JP2020078320A - Method for producing sheet-like cell culture - Google Patents

Abstract

To provide sheet-like cell culture having excellent strength, safety and effectiveness.SOLUTION: A method for producing sheet-like cell culture includes a process of disseminating cells with density by which sheet-like cell culture can be formed without substantially propagating the cells on a culture substrate coated with serum, and sheet-like cell culture produced by the producing method. More specifically, the method includes: a process of disseminating cells on the culture substrate coated with serum at the cell density of 1.0×10to 1.7×10pcs./cm; and a process of culturing disseminated cells to form a sheet-like cell culture without substantially propagating the cells.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a sheet-shaped cell culture and a sheet-shaped cell culture produced by the production method.

The heart transports oxygen throughout the body by its pump function, and plays an essential role in sustaining life. Therefore, heart disease is the leading cause of death, and in ischemic heart diseases such as angina and myocardial infarction, sufficient oxygen is not distributed to myocardial tissues, and if this state continues for a long time, necrosis occurs. Since adult cardiomyocytes have poor self-renewal ability, once necrotized, they do not regenerate and fall into heart failure. When such a condition is reached, the only treatment currently available is to wear a left ventricular assist artificial heart or finally receive a heart transplant.
Under such circumstances, cell transplantation to the heart is being researched as a new treatment method for heart failure. Up to now, utilization of fetal cardiomyocytes, skeletal myoblasts, ES cells, bone marrow-derived cells, etc. has been reported in experiments using animals.

Of these, skeletal myoblasts are present in skeletal muscle tissue, are activated when the muscle tissue is damaged, and proliferate and fuse to regenerate skeletal muscle tissue. Skeletal myoblasts have many parts similar in structure and function to the myocardium, and instead of proliferating myocardial cells that cannot repair infarct lesions, they can physically fill the lesions and improve the pumping function of the heart. Adaptation to heart disease has been expected from the idea of whether or not there is. On the other hand, in recent years, despite its effectiveness, the engraftment rate of transplanted cells is low, so that the humoral factor produced by transplanted cells acts on myocardial tissue to suppress its effect on cell death and angiogenesis. It is also considered to show sex. Further, in the case of cell transplantation using skeletal myoblasts, there is an advantage that it is possible to avoid the problem of immunocompatibility by using the cells of the patient himself, and clinical trials have been conducted overseas.
However, when transplanted cells are administered to a tissue in the state of a cell suspension, there are problems such as poor injection efficiency of transplanted cells, obstacles due to puncture of recipient tissue, and difficulty in extensive tissue repair. It has been pointed out that, in order to overcome this, cell constructs using scaffolds and cell sheets in which cells are formed into sheets have been developed.

It is known that skeletal myoblasts can also be formed into a cell sheet by growing them in an appropriate culture medium for a certain period of time (Patent Document 1). In the same document, the skeletal myoblast sheet is formed by seeding cells at a density of about 9×10 ⁴ cells/cm ² and proliferating for a sufficient period in a culture medium containing growth factors and the like. However, in such a production method, there is a safety problem that additional factors such as a growth factor, a steroid agent, and a heterologous serum contained in the culture medium may remain in the product. Further, in the conventional manufacturing method, there is a problem that the formed cell sheet has poor physical strength and is difficult to handle during washing operation, transplant operation, and transportation. Furthermore, in recent years, the action of humoral factors produced from transplanted cells has been attracting attention as an action mechanism of cell transplantation as a therapeutic method. From transplanted cells, in addition to factors related to the effectiveness of treatment, inflammatory cytokines Since there is a possibility that factors that have a detrimental effect on the recipient, etc. may also be produced, many factors related to efficacy, such as inhibition of angiogenesis and cell death, for clinical application of cell sheets. It is desirable to obtain a cell sheet that produces and has suppressed inflammatory cytokine production. Therefore, there has been a demand for the production of a cell sheet having high strength, safety and efficacy, which does not have the above-mentioned problems of the conventional techniques.

Special Table 2007-528755 Publication

  The present invention aims to produce a sheet-shaped cell culture excellent in strength, safety and effectiveness, which does not have the problems of the prior art.

The present inventors have conducted diligent research to solve the above problems, and when skeletal myoblasts are seeded at a density of more than 1.0×10 ⁶ cells/cm ² , the seeded cells are completely formed into a sheet. It was impossible to form a sheet-like cell culture with sufficient strength before the formation of the cells, but as a pretreatment, the culture substrate was coated with serum and the high-density cells were seeded. , It has been found that it is possible to form a sheet-shaped cell culture without spontaneous exfoliation. Further, in the case of a lower seeding density of 3.5×10 ⁵ cells/cm ² , the use of the serum-coated culture dish makes it possible to compare with the case where the culture dish without such treatment is used. It has been found that the number of cells constituting the sheet increases, that is, the cell recovery rate increases and the strength of the sheet also increases. After further research, sheet-like cell cultures of non-nucleated skeletal myoblasts showed more angiogenesis than sheet-like cell cultures of skeletal myoblasts that had undergone differentiation. Although the production of factors having an effect on efficacy is low, the production of inflammatory cytokines is low, and the degree of differentiation was evaluated in the sheet-shaped cell culture prepared by the above-mentioned production method. The present invention has been completed by finding that no stain is observed.

Therefore, the present invention is shown in the following (1) to (10).
(1) A method for producing a sheet-shaped cell culture, which comprises a step of seeding a cell having a density capable of forming a sheet-shaped cell culture without substantially growing on a culture substrate coated with serum.
(2) The production method according to (1) above, wherein the cell density is 3.5×10 ⁵ or more and less than 3.4×10 ⁶ cells/cm ² .
(3) The production method according to (1) or (2) above, further including the step of isolating the sheet-shaped cell culture from the culture substrate.
(4) The production method according to any one of (1) to (3) above, which further comprises a step of washing the isolated sheet-shaped cell culture.
(5) The production method according to any of (1) to (4) above, wherein the sheet-shaped cell culture contains skeletal myoblasts whose differentiation into myotubes is suppressed.
(6) A sheet-shaped cell culture produced by the method according to any one of (1) to (5) above.
(7) The sheet-shaped cell culture according to (6) above, which has a sufficient physical strength for washing operation.
(8) The sheet-shaped cell culture according to the above (6) or (7), which has a physical strength capable of withstanding a transplantation operation.
(9) The sheet-shaped cell culture according to any of (6) to (8) above, which is used for treating a disease or injury.
(10) The sheet-shaped cell culture according to any of (6) to (9) above, which is substantially free from production-derived impurities.

  According to the production method of the present invention, a sheet-shaped cell culture having sufficient physical strength to withstand a washing operation such as immersion stirring and a transplanting operation such as taking out, holding and transferring can be obtained by a conventional production method. It is possible to perform the manufacturing/transplanting operation more easily and surely than the cell sheet. Moreover, since the sheet-shaped cell culture can be obtained with a high cell recovery rate by the production method of the present invention, the cells to be used can be effectively used, which contributes to cost reduction. Further, the sheet-shaped cell culture obtained by the production method of the present invention produces a large amount of factors having an action relating to the efficacy of angiogenesis and the like, while producing a small amount of inflammatory cytokines, and therefore has a therapeutic effect. It is extremely useful and clinically extremely useful as well as safe for recipients. Therefore, a great contribution can be expected in human medicine and veterinary medicine.

FIG. 1 is a graph showing the measured CK activity when cells were seeded at various densities. FIG. 2 is a graph comparing the amounts of various inflammatory cytokines in undifferentiated sheet-shaped cell culture supernatant and multinucleated sheet-shaped cell culture supernatant. FIG. 3 is a graph comparing the amounts of various humoral factors in the undifferentiated sheet-shaped cell culture supernatant and the multinucleated sheet-shaped cell culture supernatant.

The present invention relates to a method for producing a sheet-shaped cell culture, which comprises a step of seeding a cell having a density capable of forming a sheet-shaped cell culture without substantially growing on a culture substrate coated with serum. ..
In the present invention, the “culture substrate” is not particularly limited as long as cells can form a sheet-like cell culture thereon, and examples thereof include containers of various materials, solid or semi-solid containers in the container. Including the surface. The container preferably has a structure/material that does not allow liquid such as a culture solution to pass through. Examples of such a material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metal (for example, 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 containers include, but are not limited to, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside. Examples of the solid surface include plates and containers made of various materials as described above, and examples of the semi-solid surface include gel and a soft polymer matrix.

The surface of 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 (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl Amide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivative (for example, N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), having a cyclic group ( (Meth)acrylamide derivative (for example, 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 consisting of a homopolymer or copolymer of vinyl ether derivative (eg, methyl vinyl ether), a light-absorbing polymer having an azobenzene group, a vinyl derivative of triphenylmethane leuco hydroxide and an acrylamide monomer Known materials such as copolymers with the body and photoresponsive materials such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-211865 and JP-A-2003-33177). ). By imparting a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity and hydrophobicity, can be changed, and the detachment of the cell culture adhered onto the materials can be promoted.
The culture substrate may have various shapes, but is preferably flat. The area is not particularly limited, but is typically 1 to ²⁰⁰ cm ² , preferably ² to 100 cm ² , and more preferably 3 to 50 cm ² .

In the present invention, the culture substrate is coated (coated or coated) with serum. "Coated with serum" means a state in which serum components are attached to the surface of the culture substrate. Such a state is not limited and can be obtained, for example, by incubating a culture substrate with serum. Incubation includes contacting serum with the culture substrate. Heterologous serum and homologous serum can be used as serum. Heterologous serum means, when transplanting a cell culture, serum derived from an organism of a different species than the recipient. For example, when the recipient is human, serum derived from bovine or equine, such as fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc., corresponds to the heterologous serum. In addition, "allogeneic serum" means serum derived from an organism of the same species as the recipient. For example, when the recipient is human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum, ie serum derived from the recipient.
Serum for coating a culture substrate is commercially available or can be prepared by a standard method from blood collected from a desired organism. Specifically, for example, a method may be mentioned in which the collected blood is allowed to stand at room temperature for about 20 to 60 minutes to coagulate, this is centrifuged at about 1000 to 1200×g, and the supernatant is collected.

When incubating on a culture substrate, the serum may be used as a stock solution or diluted. Dilution may be any medium, including but not limited to water, saline, various buffers (eg PBS, HBS, etc.), various liquid media (eg DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199 etc.), L15, SkBM, RITC80-7, DMEM/F12 etc.) can be performed. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, 0.5 to 90% (v/v), preferably 1 to 60% (v/v), and more preferably Is 5 to 40% (v/v).
The incubation time is not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, and further preferably 6 to 12 hours. It's time. The incubation temperature is also not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, 0 to 60°C, preferably 4 to 45°C, more preferably room temperature to 40°C.

  Serum may be discarded after incubation. As a method for discarding serum, a conventional liquid discarding method such as suction with a pipette or decantation can be used. In one aspect of the present invention, the culture substrate may be washed with a serum-free washing solution after the serum is discarded. The serum-free washing liquid 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, and examples thereof include, without limitation, water, physiological saline, various buffers. Liquid (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7. , DMEM/F12, etc.) and the like. As the washing method, a conventional culture substrate washing method, for example, without limitation, a method of adding a serum-free washing solution on the culture substrate, stirring the mixture for a predetermined time (for example, 5 to 60 seconds), and then discarding it is used. be able to.

  In the present invention, the culture substrate may be coated with a growth factor. Here, "growth factor" means any substance that promotes the proliferation of cells as compared with the absence thereof, and examples thereof include epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and fibroblast. Includes cell growth factor (FGF) and the like. In the culture substrate coating method using a growth factor, the discarding method, and the washing method, the dilution concentration at the time of incubation is, for example, 0.0001 μg/mL to 1 μg/mL, preferably 0.0005 μg/mL to 0.05 μg/mL, It is basically the same as serum except that it is more preferably 0.001 μg/mL to 0.01 μg/mL.

  In the present invention, the culture substrate may be coated with a steroid agent. Here, the "steroid drug component" refers to a compound having a steroid nucleus, which can exert an adverse effect on the living body such as adrenocortical dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the method for coating a culture substrate with a steroid agent, the method for discarding and the method for washing, the dilution concentration during incubation is, for example, 0.1 μg/mL to 100 μg/mL, preferably 0.4 μg/mL to 40 μg/mL as dexamethasone. , And more preferably 1 μg/mL to 10 μg/mL except that it is basically the same as serum.

  The culture substrate may be coated with any one of serum, growth factor and steroid, but any combination of these, ie serum and growth factor, serum and steroid, or serum and growth factor and steroid. You may coat with the combination of. When coating with a plurality of components, these components may be mixed and coated simultaneously, or may be coated 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 4° C. or lower, preferably −20° C. or lower, more preferably −80° C. or lower.

In the production method of the present invention, cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing. "Density capable of forming a sheet-like cell culture without substantially growing" means forming a sheet-like cell culture when cultured in a non-proliferating culture medium containing substantially no growth factor It means the cell density that can be achieved. For example, in the case of skeletal myoblasts, in the conventional method using a culture solution containing growth factors, cells having a density of about 6,500 cells/cm ² are seeded on a plate to form a sheet-shaped cell culture. However, even if cells having such a density are cultured in a culture medium containing no growth factor, a sheet-shaped cell culture cannot be formed. Therefore, the seeding density in the production method of the present invention is higher than that in the conventional method using the culture solution containing the growth factor. Therefore, in the present specification, the seeding density may be simply referred to as “high density”. Specifically, for example, the density is typically 300,000 cells/cm ² or more for skeletal myoblasts. The upper limit of the seeding density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not differentiate, but for skeletal myoblasts, it is less than 3.4×10 ⁶ cells/cm ² .

Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is 3.0×10 ^{5 to} 3.4×10 ⁶ cells/cm ² in one aspect, and 3.5×10 ^{5 to} 3 in another aspect. .4 × 10 ⁶ cells / cm ^2, yet another embodiment 1.0 × ^{10 6} ~3.4 × ¹⁰ 6 cells / cm ^2, in yet another aspect 3.0 × ¹⁰ 5 ~1.7 × ^{10 6} / cm ^2, in another embodiment 3.5 × ¹⁰ 5 ~1.7 × ^{10 6} cells / cm ^2, in yet another aspect 1.0 × ^{10 6} ~1.7 × ¹⁰ 6 cells / cm ² Is. The above range may include both the upper limit and the lower limit, or either one of them, as long as the upper limit is less than 3.4×10 ⁶ pieces/cm ² . Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0×10 ⁵ cells/cm ² or more and less than 3.4×10 ⁶ cells/cm ² (including the lower limit and not the upper limit. ), 3.5×10 ⁵ pieces/cm ² or more and less than 3.4×10 ⁶ pieces/cm ² (including the lower limit and not including the upper limit), 1.0×10 ⁶ pieces/cm ² or more and 3.4× 10 less than ⁶ / cm ² (including the lower and the upper limit is not included), 1.0 × 10 ⁶ cells / cm ² ultra 3.4 × 10 than ⁶ / cm ² (lower limit also contains no upper limit), 1 It may be more than 0.0×10 ⁶ pieces/cm ^{2 and} 1.7×10 ⁶ pieces/cm ² or less (the lower limit is not included, but the upper limit is included). Those skilled in the art can appropriately determine the cell density suitable for the present invention by experimenting with cells other than skeletal myoblasts according to the teaching of the present specification.

  During the culture period, the cells may or may not proliferate, but even if they proliferate, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start differentiation when they become confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a cell culture but does not transfer to differentiation. In a preferred embodiment of the present invention, cells do not grow beyond the bounds of measurement error. Whether or not the cells have proliferated can be evaluated, for example, by comparing the number of cells at the time of seeding with the number of cells after formation of the cell culture. In this embodiment, the number of cells after formation of the cell culture is typically 300% or less, preferably 200% or less, more preferably 150% or less, still more preferably 125% or less, particularly preferably the number of cells at the time of seeding. Is 100% or less.

In the present invention, culturing of cells is performed under conditions suitable for the target cells to form a sheet-shaped cell culture.
In the present invention, the “sheet-shaped cell culture” refers to cells in which cells are connected to each other to form a sheet, which is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are done. The cells may be linked to each other directly and/or via intervening substances. The intervening substance is not particularly limited as long as it is a substance capable of mechanically connecting cells at least, and examples thereof include extracellular matrix. The intervening substance is preferably derived from cells, particularly derived from cells constituting a cell culture. The cells are at least mechanically linked, but may also be functionally linked, eg chemically, electrically linked.

  The sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells to and/or within their surface and maintain the physical integrity of cell cultures, such as polyvinylidene difluoride (PVDF). Although membranes made of the same are known, the cell culture of the present invention can maintain its physical integrity without such a scaffold. In addition, the cell culture of the present invention preferably consists only of substances derived from cells constituting the cell culture and does not contain substances other than those.

  In one embodiment of the present invention, the cell culture is performed within a predetermined period, preferably within a period in which the cells do not undergo differentiation. Therefore, in this aspect, the cells are maintained in an undifferentiated state during the culture period. The transition of cells to differentiation can be evaluated by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, multinucleation of cells, formation of myotubes and the like can be used as an indicator of differentiation. In a preferred embodiment of the present invention, the culture period is 48 hours or less, more preferably 40 hours or less, and further preferably 24 hours or less.

  The cell culture medium used for culturing (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes It is possible to use those containing as a main component. In one aspect of the invention, the culture medium is based on a basal medium for cell culture. Such basal medium includes, but is not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc. .. Many of these basal media are commercially available and their compositions are known. As an example, Table 1 below shows the compositions of MCDB131 and DMEM.

The basal medium may be used in a standard composition as it is (for example, in a state of being commercially available), or its composition may be appropriately changed depending on cell types and cell conditions. Therefore, the basal medium used in the present invention is not limited to one having a known composition, and includes one in which one or more components are added, removed, increased or decreased.

The amino acid contained in the basal medium is not limited and includes, for example, 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 and the like are not limited as vitamins, and examples thereof include calcium D-pantothenate, choline chloride, folic acid, i. - inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin _{B 12,} adenine, thymidine and then, as the electrolyte, without limitation, for _{example, CaCl 2, KCl, MgSO 4} , NaCl, NaH ₂ PO ₄ , NaHCO ₃ , Fe(NO ₃ ) ₃ , FeSO ₄ , CuSO ₄ , MnSO ₄ , Na ₂ SiO ₃ , (NH ₄ )6Mo ₇ O ₂₄ , NaVO ₃ , NiCl ₂ , ZnSO _4, etc. are included, respectively. Be done. In addition to these components, the basal medium may contain saccharides such as D-glucose, sodium pyruvate, pH indicators such as phenol red, putrescine and the like.

In one embodiment of the present invention, the concentration of amino acids contained in the basal medium is L-arginine: 63.2-84 mg/L, L-cystine: 35-63 mg/L, L-glutamine: 4.4-584 mg/L. , Glycine: 2.3 to 30 mg/L, L-histidine: 42 mg/L, L-isoleucine: 66 to 105 mg/L, L-leucine: 105 to 131 mg/L, L-lysine: 146 to 182 mg/L, L -Methionine: 15-30 mg/L, L-phenylalanine: 33-66 mg/L, L-serine: 32-42 mg/L, L-threonine: 12-95 mg/L, L-tryptophan: 4.1-16 mg/L , L-tyrosine: 18.1-104 mg/L, L-valine: 94-117 mg/L.
Moreover, in one aspect of the present invention, the concentration of the vitamin preparation contained in the basal medium is D-calcium pantothenate: 4 to 12 mg/L, choline chloride: 4 to 14 mg/L, folic acid: 0.6 to 4 mg/L. , I-inositol: 7.2 mg/L, niacinamide: 4-6.1 mg/L, riboflavin: 0.0038-0.4 mg/L, thiamine: 3.4-4 mg/L, pyridoxine: 2.1-. It is 4 mg/L.

  In addition to the above, the cell culture medium may contain one or more additives such as serum, growth factor, steroid drug component, and selenium component. However, these components are impurities derived from the manufacturing process that cannot be ruled out as a side effect factor such as anaphylactic shock to the recipient in clinical practice, and are components to be excluded in clinical application. Therefore, in a preferred embodiment of the present invention, the cell culture medium does not contain an effective amount of at least one of these additives. Further, in a more preferred embodiment of the present invention, the cell culture medium is substantially free of at least one of these additives. Furthermore, in a particularly preferred embodiment of the present invention, the cell culture medium is substantially free of additives. Therefore, the cell culture medium may contain only the basal medium.

  The serum contained in the cell culture medium includes heterologous serum and allogeneic serum. Here, "heterologous serum" means serum derived from an organism of a species different from that of the recipient when the cell culture is used for transplantation. means. For example, when the recipient is human, serum derived from bovine or equine, such as fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc., corresponds to the heterologous serum. In addition, "allogeneic serum" means serum derived from an organism of the same species as the recipient. For example, when the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera, ie, sera from the recipient, and allogeneic allogeneic sera from non-recipient allogeneic individuals. In the present specification, sera other than autologous serum, that is, xenogeneic serum and allogeneic allogeneic serum may be collectively referred to as non-autologous serum. In the present invention, allogeneic serum is preferred, and autologous serum is more preferred, in order to avoid side effects during transplantation.

  In one aspect of the invention, the cell culture medium is substantially free of serum. The cell culture medium substantially free of serum may be referred to as “serum-free medium” in the present specification. Here, "substantially free of serum" means that the content of serum in the culture medium does not have an adverse effect when the cell culture is applied to a living body (for example, the content of serum albumin in the cell culture is The amount is less than 50 ng), and preferably means that these substances are not actively added to the culture solution. In the present invention, in order to avoid side effects at the time of transplantation, it is preferable that the cell culture medium is substantially free of heterologous serum, and further preferably substantially free of non-self serum.

  In one aspect of the invention, the cell culture medium does not contain an effective amount of growth factors. Here, "growth factor" means any substance that promotes the proliferation of cells as compared with the absence thereof, and examples thereof include epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and fibroblast. Includes cell growth factor (FGF) and the like. Further, the "effective amount of growth factor" means the amount of the growth factor that significantly promotes the growth of cells as compared with the case where there is no growth factor, or, for convenience, the purpose of the growth of cells in the art. Means the amount usually added. The significance of cell growth promotion can be appropriately evaluated by, for example, any statistical method known in the art, such as t-test, and the usual addition amount can be various levels in the art. It can be known from publicly known documents. Specifically, the effective amount of EGF in skeletal myoblast culture is, for example, 0.005 μg/mL or more.

  Therefore, “does not contain an effective amount of growth factor” means that the concentration of the growth factor in the culture medium of the present invention is less than the effective amount. For example, the concentration of EGF in the culture solution in the culture of skeletal myoblasts is preferably less than 0.005 μg/mL, more preferably less than 0.001 μg/mL. In a preferred embodiment of the present invention, the concentration of the growth factor in the culture medium is lower than the normal concentration in the living body. In such an embodiment, for example, the concentration of EGF in the culture solution in the culture of skeletal myoblasts is preferably less than 5.5 ng/mL, more preferably less than 1.3 ng/mL, and further preferably 0.5 ng/mL. Less than mL. In a further preferred embodiment, the culture medium of the present invention contains substantially no growth factor. Here, the term "substantially free from" means that the content of the growth factor in the culture medium is such that it does not have an adverse effect when the cell culture is applied to a living body, and preferably the growth factor is positively added to the culture medium. It means that it is not added. Therefore, in this embodiment, the culture medium does not contain a growth factor at a concentration higher than that contained in other components therein, such as serum.

  In one aspect of the invention, the cell culture fluid is substantially free of steroidal drug components. Here, the "steroid drug component" refers to a compound having a steroid nucleus, which can exert an adverse effect on the living body such as adrenocortical dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, "substantially free of steroid drug component" means that the content of these compounds in the culture medium is such that the cell culture is not adversely applied to the living body, preferably the culture medium. Means that these compounds are not positively added to, that is, the culture medium does not contain other components therein, for example, steroid drug components in concentrations higher than those contained in serum and the like.

  In one aspect of the present invention, the cell culture medium is substantially free of selenium components. Here, the "selenium component" includes a selenium molecule and a selenium-containing compound, particularly a selenium-containing compound capable of liberating the selenium molecule in vivo, such as selenious acid. Therefore, "substantially free of selenium components" means that the content of these substances in the culture medium is such that it does not have an adverse effect when the cell culture is applied to a living body, preferably the culture medium. This means that these substances are not positively added, that is, the culture medium does not contain other components therein, for example, selenium components at concentrations higher than those contained in serum and the like. Specifically, for example, in the case of human, the selenium concentration in the culture medium is the normal value in human serum (for example, 10.6 to 17.4 μg/dL), and the ratio of human serum contained in the medium is It is lower than the multiplied value (ie, if the content of human serum is 10%, the selenium concentration is, for example, 1.0 to less than 1.7 μg/dL).

In the above preferred embodiment of the present invention, impurities derived from the manufacturing process such as a growth factor, a steroid agent component, and a heterologous serum component, which have been conventionally required when preparing a cell culture to be applied to a living body, are removed by washing or the like. No process is required. Therefore, one aspect of the method of the present invention does not include the step of removing impurities derived from the manufacturing process.
Here, the “manufacturing process-derived impurities” typically include those listed below derived from each manufacturing process. That is, those derived from cell substrates (eg, host cell-derived proteins, host cell-derived DNA), those derived from cell culture medium (eg, inducers, antibiotics, medium components), or in the steps after cell culture. For example, those derived from the extraction, separation, processing, and purification steps of a target substance (see, for example, Pharmaceutical Prosecution No. 571).

  Further, since the sheet-shaped cell culture produced by the method of the present invention has a physical strength that can withstand the washing operation, the method of the present invention removes impurities and the like derived from the above-mentioned production steps by washing or the like. You may include a process. Accordingly, one aspect of the present invention further comprises the step of washing the isolated sheet cell culture. By adding such a step, it becomes possible to obtain a sheet-shaped cell culture that does not substantially contain such impurities, even if a cell culture solution that substantially contains the impurities derived from the above manufacturing steps is used.

Culturing of cells can be performed under the conditions generally used in the art. For example, typical culturing conditions include culturing at 37° C. and 5% CO ₂ . The culture period is preferably 48 hours or less, more preferably 40 hours or less, and further preferably 24 hours or less, from the viewpoint of sufficient formation of cell culture and prevention of cell differentiation. The culture can be performed in a container of any size and shape. In the method of the present invention, since cells do not substantially grow, a cell culture of a desired size and shape can be obtained in a short period of time without waiting for the cell culture to grow to a desired size as in the conventional method. It will be possible to get in between. For the size and shape of the cell culture, adjust the size and shape of the cell attachment surface of the culture container, or install a mold of the desired size and shape on the cell attachment surface of the culture container and It can be optionally adjusted by culturing the cells in.

  The method may further include the step of isolating the cell culture from the culture substrate. Isolation of the cell culture from the substrate is not particularly limited as long as the cell culture can be released (exfoliated) from the substrate serving as the scaffold while maintaining the sheet structure at least partially. It can be carried out by enzyme treatment with an enzyme such as trypsin and/or mechanical treatment such as immersion stirring, pipetting and the like. In addition, by culturing cells on a culture substrate whose surface is coated with a material whose physical properties change in response to a stimulus, for example, temperature or light, to form a cell culture, by applying a predetermined stimulus, It can also be released non-enzymatically.

The method of the present invention can also be positioned as one step of regenerative treatment from collection of cells, growth of cells and production of cell cultures to application of cell cultures. Therefore, the present invention
(I) isolating desired cells from tissue or biological fluid collected from the subject,
(Ii) growing the isolated cells,
(Iii) seeding a cell having a density capable of forming a sheet-shaped cell culture without substantially growing on a culture substrate coated with serum,
(Iv) culturing cells to form a sheet-shaped cell culture, and (v) isolating the formed culture from a substrate, and optionally (vi) isolated sheet-shaped cell culture The present invention also relates to a method for producing a sheet cell culture for regenerative treatment, which comprises a step of washing the product.

The present invention also relates to a sheet-shaped cell culture produced by the above production method.
In a preferred embodiment, the sheet-shaped cell culture of the present invention has sufficient physical strength for washing operations such as immersion stirring and transplantation operations such as taking out, holding and transferring. Having sufficient physical strength means that the sheet-like structure of the cell culture is not impaired by performing the above-mentioned operation, which means that, for example, the above-mentioned operation is actually performed on the obtained sheet-like cell culture. It is possible to confirm that the sheet-like structure is maintained by performing a macroscopic examination or a microscopic examination. Here, the washing operation by immersion stirring typically means adding a sufficient amount of buffer solution to the culture container containing the sheet-shaped cell culture to sufficiently immerse the culture, and stirring the whole culture container with shaking. Say. Therefore, having sufficient physical strength can also be confirmed by applying the same physical stress to the sheet-shaped cell culture as the washing operation by such immersion stirring. In addition, the sheet-shaped cell culture produced by the above-mentioned production method usually has such sufficient physical strength.

  In a preferred embodiment of the cell culture of the present invention, the cell culture is substantially free of non-autologous serum, growth factors, steroids and/or selenium components. In a more preferred embodiment, the cell culture of the present invention is substantially free of manufacturing process-derived impurities containing the above components. This cell culture can be prepared by culturing cells in a culture medium substantially free of non-autologous serum, growth factors, steroids and/or selenium components to form cell cultures. Here, the cell culture is substantially free of non-autologous serum, growth factors, steroids and/or selenium components, as long as the cell culture does not contain these components at a concentration that adversely affects the recipient. By at least the above, it is possible to satisfy such a condition by carrying out the formation of the cell culture in a culture medium that is substantially free of non-autologous serum, growth factors, steroids and/or selenium components. In a further preferred embodiment, the cell culture of the present invention is substantially free of autologous serum in addition to impurities derived from the manufacturing process. Here, the meaning of “not substantially including” is the same as above.

  In a preferred embodiment of the cell culture of the present invention, the cell culture has reduced inflammatory cytokines. The inflammatory cytokine is a general term for cytokines produced with inflammation, and includes, for example, without limitation, TNF-α, IL-1 (IL-1α, IL-1β, etc.), IL-4, IL-5. , IL-6, IL-13 and the like. Therefore, “the inflammatory cytokines are reduced” means that the abundance or production amount (secretion amount) of these cytokines is reduced as compared with the differentiated cell culture. Therefore, the cytokine may exist in any form in the process from the transcription of the gene to the secretion of the protein, for example, in the form of a transcript such as mRNA or in the form of protein. The degree of reduction is not particularly limited as long as it exceeds the range of error, but for example, it is 15% or more, preferably 25% or more, more preferably 50% or more, still more preferably 60% compared to the differentiated cell culture. % Or more, particularly preferably 70% or more.

  At the gene level, the amount of cytokine can be any known method such as Northern blotting method, Southern blotting method, RNase protection assay, RT-PCR, PCR method such as real-time PCR, in situ hybridization method, in vitro transcription method and the like. It can be detected by a gene expression analysis method and at the protein level by any known protein detection method such as immunoprecipitation method, western blotting method, EIA, ELISA, RIA, immunohistochemistry method, immunocytochemistry method and the like. .. A medium impregnated with the cell culture, a part of the cell culture, or the like can be used as the sample.

  In a preferred embodiment of the present invention, the cell culture contains a therapeutically useful physiologically active substance. Such substances are not particularly limited as long as they have some therapeutic utility, and include, for example, substances involved in angiogenesis, suppression of cell death, tissue remodeling, cardiac development, induction of stem cells, and the like, and more specifically, Specifically, without limitation, for example, VEGF, PIGF, angiogenin, angiopoietin, HGF, PDGF (PDGF-AB, PDGF-BB, etc.), BAG-1, BCL-2, MMP (MMP-2, MMP-3, MMP-9, MMP-10, etc.), TIMP, TNNT2, TNNC1, IGF-1 and the like.

  By “comprising” these substances is meant that their abundance or production (secretion) can be detected in cell culture. In a preferred embodiment of the present invention, the cell culture contains more of these substances than the differentiated cell culture. The degree of increase is not particularly limited as long as it exceeds the error range, but for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, as compared with the differentiated cell culture. More preferably, it is 3 times or more. These substances may be present in any form in the process from the transcription of a gene to the secretion of a protein, and may be in the form of a transcript such as mRNA or the form of a protein. The presence of these substances can be detected and/or quantified similarly to the cytokines mentioned above.

  In a preferred embodiment of the invention, the cell culture is in an undifferentiated state. The transition of cells to differentiation can be evaluated by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, multinucleation of cells and the like can be used as an indicator of differentiation. In addition, since undifferentiated cells have less inflammatory cytokines and more physiologically useful therapeutically useful substances than differentiated cells, the presence or absence of differentiation can be determined using this as an index. Examples of the method for obtaining an undifferentiated cell culture include culturing cells according to the method of the present invention for a period of 48 hours or less, more preferably 40 hours or less, and further preferably 24 hours or less.

  The cell culture of the present invention can be used for treatment of a target disease or injury. For example, cell cultures of skeletal myoblasts can be used in the form of transplants for heart diseases such as myocardial infarction and dilated cardiomyopathy. Therefore, another aspect of the present invention relates to an implant comprising the above cell culture.

The invention also relates to culture substrates coated with serum and/or growth factors and/or steroids. The culture substrate of the present invention is preferably used for producing the sheet-shaped cell culture according to the present invention. The coating method and the like are as described above for the method for producing a sheet-shaped cell culture of the present invention.
The present invention further relates to the above-mentioned method for producing a culture substrate, which comprises (i) incubating the culture substrate with serum, and (ii) discarding the serum. The specific manufacturing method is as described above for the method for manufacturing the sheet-shaped cell culture of the present invention. In one aspect of the present invention, after the step (ii), a step of (iii) washing the culture substrate with a serum-free washing solution may be added.

  The present invention further relates to the above-mentioned kit for producing a culture substrate, which comprises the culture substrate and a coating agent selected from the group consisting of serum, growth factors and steroids. In the kit, the coating agent is in various storable forms, for example, in a refrigerated, frozen, or freeze-dried state, and in a suitable container, for example, vials, ampoules, bottles of various materials (glass, plastic, etc.), It may be stored in a tube or the like. In addition to the above, the kit of the present invention is a medium for diluting and/or reconstituting the coating agent, such as water, physiological saline, various buffers (eg, PBS, HBS, etc.), various liquid media ( For example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM/F12, etc.) and a culture substrate coating method. It may include an instruction manual including information about the information, an electronic recording medium such as a CD or a DVD, and the like. The culture substrate, coating agent and the like contained in the kit can be provided preferably in a sterilized state. The sterilization method is not limited, and examples thereof include gas sterilization with ethylene oxide gas and the like, sterilization with ultraviolet rays and radiation (eg, γ rays), and the like.

  The invention also relates to methods of reducing inflammatory cytokines in cell culture by maintaining the cell culture in an undifferentiated state. The present invention further relates to a method for suppressing the reduction of therapeutically useful physiologically active substances contained in a cell culture by maintaining the cell culture in an undifferentiated state. In these methods, examples of the method for maintaining the cell culture in an undifferentiated state include culturing the cells within a predetermined period, preferably within a period during which the cells do not undergo differentiation. In a preferred embodiment of these methods, the culture period is 48 hours or less, more preferably 40 hours or less, and further preferably 24 hours or less. The steps, inflammatory cytokines and therapeutically useful physiologically active substances in these methods are basically as described above for the method for producing a sheet cell culture and the sheet cell culture of the present invention. In these methods, “reducing inflammatory cytokines” means reducing the abundance or production (secretion) of these cytokines as compared to differentiated cell cultures. The degree of reduction is not particularly limited as long as it exceeds the range of error, but for example, it is 15% or more, preferably 25% or more, more preferably 50% or more, still more preferably 60% compared to the differentiated cell culture. % Or more, particularly preferably 70% or more. In addition, “suppressing the reduction of therapeutically useful physiologically active substances” means maintaining the abundance or production amount (secretion amount) of these substances at an increased level as compared with the differentiated cell culture. means. The degree of increase is not particularly limited as long as it exceeds the error range, but for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, as compared with the differentiated cell culture. More preferably, it is 3 times or more.

  At the gene level, the amount of inflammatory cytokine or therapeutically useful physiologically active substance is, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, real-time PCR, in situ hybridization method, By any known gene expression analysis method such as in vitro transcription method, and at the protein level, any known method such as immunoprecipitation method, Western blotting method, EIA, ELISA, RIA, immunohistochemistry method, immunocytochemistry method, etc. It can be detected by the protein detection method. A medium impregnated with the cell culture, a part of the cell culture, or the like can be used as the sample.

Hereinafter, the present invention will be further described based on examples, but the examples are examples of the present invention and do not limit the present invention. In the following examples, unless otherwise specified, skeletal myoblasts are of human origin, and the cells are cultured under the conditions of 37°C and 5% CO ² .

Example 1 Effect of seeding density/serum coating treatment on strength of sheet-shaped cell culture A temperature-responsive culture dish (24-well plate, made by Cell Seed) was prepared, and a serum-containing medium (DMEM) was used in some wells. /F12 was added with 20% human serum (produced by Cambrex or derived from research blood, hereinafter referred to as serum-containing medium) and incubated at 37° C. under 5% CO ² to perform serum coating treatment. Was applied. Next, skeletal myoblasts (manufactured by Lonza) were suspended in a serum-containing medium, and 3.5×10 ⁵ , 1.0×10 ⁶ , 1.4× were added to a culture dish coated with serum or an untreated culture dish. Seeds were seeded at a density of 10 ⁶ , 1.7×10 ⁶ , 3.4×10 ⁶ cells/cm ² . After culturing for 24 hours, the cell culture was peeled from the temperature-responsive culture dish, and the possibility of sheet formation and its strength were evaluated. As a result, in the case of using a non-treated culture dish, when seeded at a density of more than 1.0×10 ⁶ cells/cm ² , peeling occurred before the seeded cells completely formed a sheet, It was not possible to form a sheet-like cell culture with sufficient strength. On the other hand, when a culture dish coated with serum was used, it was possible to form a sheet-shaped cell culture without spontaneously peeling. Note that a sheet-shaped cell culture could not be obtained at a cell seeding density of 3.4×10 ⁶ cells/cm ² .

Furthermore, a temperature-responsive culture dish (φ3.5 cm, made by Cell Seed) with or without serum coating treatment was prepared in the same manner as above, and skeletal myoblasts were suspended in a serum-containing medium to give 3.5×10 5. Seeding was performed at a density of ⁵ cells/cm ² . After culturing for 24 hours, the sheet-shaped cell culture was peeled from the temperature-responsive culture dish, and its strength was evaluated based on the degree of damage by immersion and stirring, and it was found that the strength was higher when the culture dish coated with serum was used. It was possible to obtain an excellent sheet-shaped cell culture. In addition, when the number of cells that compose the exfoliated sheet-shaped cell culture was examined, it was found that when the serum-coated culture dish was used, more cells were formed from the sheet-shaped cells than when the untreated culture dish was used. It was revealed that a cell culture was formed and the cell recovery rate was significantly higher at 96.3% compared to 69.8% when the untreated culture dish was used (Table 3).

Further, when the strength of the formed sheet-shaped cell culture was evaluated by the seeding density, it was possible to obtain a sheet-shaped cell culture having higher strength by increasing the seeding density.

Example 2 Evaluation of Differentiation A temperature-responsive culture dish (24-well plate, made by Cell Seed) was prepared, and some wells were coated with serum in the same manner as in Example 1. Next, skeletal myoblasts were suspended in a serum-containing medium, and 1.0×10 ⁶ , 1.4×10 ⁶ , 1.7×10 ⁶ cells/cm ² were added to wells coated with serum or untreated wells. Seed at each density. After culturing for 24 hours, protein was extracted from the formed sheet-shaped cell culture, and the activity of creatine kinase (CK) was measured. CK is one of the indicators of skeletal muscle differentiation, and the higher the differentiation, the higher the CK activity. Specifically, first, a cell solubilizing reagent (CelLyticM, manufactured by sigma) and a protease inhibitor (Protease Inhibitor Cocktail, manufactured by sigma) are added to the formed sheet-shaped cell culture, and the mixture is stirred at room temperature while shaking with a shaker. Incubated for 15 minutes. The cell lysate was collected, centrifuged at 4° C. and 15,000 g for 15 minutes, and the supernatant was collected as a protein extract for measurement. The CK activity was measured using EnzyChrom ^™ Creatine Kinase Assay Kit (BioAssay Systems) according to the attached protocol. In addition, the protein concentration of the extract was measured by the BCA method, and the CK activity value per protein concentration was calculated. As a result of the measurement, in the sheet-shaped cell culture using a serum-coated culture dish, no increase in activity was observed even when the seeded cell density was increased, and no differentiation was observed (FIG. 1).

Example 3 Measurement of Humoral Factors Skeletal myoblasts were seeded at a density of 3.5×10 ⁵ cells/cm ^{2 in} a temperature-responsive culture dish (φ3.5 cm, made by Cell Seed), and cultured for 4 days, and then cultured. The supernatant was collected (undifferentiated sheet cell culture supernatant). In addition, skeletal myoblasts seeded in a temperature-responsive culture dish were mixed with a differentiation-inducing medium (100 μl of 100 μg/ml IGF-1 in 2.6 ml of DMEM and 2.6 ml of 7.5% BSA solution were mixed) for 4 days. After culturing to give multinucleated cells (differentiated cells), the cells were further cultured for 4 days to recover the culture supernatant of the multinucleated cells (multinucleated sheet-like cell culture supernatant). Measurement of humoral factors contained in the culture supernatant of undifferentiated sheet cell cultures and multinucleated sheet cell cultures using an antibody array (RayBio ^(R) Human Cytokine Antibody Array G Series 2000, manufactured by RayBiotech) I went. As a result, it was revealed that the multinucleated sheet-like cell culture produced more inflammatory cytokines such as IL-1α, IL-4, IL-5, and IL-13 than the skeletal myoblast sheet-like cell culture. (Figure 2). IL-1α is involved in the induction of inflammatory response and T cell and B cell activation, IL-4 and IL-13 induce B cell activation and antibody production, and IL-5 is mainly eosinophil-derived. Both are cytokines that act on activation but are deeply involved in the induction of allergic reactions.

Regarding various humoral factors that are considered to be one of the efficacy mechanisms in stem cell transplantation, PDGF-AB, PDGF-BB in undifferentiated sheet-like cell culture as compared with multinucleated sheet-like cell culture It was revealed that the production of factors having an effect on efficacy was significantly increased (Fig. 3).
Therefore, when the surface of the culture dish is coated with serum, it is possible to form a sheet-shaped cell culture even if the number of cells seeded is increased, and it is expected that a factor that acts therapeutically effectively is produced.

Claims (8)

Seeding cells on a culture substrate coated with serum at a cell density of 1.0×10 ^{6 to} 1.7×10 ⁶ cells/cm ² , and culturing the seeded cells to Forming a sheet-shaped cell culture without substantially growing the sheet-shaped cell culture.   The method of claim 1, wherein the serum is autologous serum.   A sheet-shaped cell culture produced by the method according to claim 1.   The sheet-shaped cell culture according to claim 3, which is used for treating a disease or injury. Including serum, and culture substrate, and the number of cells leading to cell density of 1.0 × ^{10 6} ~1.7 × ¹⁰ 6 cells / cm ² when seeding into the culture substrate according to claim 1 or 2 A combination for use in the method according to. Including serum, and culture substrate, and the number of cells leading to cell density of 1.0 × ^{10 6} ~1.7 × ¹⁰ 6 cells / cm ² when seeding into the culture substrate according to claim 1 or 2 A set for use in the method described in 1. Including serum, and culture substrate, and the number of cells leading to cell density of 1.0 × ^{10 6} ~1.7 × ¹⁰ 6 cells / cm ² when seeding into the culture substrate according to claim 1 or 2 A set of manufacturing elements for use in the method described in. Including serum, and culture substrate, and the number of cells leading to cell density of 1.0 × ^{10 6} ~1.7 × ¹⁰ 6 cells / cm ² when seeding into the culture substrate according to claim 1 or 2 A kit for use in the method described in 1.