JP2021132584A - Evaluation method regarding detachment of sheet-like cell culture - Google Patents

Abstract

To provide a method for evaluating possibility of detaching sheet-like cell culture from a cell culture substrate while culturing sheet-like cell culture, culture solution for preparing sheet-like cell culture related to the method, a kit for preparing sheet-like cell culture, or the like.SOLUTION: A method for evaluating possibility of detaching sheet-like cell culture from a cell culture substrate while culturing the sheet-like cell culture includes: a step of measuring the concentration of cell adhesion component in culture solution; and a step of comparing a measured value and a predetermined threshold value.SELECTED DRAWING: None

Description

The present invention is a method for evaluating the possibility that a sheet-shaped cell culture is exfoliated from a cell culture substrate during culturing of a sheet-shaped cell culture, and for preparing a sheet-shaped cell culture related to the method. The present invention relates to a culture solution, a kit for preparing a sheet-shaped cell culture, and the like.

Sheet-shaped cell culture is known as one of the therapeutic means for regenerative medicine. In addition to being able to colonize a large amount of desired cells at the injured site, the sheet-shaped cell culture can be transplanted with an appropriately organized cell population according to the characteristics of the recipient tissue, and thus can be regenerated. Very useful for medical treatment.
The sheet-shaped cell culture is a cell culture group obtained by culturing desired cells on the surface of a cell culture base material and forming a layered structure by cells so as not to destroy the structure. Manufactured by peeling from the material. Generally, in order to form a sheet-like structure by a cell culture, cell culture is performed on the surface of a cell culture base material coated with a component having a function of adhering cells to the base material.
Although there are various such components, particularly when cells are cultured on a cell culture substrate whose surface is coated with vitronectin to form a sheet-like cell culture, the sheet-like cell culture is easily peeled off from the cell culture substrate. It is known that the cells can be recovered without damaging the sheet-like structure (Patent Document 1). According to this, a sheet-shaped cell culture can be obtained with a high recovery rate.

Japanese Unexamined Patent Publication No. 2014-143970

The present invention is a method for evaluating the possibility that a sheet-shaped cell culture is exfoliated from a cell culture substrate during culturing of a sheet-shaped cell culture, and for preparing a sheet-shaped cell culture related to the method. An object of the present invention is to provide a culture solution, a kit for preparing a sheet-shaped cell culture, and the like.

The present inventors confirm a phenomenon in which a sheet-shaped cell culture spontaneously exfoliates from a cell culture substrate during culturing of a sheet-shaped cell culture, and evaluate the possibility before such a phenomenon occurs. As a result of diligent research to develop the above, it was found that such a phenomenon occurs when the concentration of the cell-adhesive component in the culture solution is less than a predetermined threshold value, and the present invention has been completed.

That is, the present invention relates to the following.
[1] During culturing of a sheet-like cell culture, the sheet-like cell culture contains cells, which comprises a step of measuring the concentration of cell-adhesive components in the culture solution and a step of comparing the measured value with a predetermined threshold. A method for assessing the potential for exfoliation from a culture substrate.
[2] The method according to [1], wherein the step of measuring the concentration of the cell adhesive component in the culture solution is performed before the cell culture.
[3] The method according to [1] or [2], wherein the culture solution contains serum.
[4] The method according to any one of [1] to [3], wherein the sheet-shaped cell culture contains CD56-positive cells.
[5] The method according to any one of [1] to [4], wherein the cell adhesion component is fibronectin, vitronectin and / or thrombospondin 1.
[6] (i) The cell adhesion component is fibronectin and the predetermined threshold is 5 μg / mL, (ii) the cell adhesion component is vitronectin and the predetermined threshold is 3 μg / mL, and / or (Iii) The method according to any one of [1] to [5], wherein the cell adhesion component is thrombospondin 1 and a predetermined threshold value is 0.1 μg / mL.
[7] The method according to any one of [1] to [6], further comprising the step of adding the cell adhesive component to the culture solution so as to contain the cell adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold value. the method of.
[8] (i) The cell adhesion component is fibronectin and the predetermined concentration range is 5 to 40 μg / mL, and (ii) the cell adhesion component is vitronectin and the predetermined concentration range is 3 to 40 μg / mL. And / or (iii) the method according to [7], wherein the cell adhesion component is thrombospondin 1 and the predetermined concentration range is 0.1 to 5.7 μg / mL.
[9] (i) Fibronectin is contained in a concentration range of 5 to 40 μg / mL, (ii) Vitronectin is contained in a concentration range of 3 to 40 μg / mL, and / or (iii) Thrombospondin 1 is 0.1 to 0.1. A culture medium for preparing a sheet-like cell culture containing a concentration range of 5.7 μg / mL.
[10] A kit for preparing a sheet-shaped cell culture containing the culture medium and cell culture substrate according to [9].
[11] A method for producing a sheet-shaped cell culture, in which a step of measuring the concentration of a cell-adhesive component in a culture medium, a step of comparing the measured value with a predetermined threshold value, and a step of comparing cells in the culture medium. It includes the step of culturing to form a sheet-like cell culture, and further includes the step of adding the cell-adhesive component to the culture medium so as to contain the cell-adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold. The method.
[12] A sheet-like cell culture produced by the method according to [11].
[13] A method for treating a disease in a subject, comprising the step of administering a therapeutically effective amount of the sheet-shaped cell culture according to [12] to the subject in need thereof.

The sheet-shaped cell culture during the culture of the sheet-shaped cell culture by a method for evaluating the possibility of the sheet-shaped cell culture peeling from the cell culture substrate during the culture of the sheet-shaped cell culture of the present invention. It is possible to evaluate the possibility of exfoliation from the cell culture substrate, and further, when the measured value is less than a predetermined threshold value, the culture solution contains the cell-adhesive component in a predetermined concentration range. By adding it, it is possible to prevent such peeling. Further, by preventing such exfoliation, it is possible to prevent the sheet-shaped cell culture from curling up into a mass after exfoliation. As a result, the sheet-shaped cell culture can be stably formed and recovered on the cell culture substrate.

FIGS. 1A to 1C show the presence or absence of exfoliation of the sheet-shaped cell culture at each concentration of each cell adhesive component (fibronectin, vitronectin, thrombospondin 1) in the culture medium.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety.

[Method for evaluating the possibility that the sheet-shaped cell culture is detached from the cell culture substrate during the cultivation of the sheet-shaped cell culture]
One aspect of the invention includes sheet cells during culture of a sheet cell culture, comprising measuring the concentration of cell adhesive components in the culture and comparing the measured values with a predetermined threshold. The present invention relates to a method for evaluating the possibility of a culture being exfoliated from a cell culture substrate (hereinafter, may be referred to as “the method of the present invention”).
In the present invention, when the measured value is less than a predetermined threshold value, it is determined that the sheet-shaped cell culture may be exfoliated from the cell culture substrate during the culture of the sheet-shaped cell culture. On the other hand, when the measured value is equal to or higher than a predetermined threshold value, it is determined that there is no possibility that the sheet-shaped cell culture is exfoliated from the cell culture substrate during the culture of the sheet-shaped cell culture.
The method of the present invention may further include the step of adding the cell adhesive component to the culture medium so as to contain the cell adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold value. Thereby, the peeling can be prevented. Further, by preventing such exfoliation, it is possible to prevent the sheet-shaped cell culture from curling up into a mass after exfoliation.
In the present invention, "evaluation" includes predicting.

In the present invention, the "culture solution" is not particularly limited as long as it can maintain the survival of cells, but typically, those containing amino acids, vitamins, and electrolytes as main components can be used. In one aspect of the invention, the culture medium is based on a basal medium for cell culture. Such basal medium is not limited to, for example, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80. -7 etc. are included. Such a basal medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to that having a known composition, and may include one or two or more components added, removed, increased or decreased.

The concentration of the cell adhesive component in the culture medium is measured before or during the cell culture. Before cell culture, if the possibility of sheet-like cell culture peeling from the cell culture substrate during culture of sheet-like cell culture is predicted, measuring the concentration of cell-adhesive components in the culture medium is cell culture. It is done before. On the other hand, in the case of predicting the possibility that the sheet-shaped cell culture may peel off from the cell culture substrate during the culture of the sheet-shaped cell culture during the cell culture, the concentration measurement of the cell adhesive component in the culture solution is performed. Performed during cell culture. In addition, the culture medium may contain serum.
In the present invention, the "serum" can be derived from any organism. Such organisms include, but are not limited to, for example, humans, non-human primates, rodents (mouse, rat, hamster, guinea pig, etc.), dogs, cats, pigs, horses, cows, goats, sheep and the like. ..

In the present invention, the "cell adhesion component" may be any component normally used for adhering cells to the culture surface in cell culture technology, for example, collagen, fibronectin, laminin, vitronectin, proteoglycan. , Extracellular matrix such as glycosaminoglycan, cadoherin family, selectin family, integrin family, thrombospondin 1 and the like, but fibronectin, vitronectin and / or thrombospondin 1 are preferable.

The concentration of the cell adhesion component can be measured by using any general protein quantification method, for example, absorptiometry, Biuret method, Lowry method, fluorescence method, Bradford method, WST method and the like can be used. can. In addition, a protein quantification method using an antibody such as ELISA or Western blotting can also be used. Those skilled in the art can appropriately select a protein quantification method to measure the concentration of cell adhesion components.
In the present invention, the "predetermined threshold" refers to the lower limit concentration of the cell-adhesive component in the culture medium in which the sheet-shaped cell culture does not peel off from the cell culture substrate during the culture of the sheet-shaped cell culture. For example, the predetermined threshold is 5 μg / mL when the cell adhesion component is fibronectin, 3 μg / mL when the cell adhesion component is vitronectin, and the cell adhesion component is thrombospondin 1. The case is 0.1 μg / mL.

In the present invention, the "sheet-like cell culture" refers to cells connected to each other to form a sheet. Cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. 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 derived from cells, particularly from the cells that make up the sheet-like cell culture. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminate (multilayer), for example, two layers, three layers, etc. 4 layers, 5 layers, 6 layers, etc.) may be used. Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without showing a clear layered structure of the cells. For example, in the vertical cross section of a sheet-shaped cell culture, cells may be present in a non-uniformly (for example, mosaic-like) arrangement without being uniformly aligned in the horizontal direction. Sheet cell cultures include, for example, CD56 positive cells.

In the present invention, the "cell" can include any cell isolated from a living tissue. Non-limitingly, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin cells, synovial cells, chondrocytes, Examples include stem cells, myoblasts (eg, skeletal myoblasts), muscle satellite cells and the like. The cells preferably include CD56-positive cells in skeletal muscle tissue such as skeletal myoblasts and muscle satellite cells.
In the present invention, the "living body-derived tissue" can be derived from any organism. Such organisms include, but are not limited to, for example, humans, non-human primates, rodents (mouse, rat, hamster, guinea pig, etc.), dogs, cats, pigs, horses, cows, goats, sheep and the like. .. When a cell isolated from a biological tissue is used for transplantation, the biological tissue causes rejection by using an autologous cell isolated from the biological tissue collected from the transplant target (recipient) itself. It can be avoided. On the other hand, it is also possible to use heterologous cells or allogeneic non-self-derived cells isolated using a heterologous or allogeneic non-self-derived tissue.

The cell may be a heterologous cell or an allogeneic cell. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not cause rejection when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cells are autologous cells or allogeneic cells.

In the present invention, the "cell culture substrate" is not particularly limited as long as the cells can form a cell culture on the cells, and for example, containers of various materials and / or shapes, solids in the containers, or Includes semi-solid surface and the like. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such a container include, without limitation, a culture container having a bottom surface made of a cell culture substrate capable of forming a cell culture and a liquid-impermeable side surface. Specific examples of such a culture vessel include, but are not limited to, a cell culture dish, a cell culture bottle, and the like. The bottom surface 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. Further, the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices. The cell culture substrate may be prepared using the above-mentioned materials, or a commercially available one may be used.

Preferred cell culture substrates include, but are not limited to, for example, substrates with an adhesive surface suitable for the formation of sheet-like cell cultures, groups with a low adhesive surface suitable for the formation of spheroids. Examples include materials and / or substrates having a uniform well-like structure. Specifically, in the case of forming a sheet-like cell culture, for example, a substrate coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or a hydrophilic polymer on the surface thereof, and further cells. Examples thereof include a base material whose surface is coated with an adhesive component or the like. In addition, such a base material is commercially available (for example, Corning (registered trademark) TC-Treated Culture Dish, Corning, etc.). In the case of spheroid formation, for example, soft agar, temperature-responsive gel obtained by cross-linking poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG) (trade name: Mobiol gel), polyhydroxyethyl methacrylate (polymethacrylate) ( Examples include a base material coated with a non-cell adhesive compound such as hydrogel such as poly-HEMA) and 2-methacryloyloxyethyl phosphorischoline (MPC) polymer and / or a base material having a uniform uneven structure on the surface. Be done. Such substrates are also commercially available (eg, EZSPHERE®, etc.). The cell culture substrate may be transparent or opaque in whole or in part.

The surface of the cell culture substrate may be coated with a material whose physical properties change in response to irritation, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl (Amid, etc.), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), having cyclic groups (eg, N, N-dialkylacrylamide, N, N-diethylacrylamide, etc.) Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidin, 4- (1-oxo-2-propenyl) -morpholin, 1, -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether) or a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether), a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-21186 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell®) and can be used in the production methods of the present disclosure.

The cell culture substrate may have various shapes. The area thereof 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 , and the like. For example, a circular culture dish having a diameter of 10 cm can be mentioned as a cell culture substrate. In this case, the area is 56.7 cm ² . The culture surface may be flat or may have an uneven structure. When it has a concavo-convex structure, it is preferable that it has a uniform concavo-convex structure.

In order to form a denser sheet-like cell culture, the cell culture substrate may be coated with blood-derived components and / or cell adhesion components. "Coated with blood-derived components and / or cell-adhesive components" means a state in which blood-derived components such as serum and / or cell-adhesive components are attached to the surface of the cell culture substrate. Such a state can be obtained without limitation, for example, by treating the cell culture substrate with a blood-derived component and / or a cell adhesion component. Treatment with blood-derived components and / or cell-adherent components includes, for example, contacting serum and / or cell-adhesive components with a cell culture substrate and, if necessary, incubating for a predetermined period of time. The serum and / or cell-adhesive component used for coating may be the same type of serum as the seeded cell origin (homogeneous serum) or a different type of serum (heterologous serum), for example FBS, but is preferable. It is an allogeneic serum, more preferably a serum (autologous serum) obtained from an individual from which the seeded cells are derived. Other blood-derived components include albumin and platelet lysates.

Seeding of cells into a cell culture substrate can be performed by any known method and condition. Seeding of cells into a cell culture substrate may be performed, for example, by injecting a cell suspension in which cells are suspended in a culture medium into a cell culture substrate (culture container). For injection of the cell suspension, an instrument suitable for the injection operation of the cell suspension, such as a dropper or a pipette, can be used. The seeding density of the cells is set to a density capable of forming a sheet-shaped cell culture, and the density may vary depending on the desired cells, but those skilled in the art select an appropriate density from methods known in the art. can do.

Examples of higher densities include, for example, a density that reaches confluence, that is, a density at which cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, cells come into contact with each other when seeded. It can be as dense as expected, the density at which contact inhibition occurs, or the density at which cell proliferation is substantially stopped by contact inhibition or higher. The upper limit of the seeding density is not particularly limited, but if the seeding density is excessively high, more cells will die, resulting in inefficiency. In one aspect of the present disclosure, the seeding densities are about 5.0 × 10 ⁵ pcs / cm ² to about 1.0 × 10 ⁷ pcs / cm ² , about 5.0 × 10 ⁵ pcs / cm ² to about 5. 0 x 10 ⁶ pieces / cm ² , about 5.0 x 10 ⁵ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁶ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 3.0 x 10 ⁶ Pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁶ pieces / cm cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , about 2.0 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , Approximately 2.0 x 10 ⁶ pcs / cm ² to Approximately 5.0 x 10 ⁶ pcs / cm ² , Approximately 2.0 x 10 ⁶ pcs / cm ² to Approximately 3.0 x 10 ⁶ pcs / cm ² etc. could be. In one preferred embodiment, it is about 7.5 × 10 ⁵ pieces / cm ^{2 to} 3.0 × 10 ⁶ pieces / cm ² , and in another preferred aspect, it is about 1.76 × 10 ⁶ pieces / cm ² to. Approximately 2.33 × 10 ⁶ pieces / cm ² .

The seeded cell population may contain other cells (fibroblasts) as long as they contain the desired cells, and if the desired cells are skeletal myoblasts or muscle satellite cells, for example fibers. It may further include blast cells, vascular endothelial cells and the like. As the cell population, the cell population collected from the tissue may be used as it is, or may be used after cryopreservation, pre-culture, removal of fibroblasts, or the like. In a preferred embodiment, the seeded cell population was separated from the biological tissue, seeded on a cell culture substrate (preferably on a planar cell culture substrate), adherently cultured, and then recovered. It is a cell population. Cryopreservation and thawing may be performed before or after such adhesive culture.
In such an adhesive culture step, the culture conditions and the like may be the same as those for normal adhesive culture. For example, a commercially available culture vessel for adhesive culture may be used for culturing _{under 37 ° C. and 5% CO 2} conditions. The seeding density of the cells may be any density as long as it does not interfere with the adhesion between the cells and / or the formation of the adhesion between the cells and the cell culture substrate, and may be, for example, a subconfluent density. , The density to reach confluence or higher. The culturing time may be such that adhesion between cells and / or adhesion between cells and a cell culture substrate is formed, and specifically, for example, 2 to 24 hours, 2 to 12 hours, and 2 to 6 hours. The time may be about 2 to 4 hours.

In the present invention, "peeling" refers to a state in which all or part of the sheet-shaped cell culture is physically separated from the surface of the cell culture substrate, and the sheet-shaped cell culture forms a sheet on the cell culture substrate. It also includes a state in which it is fragmented without forming and floats in the culture medium.
In the present invention, the "predetermined concentration range" is the culture of the sheet-shaped cell culture from the lower limit concentration of the cell-adhesive component in which the sheet-shaped cell culture does not peel off from the cell culture substrate during the culture of the sheet-shaped cell culture. It refers to the range up to the upper limit concentration of the cell adhesive component that can maintain the state in which the sheet-shaped cell culture does not peel off from the cell culture substrate. For example, the predetermined concentration range may be 5 μg / mL or more when the cell adhesion component is fibronectin, preferably 5 to 40 μg / mL, particularly preferably 10 to 35 μg / mL, and further. It is preferably 15 to 30 μg / mL. For example, the predetermined concentration range may be 3 μg / mL or more when the cell adhesion component is vitronectin, preferably 3 to 40 μg / mL, particularly preferably 8 to 35 μg / mL, and further. It is preferably 13 to 30 μg / mL. For example, the predetermined concentration range may be 0.1 μg / mL or more when the cell adhesion component is thrombospondin 1, preferably 0.1 to 5.7 μg / mL, and particularly preferably 0.1 μg / mL. It is 0.2 to 4.0 μg / mL, more preferably 0.3 to 3.0 μg / mL.

In a preferred embodiment of the method of the present invention, the concentration measurement of the cell adhesive component in the culture medium is performed before the cell culture, the culture medium contains serum, and the sheet-like cell culture contains CD56-positive cells.
In a more preferred embodiment of the method of the invention, the concentration of cell adhesion components in the culture medium is measured prior to cell culture, the culture medium contains serum, the sheet cell culture contains CD56 positive cells, and (I) The cell adhesion component is fibronectin and the predetermined threshold is 5 μg / mL, (ii) the cell adhesion component is bitronectin and the predetermined threshold is 3 μg / mL, and / or (iii). The cell adhesion component is thrombospondin 1 and the predetermined threshold is 0.1 μg / mL.

[Culture solution for preparing sheet-shaped cell culture]
Another aspect of the invention is that (i) fibronectin is contained in a concentration range of 5-40 μg / mL, (ii) vitronectin is contained in a concentration range of 3-40 μg / mL, and / or (iii) thrombospondin 1 The present invention relates to a culture solution for preparing a sheet-like cell culture (hereinafter, may be referred to as “culture solution of the present invention”) containing a concentration range of 0.1 to 5.7 μg / mL.

[Kit for preparing sheet cell culture]
Another aspect of the present invention relates to a kit for preparing a sheet-like cell culture containing the culture medium and cell culture substrate of the present invention (hereinafter, may be referred to as "kit of the present invention").
In the present invention, "kit" refers to a kit containing various components for preparing a sheet-shaped cell culture, for example, the culture medium, cell culture substrate, instruments (for example, pipette, dropper) of the present invention. , Tweezers, etc.), instructions (eg, instructions for use, media on which information about the method of the invention is recorded, such as flexible discs, CDs, DVDs, Blu-ray discs, memory cards, USB memories, etc.) and the like. The kit of the present invention may be disposable.

[Method for producing sheet-shaped cell culture]
Another aspect of the present invention is the step of measuring the concentration of cell-adhesive components in the culture medium, the step of comparing the measured value with a predetermined threshold, and the sheet-like cell culture by culturing the cells in the culture medium. Production of sheet-like cell cultures comprising the step of forming a substance and further comprising the step of adding a cell-adhesive component to the culture medium so as to contain a cell-adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold. The present invention relates to a method (hereinafter, may be referred to as “the production method of the present invention”).

[Sheet-shaped cell culture]
Another aspect of the present invention relates to a sheet-shaped cell culture (hereinafter, may be referred to as "sheet-shaped cell culture of the present invention") produced by the production method of the present invention.

[How to treat the disease in the subject]
Another aspect of the present invention is a method of treating a disease in a subject, comprising the step of administering a therapeutically effective amount of the sheet cell culture of the present invention to a subject in need thereof (hereinafter, "therapeutic method of the present invention"). May be written).

In the present invention, the "therapeutically effective amount" is an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-shaped cell cultures, etc.). It is an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, the therapeutically effective amount is preferably an amount that does not cause an adverse effect exceeding the benefit of administration. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, a disease model animal, or the like, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining a therapeutically effective amount.
In the present invention, the "object" can be any organism. Such organisms include, but are not limited to, for example, humans, non-human primates, rodents (mouse, rat, hamster, guinea pig, etc.), dogs, cats, pigs, horses, cows, goats, sheep and the like. ..

In the present invention, "treatment" refers to those comprising all kinds of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, transient remission or prevention of a disease, eg, tissue. Includes medically acceptable interventions for a variety of purposes, including delaying or stopping the progression of a disease associated with an abnormality, regressing or eliminating a lesion, preventing the onset or recurrence of the disease, and the like.
In the treatment method of the present invention, a component that enhances the viability, engraftment and / or function of the sheet-shaped cell culture, other active ingredients useful for treating the target disease, and the like are used for the sheet-shaped cell of the present invention. It can be used in combination with cultures and the like.

The treatment method of the present invention may further include the step of producing the sheet-shaped cell culture of the present invention according to the production method of the present invention. The treatment method of the present invention is a biological tissue that is a source of skeletal myoblasts and / or myosatellite cells for producing a sheet-like cell culture from a subject before the step of producing the sheet-like cell culture. May include an additional step of harvesting. For example, the subject from which the cells or skeletal myoblasts and / or the tissue from which the muscle satellite cells are supplied is collected is the same individual as the subject to which the cell culture, composition, or sheet-like cell culture is administered. be. For example, the target for collecting skeletal myoblasts and / or myoblasts or tissues from which skeletal myoblasts and / or muscle satellite cells are supplied may be cell cultures, compositions, sheet-like cell cultures, or the like. The subject to be administered is a separate body of the same species. For example, the subject from which the skeletal myoblasts and / or myoblasts or the tissue that is the source of the skeletal myoblasts and / or muscle satellite cells is collected is different from the subject to which the sheet-like cell culture or the like is administered. It is an individual.

Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, direct application to tissues, and the like. The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained. When applied to a tissue, a cell culture, a composition, a sheet-like cell culture, or the like may be fixed to the target tissue by a locking means such as a suture or a staple.
The present invention will be described in more detail with reference to the following examples, but these show specific specific examples of the present invention, and the present invention is not limited thereto.

Example 1. Specific factors involved in the formation and exfoliation of sheet-shaped cell cultures Human skeletal myoblasts were proliferated in MCDB131 medium (manufactured by Life Technologies Japan) containing 20% (v / v) FBS, and then recovered and collected. It was cryopreserved using a cryopreservation solution containing% DMSO (manufactured by Genuine Chemicals). The cryopreserved cells were thawed in a water bath set at about 37 ° C., washed with a buffer solution containing 0.5% human serum albumin, and used for the formation of the following sheet-shaped cell culture.

Each culture medium prepared by mixing the adhesion factors (FN: fibronectin, VN: vitronectin, TSP-1: thrombospondin-1, CO-4: collagen type 4, ALB: albumin) shown in Table 1 in DMEM / F12 medium was added. , 0.78 mL each was added to a temperature-responsive culture medium (UpCell® Φ3.5 cm, Cellseed) and incubated for 12 to 72 hours (set value: 37 ° C., 5% CO ₂ ). After removal of the culture medium from the temperature-responsive culture dish, were seeded such that 9.3 × 10 ⁶ pieces of skeletal myoblasts, 37 ° C., 12 to 26 hours sheeting cultured under 5% CO ₂ went. After sheet-forming culture, the medium was removed, 1.55 mL of HBSS (+) cooled to 4 ° C. (Thermo Fisher Scientific) was added, and the cells were allowed to stand for 10 minutes, and then gently pipetting to form sheet-like cells. The culture was completely exfoliated.

As a result, it was found that the sheet-like cell culture was not formed in the absence of vitronectin, or the cultured cells spontaneously exfoliated during the culture. It was also found that a sheet-like cell culture can be formed by the presence of vitronectin regardless of the presence or absence of other adhesion factors.

Example 2. Identification of Vitronectin Concentration that Causes Spontaneous Peeling of Sheet Cell Culture (1) Production of Sheet Cell Culture A sheet cell culture was prepared using skeletal myoblasts prepared by a conventional method from human skeletal muscle. 20v / v% human-derived serum-containing DMEM / F12 medium is prepared, and each medium is diluted 2-fold with DMEM / F12 medium to obtain 10v / v% human-derived serum-containing DMEM / F12 medium and 5v / v%. Human-derived serum-containing DMEM / F12 medium was prepared. 0.78 mL of each of the prepared media was added to a temperature-responsive culture dish (UpCell® Φ3.5 cm, CellSeed) and incubated for 12 to 72 hours (set value: 37 ° C., 5% CO ₂ ). .. After removing each culture medium from the temperature-responsive culture dish, skeletal myoblasts were suspended and seeded in a medium having a concentration of ^{9.3 × 10 6} _{at 37 ° C. and 5% CO 2} . Sheet culture was performed for 12 to 26 hours. After sheet-forming culture, the medium was removed, 1.55 mL of HBSS (+) cooled to 4 ° C. (Thermo Fisher Scientific) was added, and the cells were allowed to stand for 10 minutes, and then gently pipetting to form sheet-like cells. The culture was completely exfoliated.

Further, as a comparative example, a medium having a composition obtained by removing the adhesive factor from the above medium (hereinafter referred to as “control medium”) was placed in a temperature-responsive culture medium (UpCell® Φ3.5 cm, CellSeed). 78 mL was added and incubated for 12 to 72 hours (set value: 37 ° C., 5% CO ₂ ). After removing the culture medium from the temperature-responsive culture dish, suspended and seeded in control medium so that 9.3 × 10 ⁶ pieces of skeletal myoblasts, 37 ℃, 5% CO ₂ under 12 to 26 Time sheet culture was performed.

As a result, detachment of cultured cells was observed during culturing in a part of DMEM / F12 medium containing 5v / v% human serum having a low human serum content. On the other hand, sheet-like cell cultures could be formed in media other than the above. Since the sheet-shaped cell culture was not exfoliated, it did not curl up into a mass and was obtained as a flat structure. In addition, it was confirmed that in the sheet-shaped cell culture of the comparative example obtained using the control medium, the cultured cells were exfoliated during the culture and curled up into a mass.

(2) Measurement of adhesion factor in sheet preparation medium Regarding the human-derived serum used for the medium preparation in (1), the concentrations of fibronectin, vitronectin and thrombospondin-1 contained in the serum were determined by the ELISA method and collagen type 4. The concentration was measured by the latex suspension turbidimetry method. From the measurement results of each adhesion factor contained in human-derived serum, the concentration of each adhesion factor contained in the medium prepared in (1) was calculated. When the vitronectin concentration (n = 3) in the medium in which the cultured cells were detached during the culture of (1) and the vitronectin concentration in the medium in which the sheet-like cell culture was formed were compared, the culture cells were cultured. The peeled / non-peeled boundary concentration was 3 μg / mL. From these results, it was found that the presence of vitronectin in the medium of 3 μg / mL or more enables the formation of a sheet-like cell culture without exfoliation of the cultured cells during the culture. Since the sheet-shaped cell culture did not peel off, it was found that the sheet-like cell culture did not curl up into a mass and was obtained as a flat structure.

The various features of the invention described herein can be combined in various ways, and all aspects obtained by such combinations, including combinations not specifically described herein, are of the present invention. It is within the range. Those skilled in the art also understand that a number of various modifications that do not deviate from the spirit of the present invention are possible, and equivalents containing such modifications are also included in the scope of the present invention. Therefore, it should be understood that the embodiments described herein are merely exemplary and are not intended to limit the scope of the invention.

Claims (13)

During the culture of the sheet-like cell culture, the sheet-like cell culture is a cell culture substrate, which comprises a step of measuring the concentration of the cell-adhesive component in the culture medium and a step of comparing the measured value with a predetermined threshold. A method for assessing the likelihood of peeling from. The method according to claim 1, wherein the step of measuring the concentration of the cell adhesive component in the culture solution is performed before the cell culture. The method according to claim 1 or 2, wherein the culture medium contains serum. The method according to any one of claims 1 to 3, wherein the sheet-like cell culture contains CD56-positive cells. The method according to any one of claims 1 to 4, wherein the cell adhesive component is fibronectin, vitronectin and / or thrombospondin 1. (I) The cell adhesion component is fibronectin and the predetermined threshold is 5 μg / mL, (ii) the cell adhesion component is vitronectin and the predetermined threshold is 3 μg / mL, and / or (iii). The method according to any one of claims 1 to 5, wherein the cell adhesion component is thrombospondin 1 and a predetermined threshold value is 0.1 μg / mL. The method according to any one of claims 1 to 6, further comprising the step of adding the cell adhesive component to the culture medium so as to contain the cell adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold value. (I) The cell adhesion component is fibronectin and the predetermined concentration range is 5 to 40 μg / mL, and (ii) the cell adhesion component is vitronectin and the predetermined concentration range is 3 to 40 μg / mL. The method according to claim 7, wherein the (iii) cell adhesion component is thrombospondin 1 and the predetermined concentration range is 0.1 to 5.7 μg / mL. (I) Fibronectin in a concentration range of 5-40 μg / mL, (ii) Vitronectin in a concentration range of 3-40 μg / mL, and / or (iii) Thrombospondin 1 in a concentration range of 0.1 to 5.7 μg. A culture medium for preparing a sheet-like cell culture containing a concentration range of / mL. A kit for preparing a sheet-like cell culture containing the culture medium and cell culture substrate according to claim 9. A method for producing a sheet-shaped cell culture, in which a step of measuring the concentration of a cell-adhesive component in a culture medium, a step of comparing the measured value with a predetermined threshold, and a step of culturing cells in the culture medium are performed. The method comprising the step of forming a sheet-like cell culture, further comprising the step of adding the cell adhesive component to the culture medium so as to contain the cell adhesive component in a predetermined concentration range when the measured value is less than a predetermined threshold. A sheet-like cell culture produced by the method according to claim 11. A method for treating a disease in a subject, comprising the step of administering a therapeutically effective amount of the sheet cell culture according to claim 12 to the subject in need thereof.

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