoid side effects during transplantation, the cell culture medium is preferably substantially free of xenogenous serum, and more preferably substantially free of non-autologous serum.

In one aspect of the present disclosure, the cell culture medium contains serum. The serum may be allogeneic or xenogeneic. In certain embodiments, the cell culture medium comprises autologous serum. When cells are cultured on a serum-coated culture substrate, the serum contained in the cell culture medium (serum used for cell culture) may be the same as or different from the serum used to coat the culture substrate. may In one aspect, the serum contained in the cell culture medium is the same serum used to coat the culture substrate, and in certain aspects, the serum is autologous serum. Serum may be for use in the production method of the present disclosure. For example, the serum may be for use in culturing cells or for coating a culture substrate.

In one aspect of the present disclosure, the cell culture medium does not contain an effective amount of growth factors. As used herein, an "effective amount of growth factor" means an amount of a growth factor that significantly promotes cell proliferation compared to the absence of the growth factor, or, for convenience in the art, It means the amount usually added for the purpose. The significance of promoting cell growth can be appropriately evaluated by any statistical method known in the art, such as the t-test, and the normal addition amount is various in the art. It can be known from publicly known literature. Specifically, an effective amount of EGF in cell culture is, for example, about 0.005 μg/mL or greater.

Thus, "free of an effective amount of growth factor" means that the concentration of growth factor in the culture medium in the present disclosure is less than such an effective amount. For example, the concentration in the medium of EGF in cell culture is preferably less than about 0.005 μg/mL, more preferably less than about 0.001 μg/mL. In preferred embodiments of the present disclosure, the concentration of growth factors in the culture medium is below normal concentrations in the body. In such embodiments, for example, the concentration of EGF in the cell culture medium is preferably less than about 5.5 ng/mL, more preferably less than about 1.3 ng/mL, and even more preferably about 0.5 ng/mL. is less than In a further preferred embodiment, the culture medium of the present disclosure is substantially free of growth factors. Here, the phrase “substantially free of growth factors” means that the content of the growth factor in the culture medium is such that the sheet-like cell culture is applied to a living organism without adversely affecting the growth factor. is not actively added. Thus, in this embodiment, the culture medium does not contain growth factors at concentrations above those contained in other components therein, such as serum.

In one aspect of the present disclosure, the cell culture medium is substantially free of steroidal drug components. As used herein, the term "steroidal agent component" refers to compounds having a steroidal nucleus that can exert adverse effects on living organisms, such as adrenal cortical dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, and the like. Therefore, the phrase "substantially free of steroidal agent components" means that the content of these compounds in the culture medium is such that the sheet-like cell culture is applied to a living body without adversely affecting it, preferably Not actively adding these compounds to the culture medium means that the culture medium does not contain steroid components at concentrations above those contained in other components therein, such as serum.

In one aspect of the present disclosure, the cell culture medium is substantially free of selenium components. Here, the "selenium component" includes selenium molecules and selenium-containing compounds, particularly selenium-containing compounds capable of liberating selenium molecules in vivo, such as selenous acid. Therefore, the phrase "substantially free of selenium components" means that the content of these substances in the culture medium is such that the sheet-like cell culture is applied to a living body without adversely affecting the body. Not actively adding these substances to the solution means that the culture solution does not contain a concentration of selenium component higher than that contained in other components therein, such as serum. Specifically, for example, in the case of humans, the selenium concentration in the culture medium is the normal value (for example, 10.6 μg/dL to 17.4 μg/dL) in human serum, and the concentration of human serum contained in the medium is lower than the value multiplied by a percentage (ie, if the content of human serum is about 10%, the selenium concentration is, for example, from about 1.0 μg/dL to less than about 1.7 μg/dL).

In the preferred embodiment of the present disclosure, impurities derived from the manufacturing process, such as growth factors, steroid drug components, and heterologous serum components, which were conventionally necessary when producing cell cultures to be applied to living organisms, are removed by washing or the like. No steps required. Accordingly, one aspect of the methods of the present disclosure does not include the step of removing impurities from this manufacturing process.
Here, the “manufacturing process-derived impurities” typically include the following listed impurities derived from each manufacturing process. That is, those derived from cell substrates (e.g., host cell-derived proteins, host cell-derived DNA), those derived from cell culture media (e.g., inducers, antibiotics, medium components), or in processes after cell culture They are those derived from the extraction, separation, processing, and purification processes of a certain target substance (see, for example, Pharmaceutical Evaluation Publication No. 571).

In (iii), the formed sheet-like cell culture is peeled off from the culture substrate.
Detachment of the sheet-like cell culture from the culture substrate is not particularly limited as long as the sheet-like cell culture can be at least partially released (peeled) from the culture substrate serving as a scaffold while maintaining the sheet structure. , for example, enzymatic treatment with a proteolytic enzyme (eg, trypsin) and/or mechanical treatment such as pipetting. In addition, when cells are cultured on a culture substrate whose surface is coated with a material whose physical properties change in response to a stimulus, such as temperature or light, to form a cell culture, a predetermined stimulus may be applied. and can also be released non-enzymatically.

For example, when cells are cultured in a temperature-responsive culture dish to form a cell culture, the temperature is set to the lower critical solution temperature (LCST) or lower or the upper critical solution temperature (UCST) or higher of the temperature-responsive material for water. The sheet-like cell culture can be released non-enzymatically by the temperature treatment. Such temperature treatment is not limited, for example, the culture substrate to which the formed sheet-like cell culture is attached, from a culture environment at a temperature higher than the LCST (e.g., in an incubator at a temperature of about 37 ° C.), It can be achieved by transferring to an environment below the LCST (for example, a room temperature environment outside an incubator). The transition to an environment below the LCST is not limited, for example, a culture medium with a temperature higher than the LCST in which the formed sheet-like cell culture exists, a medium with a temperature below the LCST (e.g., buffer solution (PBS, HBSS, etc.) or a liquid such as a culture solution, etc.). Therefore, the medium such as the buffer solution can be used in the production method of the present disclosure to non-enzymatically release the sheet-like cell culture from the culture substrate.

The sheet-like cell culture detached by the step (iii) shrinks and has a smaller area than before detachment. The sheet-like cell culture produced by the production method of the present disclosure is characterized by being less likely to shrink after detachment and having a larger area. In one aspect of the present disclosure, the sheet-like cell culture after detachment is about 20% or more, for example, about 20%, about 25%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50% , has an area of about 60%, preferably about 35% or more, such as about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50%, about 60% have an area.

The method for producing a sheet-like cell culture of the present disclosure may optionally include forming penetrating structures. Such a step may be included once or more times, and when a culture substrate without a member for forming a penetrating structure is used in step (i) If no structure was formed) is necessarily included after step (iii). As a method for forming the penetrating structure, the method described in detail in <1> above can be used.

In the method for producing a sheet-like cell culture of the present disclosure, after step (iii), a plurality of similarly prepared monolayer sheet-like cell cultures are layered to form one layered sheet-like cell culture. can include Such a lamination step may be performed after forming the penetrating structure in the monolayer sheet-like cell culture, or may include a penetrating structure forming step after the laminating step, but preferably the penetrating structure is formed after the laminating step. be done.

The production method of the present disclosure may include freezing the cells (cell population) and thawing the frozen cells prior to (i). Freezing of cells can be performed by any known technique. Such techniques include, but are not limited to, subjecting cells in a container to freezing means such as freezers, deep freezers, low-temperature media (eg, liquid nitrogen, etc.), and the like. The temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole, of the cell population in the container, but is typically about 0°C or lower, preferably about -20°C or lower, and more preferably about -20°C or lower. is about -40°C or less, more preferably about -80°C or less. In addition, the cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability or function of the cells after freezing and thawing. The cooling rate is about 1 hour to about 5 hours, preferably about 2 hours to about 4 hours, especially about 3 hours. Specifically, for example, it can be cooled at a rate of about 0.46° C./min. Such a cooling rate can be achieved by subjecting the cell-containing container directly to a freezing means set at a desired temperature, or by placing it in a freezing treatment container. The freeze processing container may have a function of controlling the rate of temperature drop in the container to a predetermined rate. Any known freezer such as BICELL ^(R) (Nippon Freezer) and program freezer can be used as the freezer.

The freezing operation may be performed while the cells are immersed in the culture solution or physiological buffer solution, but a cryoprotectant may be added to the culture solution to protect the cells from freezing and thawing, or the culture solution may be cryoprotected. It may be performed after processing such as replacing with a cryopreservation solution containing the agent. Therefore, the production method of the present disclosure including a freezing step may further include adding a cryoprotectant to the culture medium or replacing the culture medium with a cryopreservation medium. When replacing the culture medium with cryopreservation medium, if the liquid in which the cells were submerged at the time of freezing contained an effective concentration of cryoprotectant, substantially all of the culture medium should be removed before adding the cryopreservation medium. Alternatively, the cryopreservation medium may be added while part of the culture medium is left. Here, "effective concentration" means that the cryoprotectant does not exhibit toxicity and is cryoprotective, e.g. It means the concentration that shows the effect of suppressing the decrease of such as. Such concentrations are known to those skilled in the art, or can be appropriately determined by routine experiments and the like.

The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective action on cells. Examples include dimethylsulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. The cryoprotectant may be used alone or in combination of two or more.

The concentration of the cryoprotectant added to the culture medium or the concentration of the cryoprotectant in the cryopreservation medium is not particularly limited as long as it is the effective concentration defined above. About 2% to about 20% (v/v) of the total preservation solution. However, alternative known or empirically determined working concentrations outside of this concentration range for each cryoprotectant can be employed and are within the scope of this disclosure.

The step of thawing the frozen cells can be performed by any known cell thawing technique, typically e.g. It is achieved by subjecting the frozen cells to a medium (e.g., water), water bath, incubator, thermostat, etc., or immersing the frozen cells in a medium (e.g., culture solution) at a temperature higher than the freezing temperature. , but not limited to. The temperature of the thawing means or immersion medium is not particularly limited as long as the cells can be thawed within the desired time, but is typically about 4°C to about 50°C, preferably about 30°C to about 40°C, and more. Preferably from about 36°C to about 38°C. In addition, the thawing time is not particularly limited as long as it does not significantly impair the survival rate and function of the cells after thawing, but it is typically within about 2 minutes, and particularly within about 20 seconds. The decrease can be greatly suppressed. The thawing time can be adjusted by changing, for example, the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or cryopreservation solution during freezing, and the like. Frozen cells include cells that have been frozen by any technique, non-limiting examples of which include, for example, cells that have been frozen by the step of freezing cells described above. In one aspect, the frozen cells are cells that have been frozen in the presence of a cryoprotectant. In one aspect, frozen cells are for use in the manufacturing methods of the present disclosure.

In the production method of the present disclosure, the cells are washed after the step of thawing the frozen cells and before the step of forming a sheet-like cell culture, preferably before the step of seeding the cells on a culture substrate. may include steps. Washing of cells can be performed by any known technique. etc.) or in a physiological buffer (eg, PBS, HBSS, etc.), centrifugation, discarding the supernatant, and recovering the precipitated cells, but is not limited thereto. In the step of washing the cells, such suspension, centrifugation, harvesting cycles may be performed one or more times (eg, 2, 3, 4, 5, etc.). In one aspect of the present disclosure, washing the cells occurs immediately after thawing the frozen cells.

The production method of the present disclosure may further include a step of growing the cells prior to the step of freezing the cells described above. The step of growing cells may be performed by any known technique, and those skilled in the art are familiar with culture conditions suitable for growing various types of cells.

In one aspect, the manufacturing method of the present disclosure does not include the step of introducing genes into cells. In another aspect, the manufacturing method of the present disclosure includes introducing a gene into a cell. The gene to be introduced is not particularly limited as long as it is useful for treating the target disease, and may be, for example, cytokines such as HGF and VEGF. Gene introduction can be performed by any known method such as calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, adenovirus vector, method using viral vector such as retrovirus vector, microinjection method, and the like. can be performed using Introduction of genes into cells can be performed, without limitation, for example, prior to the step of freezing the cells.

In one aspect, all steps of the manufacturing method of the present disclosure are performed in vitro. In another aspect, the manufacturing methods of the present disclosure include steps performed in vivo, such as, but not limited to, removing cells or a tissue source of cells (e.g., striated muscle tissue, particularly skeletal muscle tissue) from a subject. Including the step of harvesting. In one aspect, all steps of the manufacturing method of the present disclosure are performed under aseptic conditions. In one aspect, the manufacturing method of the present disclosure is performed so that the finally obtained sheet-like cell culture is substantially sterile. In one aspect, the manufacturing method of the present disclosure is performed so that the finally obtained sheet-like cell culture is sterile.

Yet another aspect of the present disclosure includes methods of making the medical laminates of the present disclosure.
The manufacturing method of the medical laminate of the present disclosure comprises the following steps:
(i) seeding a culture substrate with a cell population comprising at least one sheet-forming cell;
(ii) sheeting the cell population seeded in (i) above in a cell culture medium to form a sheet-like cell culture;
(iii) peeling the sheet-shaped cell culture formed in (ii) above from the culture substrate; and (iv) reinforcing the sheet-shaped cell culture peeled in (iii) above with a biocompatible gel. forming a layer;
and optionally forming a penetrating structure.

Steps (i) to (iii) in the method for producing a medical laminate of the present disclosure are as described in detail for steps (i) to (iii) in the method for producing a sheet-like cell culture of the present disclosure. In addition, the sheet-like cell culture formed by such steps may optionally have penetrating structures formed therein.

In (iv), a reinforcing layer of biocompatible gel is formed on the sheet-like cell culture. The biocompatible gel that can be used in this step is as detailed in <1> above. The method for forming the reinforcing layer is as described in detail as the method for forming the reinforcing layer containing the biocompatible gel in <1> above.
The sheet-like cell culture on which the reinforcing layer is formed may be a single-layer sheet-like cell culture or a layered sheet-like cell culture. If it is a layered sheet-like cell culture, optionally after step (iii) and before step (iv), multiple layered sheet-like cell cultures are layered to form one layered sheet-like cell culture. may include steps.

Also in step (iv), the reinforcement layer may be formed on a plurality of independent sheet-like cell cultures (monolayer or laminate). In this case, a medical laminate is formed in which a plurality of independent sheet-like cell cultures (single layer or garment) are connected by one reinforcing layer.
The method of manufacturing a medical laminate of the present disclosure may optionally include forming a feedthrough structure. Such a penetrating structure is a penetrating structure that penetrates at least the reinforcing layer, preferably a penetrating structure that penetrates the reinforcing layer and the sheet-shaped cell culture.

Forming through structures may be included one or more times, and if in steps (i)-(iv) no through structures were formed during formation of the medical laminate, then step Must be included after (iv). As a method for forming the penetrating structure, the method described in detail in <1> above can be used.

<3> Treatment method using the sheet-shaped cell culture, medical laminate and/or pharmaceutical composition for transplantation of the present disclosure In another aspect of the present disclosure, the sheet-shaped cell culture and medical laminate of the present disclosure and / or applying an effective amount of a pharmaceutical composition for transplantation (hereinafter sometimes referred to as "sheet-like cell culture etc. of the present disclosure") to a subject in need thereof, It includes methods of treatment. Tissues and diseases targeted by the treatment method of the present disclosure are as described above for the sheet-like cell culture of the present disclosure. In addition, in the treatment method of the present disclosure, ingredients that enhance the viability, engraftment and/or function of sheet-like cell cultures, other active ingredients useful for treatment of target diseases, etc. are added to the sheet of the present disclosure. It can be used in combination with a similar cell culture, composition, or the like.

The treatment method of the present disclosure may further comprise producing a sheet-like cell culture according to the production method of the present disclosure. The treatment method of the present disclosure may further comprise, prior to the step of producing the sheet-like cell culture, collecting cells or tissue as a source of cells for producing the sheet-like cell culture from the subject. . In one aspect, 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 disclosure, the term "subject" means any living individual, preferably an animal, more preferably a mammal, and more preferably a human individual. In the present disclosure, 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 disclosure, an effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, alleviate symptoms, or delay or stop 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 disclosure 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 injury (trauma, burns, etc.), joint disease (e.g., osteoarthritis, etc.), cartilage disease (e.g., cartilage damage, etc.), liver disease (e.g., chronic liver disease, etc.), pancreatic disease (e.g., diabetes, etc.) ), dental disease (e.g., periodontal disease, etc.), 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, the sheet-shaped cell culture or the like of the present disclosure is applied to a diseased site, and then the sheet-shaped 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.

The invention will now be described in more detail with reference to the following examples, which are intended to be illustrative of specific embodiments of the invention and are not intended to limit the invention.

Example 1. Manufacture of medical laminate having penetrating structure (1) Manufacture of sheet-like cell culture Cells obtained from skeletal muscle tissue aseptically collected from adult femur were seeded in a culture flask, and the number of myoblasts and the number of myoblasts were determined. To adjust the ratio of fibroblast numbers, they were grown in MCDB131 medium containing 20% FBS. The proliferated cells were detached from the culture flask with a proteolytic enzyme solution, collected, and then concentrated by centrifugation.

A DMEM/F12 medium containing 20% human serum was added to the φ10 cm temperature-responsive culture dish and allowed to stand overnight. The supplemented medium was then discarded.
10 mL of a cell suspension obtained by suspending 6.0×10 ⁶ to 6.1×10 ⁶ cells per 1 mL of DMEM/F12 medium containing 20% human serum was placed in a φ10 cm temperature-responsive culture dish (UpCell ( R), manufactured by Cellseed, culture substrate area: 56.7 cm ² ). After seeding, the cells were incubated at 37° C., 5% CO ₂ and subjected to a step of detaching the sheet-like cell culture from the culture substrate after 20 hours.

(2) Formation of Reinforcement Layer and Penetration Structure A fibrinogen solution and a thrombin solution were dropped simultaneously onto the exfoliated sheet-like cell culture. After allowing to stand for 5 minutes, excess non-gelled liquid was washed with HBSS(+), and formation of a reinforcing layer containing fibrin gel was confirmed.
A hole having a diameter of about 1 mm was formed with scissors in the periphery of the sheet-like cell culture on which the reinforcing layer containing fibrin gel was formed so as to penetrate the reinforcing layer and the sheet-like cell culture (Fig. 1). It was difficult to pass the suture needle through the sheet-like cell culture without holes, but the suture needle could be easily passed through the sheet-like cell culture with holes (Fig. 2).

Example 2. Manufacture of sheet-like cell culture with penetrating structures Cells obtained from skeletal muscle tissue collected aseptically from an adult femur are seeded in culture flasks, and the ratio of myoblasts to fibroblasts is adjusted. , were grown in MCDB131 medium containing 20% FBS. The proliferated cells were detached from the culture flask with a proteolytic enzyme solution, collected, and then concentrated by centrifugation.
A DMEM/F12 medium containing 20% human serum was added to the φ10 cm temperature-responsive culture dish and allowed to stand overnight. The supplemented medium was then discarded.
A cell suspension obtained by suspending 6.0×10 ⁶ to 6.1×10 ⁶ cells per 1 mL of DMEM/F12 medium containing 20% human serum was placed as an obstacle for penetrating structure formation. Then, 10 mL was seeded in a φ10 cm temperature-responsive culture dish (UpCell (R), manufactured by Cellseed, area of culture substrate: 56.7 cm ² ) (Fig. 3A). After seeding, the cells were incubated at 37° C., 5% CO ₂ and subjected to a step of detaching the sheet-like cell culture from the culture substrate after 20 hours. A sheet-like cell culture with holes as penetrating structures was produced (before peeling: FIG. 3B, after peeling: FIG. 3C).

REFERENCE SIGNS LIST 1 medical laminate 10 sheet-like cell culture 11 reinforcing layer 101 hole 102 notch

Claims (8)

A laminate for heart transplantation comprising a sheet-like cell culture layer containing at least one monolayer sheet-like cell culture containing myoblasts or cardiomyocytes and a reinforcing layer containing a biocompatible gel, the laminate for heart transplantation The laminate includes a reinforcing layer on the sheet-like cell culture layer, and has 1 to 4 reinforcing layer penetrating structures in the peripheral portion of the sheet-like cell culture layer, and the peripheral portion has the shape of the upper surface or the lower surface. It is a portion corresponding to a distance of 50% from the outer edge on the line segment connecting the outer edge to the center , and the penetrating structure is located in the portion where the reinforcing layer and the sheet-like cell culture layer are laminated. The laminate for heart transplantation, wherein the through structure is a hole or notch with a size of 200 μm to 5 mm, and the through structure is formed before being implanted into the heart. 2. The cardiac implant laminate of claim 1, wherein the penetrating structure is present only at the periphery. 3. The heart transplant laminate according to claim 1 or 2 , wherein the peripheral portion is a portion corresponding to a distance of up to 5% from the outer edge on a line segment connecting the outer edge to the center of the shape of the upper surface or the lower surface . The laminate for heart transplantation according to any one of claims 1 to 3, wherein the shape of the incision in the penetrating structure is linear or cross-shaped. The laminate for heart transplantation according to any one of claims 1 to 4 , wherein the sheet-like cell culture layer comprises only one monolayer of sheet-like cell culture. The laminate for heart transplantation according to any one of claims 1 to 4 , wherein the sheet-like cell culture layer comprises one or at least two monolayers of sheet-like cell culture. A pharmaceutical composition for heart transplantation, comprising the heart transplantation laminate according to any one of claims 1 to 6 and a needle for suture fixation. A kit for heart transplantation, which has 1 to 4 penetrating structures in the periphery and contains a sheet-like cell culture for heart transplantation containing myoblasts or cardiomyocytes and a biocompatible gel, wherein the periphery is an upper surface Or, on the line segment connecting the outer edge to the center of the shape of the lower surface, the part corresponding to the distance from the outer edge to 50%, and the penetrating structure is located in the part where the reinforcing layer and the sheet-like cell culture layer are laminated. , the above kit, wherein the penetrating structure is a hole or notch with a size of 200 μm to 5 mm, and the penetrating structure is formed before being implanted in the heart.

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