JP7002497B2 - Method for producing sheet-shaped cell culture - Google Patents

Description

The present invention comprises a method for producing a sheet-shaped cell culture, which comprises a step of seeding cells on a culture medium coated with serum, a sheet-shaped cell culture produced by the same method, particularly impurities derived from the production process. A sheet-like cell culture substantially free of the above, a culture medium coated with serum used in the above method, a method and a production kit for producing the culture substrate, and a method for suppressing the production of inflammatory cytokines in the cell culture. Regarding.

In ischemic heart disease such as angina pectoris and myocardial infarction, sufficient oxygen is not distributed to the myocardial tissue, and if this condition continues for a long time, the myocardial tissue is damaged. Since adult cardiomyocytes originally have poor self-renewal ability, once damaged, the myocardium cannot be repaired, or even if it can be repaired, only limited recovery can be expected and eventually heart failure occurs.

Heart transplantation is an effective treatment method for heart failure, but in many cases, a left ventricular assist device is attached as a bridge while waiting for transplantation.
However, heart transplantation has serious problems such as the risk of infections associated with immunosuppressive treatment, the appearance of distant coronary atherosclerotic lesions, and an absolute shortage of donors, and the current ventricular assist device is a thromboembolism. Patient QOL is very limited due to complications such as illness and infection, and problems with the durability of the device, making long-term assistance difficult.

Under these circumstances, research is underway as a new treatment method for skeletal myoblast transplantation into myocardial tissue.
Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. Since myocardium and skeletal muscle have many similar parts in structure and function, it is considered that myoblasts derived from skeletal muscle can also repair injured myocardium. Overseas, transplantation of autologous skeletal myoblasts into the myocardium is being clinically applied.

Although the mechanism for suppressing the decline in cardiac function by transplantation of skeletal myoblasts is not clear, transplantation of myoblasts into the beating myocardium causes orientation of the cell arrangement in the place where mechanical stretch is applied. It is considered that appropriate differentiation is performed, and it is also considered that it may function as a cytokine delivery system such as VEGF (vascular endothelial growth factor).

However, transplantation of myoblast suspension as a single cell into an infarcted heart causes damage loss of the transplanted cells, tissue damage during injection of the recipient heart, efficiency of tissue supply to the recipient heart, occurrence of arrhythmia, and infarction. It has been pointed out that there are drawbacks such as difficulty in treating the entire site, and there has been a desire to sheet myoblasts in order to deal with these drawbacks.

On the other hand, artificial tissue was found that has the property that the cells are proliferated under specific culture conditions, the organization progresses more than expected, and the cells are easily detached from the culture dish. A cell sheet as a three-dimensional tissue structure applicable to the heart containing the derived cells and a method for producing the same were provided (see Patent Document 1).

By the way, the cell culture medium used in the known production method may be any medium as long as the target cells proliferate, and may be a medium to which serum such as known fetal bovine serum is added. It is said to be good (see, for example, Patent Document 1).

Further, it is known to use bovine fetal serum, horse serum, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) 2 as a cell culture medium used for proliferation of myocardial progenitor cells and the like (Patent). See Document 2).

As described above, the formation of the sheet-shaped cell culture is usually carried out by proliferating the cells, and in order to promote the proliferation of the cells, a growth factor is generally added in addition to the heterologous serum component, but on the other hand. So, for example, in skeletal myoblasts that tend to differentiate when they become confluent, it is common to add steroids to the culture medium of skeletal myoblasts in order to suppress the transition of proliferated cells to differentiation. Is. Without the addition of steroids, skeletal myoblasts begin to differentiate after about 16 hours of culture, forming tubular myotubes, making it impossible to obtain sheet-like cell cultures.
Further, selenium may be added to the culture broth for the purpose of antioxidant action.

However, for clinical application, allogeneic serum components such as heterologous serum components may contain pathogens such as viruses that can infect recipients, and growth factors are usually recombined using microorganisms. If it remains, it can be an impurity derived from the manufacturing process, so it cannot be used for transplantation as it is from the viewpoint of safety.

In addition, selenium is an essential element necessary for the synthesis of antioxidant enzymes for the living body, is incorporated into the protein as selenocysteine, works mainly as selenoprotein, and protects the living body from active oxygen and radicals in cooperation with vitamins E and C. It is believed that. However, selenium has a very narrow range between the appropriate amount and the amount of addiction, and symptoms such as nausea, nausea, diarrhea, loss of appetite, headache, immunosuppression, and decrease in high-density lipoprotein (HDL) are known as excess symptoms. There is. Furthermore, selenous acid is designated as a poisonous substance designated item in the "Poisonous and Deleterious Substances Designation Ordinance" (Cabinet Order No. 2 of January 4, 1965). On the other hand, steroids are known to have adrenal insufficiency, Cushing's syndrome and the like as their side effects, and all of them are preferable components to be removed at the time of transplantation as impurities derived from the manufacturing process.

These manufacturing process-derived impurities are usually removed by washing the cell culture with a medium that does not contain these substances, but the cell culture is mechanically extremely fragile and can be easily removed by water flow during washing. Since it is destroyed, it has been extremely difficult to wash the impurities derived from the manufacturing process in the cell culture to a level that does not cause clinical damage.
Furthermore, although self-serum that has less adverse effect on the recipient can be used instead of allogeneic serum, self-serum must be prepared by collecting blood from the recipient in advance. It imposes a physical and time burden, and it may be difficult to obtain it depending on the condition of the recipient.
Therefore, it has been required to improve the method for easily obtaining a high-quality sheet-shaped cell culture having high clinical safety.

Special Table 2007-528755 Japanese Unexamined Patent Publication No. 2008-161183

An object of the present invention is to provide a high-quality cell culture containing no impurity component derived from a manufacturing process that may hinder its clinical application, and a method for producing the same.

The present inventors continued diligent research to solve the above-mentioned problems, and found that when cells were cultured on this culture medium after coating the culture medium with serum, serum-free cells were obtained even in cell types requiring serum. It has been found that a sheet-shaped cell culture can be obtained in a medium. Furthermore, they have found that by culturing cells on such a culture substrate in a serum-free medium, the production of inflammatory cytokines can be significantly reduced as compared with cell cultures cultured in a serum-containing medium, and completed the present invention. ..

That is, the present invention relates to the following.
(1) (i) a step of seeding cells on a culture substrate coated with serum, and (ii) a step of culturing cells to form a sheet-shaped cell culture.
A method for producing a sheet-shaped cell culture.
(2) The method of (1) above, wherein the cells are seeded at a density capable of forming a sheet-like cell culture without substantially proliferating.
(3) The method of (1) or (2) above, wherein the cells include myoblasts.
(4) The method according to any one of (1) to (3) above, wherein the culture medium is further coated with a growth factor.
(5) The method according to any one of (1) to (4) above, wherein the culture medium is further coated with a steroid agent.
(6) The method according to any one of (1) to (5) above, wherein the culture substrate coated with serum is obtained by incubating the culture substrate together with serum and then discarding the serum.
(7) The culture medium coated with serum is obtained by incubating the culture medium together with the serum, then discarding the serum, and then washing the culture medium with a serum-free washing solution (1) to (1) to (1) to (1) to (1) above. 6) Any method.

(8) The method according to any one of (1) to (7) above, wherein the culture is carried out in a serum-free medium.
(9) The method according to any one of (1) to (8) above, further comprising the step of isolating the cell culture from the culture medium.
(10) A cell culture produced by any of the above methods (1) to (9).
(11) The cell culture of (10) above, which is used for treating diseases and injuries.
(12) The cell culture according to (10) or (11) above, which is substantially free of impurities derived from the manufacturing process.
(13) A graft containing the cell culture according to any one of (10) to (12) above.
(14) A culture medium coated with serum and used for any of the above methods (1) to (9).
(15) The culture medium of (14) above, further coated with a growth factor.
(16) The culture medium of (14) or (15) above, further coated with a steroid agent.

(17) (i) Incubating the culture medium with serum, and (ii) Disposing of serum,
The method for producing a culture medium according to any one of (14) to (16) above, which comprises.
(18) The method of (17) above, further comprising the step of washing the (iii) culture medium.
(19) The kit for producing a culture medium according to any one of (14) to (16) above, which comprises a culture medium and serum.
(20) (i) a step of seeding cells on a culture substrate coated with serum, and (ii) a step of culturing cells in a serum-free medium to form a cell culture.
A method of suppressing the production of inflammatory cytokines in cell cultures, including.

INDUSTRIAL APPLICABILITY According to the present invention, in the production of sheet-shaped cell culture, it is possible to culture in a non-cell proliferation type culture solution containing no serum or growth factor, which is generally added as a factor necessary for cell proliferation, and thus harmful to the serum. In addition to avoiding contamination with components and endotoxins that can be contained in growth factors, which are usually recombinant products, steroids added as differentiation inhibitors are not required. Further, the present invention eliminates the need for antioxidant selenium or its derivatives. As a result, it becomes possible to provide a cell culture that is clinically safe and does not contain impurities derived from the manufacturing process, and it is not necessary to prepare self-serum as a substitute for heterologous serum.

In addition, by enabling culturing in a serum-free medium, the production of inflammatory cytokines can be significantly reduced as compared with cell cultures cultivated in a serum-containing medium, further enhancing the clinical safety of cell cultures. Is possible.
Furthermore, when culturing in a medium that does not contain serum or growth factors, operations such as washing for the purpose of removing impurities derived from the manufacturing process, which may damage the produced cell culture, are not required, and the cell culture is more suitable. Reliable and stable production is possible.
Furthermore, since the cell culture of a desired size and shape can be produced in a short period of time by the method of the present invention, it becomes possible to more flexibly and easily perform the treatment of the living body using the cell culture.

FIG. 1 is a photograph showing a cell culture cultured in a serum-free medium on a culture substrate not coated with serum (× 200). FIG. 2 is a photograph showing a cell culture cultured in a conventional serum-containing medium on a culture substrate not coated with serum (× 100). FIG. 3 is a photograph showing a cell culture cultured in a serum-free medium on a serum-coated culture substrate (× 100). FIG. 4 shows the IL-6 concentration (pg / mL) in the culture supernatant of the cell culture cultured in the serum-containing medium (Sample 1) or the serum-free medium (Sample 2) on the serum-coated culture substrate. It is a bar graph shown.

The present invention comprises a step of seeding cells on a culture medium coated with serum and (ii) a step of culturing the cells to form a sheet-shaped cell culture. Regarding the manufacturing method of things.
The cells in the present invention include cell cultures, in particular any cells capable of forming sheet-like cell cultures. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), myoblasts, fibroblasts, synovial cells, epithelial cells, endothelial cells and the like. Of these, in the present invention, those forming a monolayer cell culture, for example, myoblasts are preferable. The cells can be derived from any organism that can be treated with cell culture. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like. Further, although only one type of cell may be used in the method of the present invention, two or more types of cells may be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the ratio (purity) of the most abundant cells is 65% or more, preferably 70% or more, more at the end of cell culture production. It is preferably 75% or more.

In the present invention, the "culture substrate" is not particularly limited as long as the cells can form a cell culture on the container, for example, a container made of various materials, a solid or semi-solid surface in the container, and the like. including. 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 containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside thereof. 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 surface of the culture substrate may be coated with a material whose physical properties change in response to stimuli, 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-Cyclopropylmethacrylate, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-Tetrahydrofurfurylacrylamide, N-Tetrahydrofurylmethacrylamide It has an N, N-dialkyl-substituted (meth) acrylamide derivative (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), and a cyclic group (such as amide). Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidin, 1- (1-oxo-2-propenyl) -piperidine, 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-2-11186 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.
The culture medium may have various shapes, but is preferably flat. The area thereof 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 medium 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 medium. Such a condition can be obtained without limitation, for example, by incubating the culture medium with the serum and then discarding the serum. Incubation involves contacting the serum with the culture medium. As the serum, heterologous serum and allogeneic serum can be used. Heterologous serum means serum derived from an organism of a different species than the recipient when transplanting a cell culture. For example, when the recipient is human, serum derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to heterologous serum. In addition, "homogeneous serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera, ie sera from the recipient.
Serum for coating the culture medium can be commercially available or prepared by routine from blood collected from the desired organism. Specific examples thereof include a method in which the collected blood is left at room temperature for about 20 to 60 minutes to coagulate, and the collected blood is centrifuged at about 1000 to 1200 × g to collect the supernatant.

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

As a serum disposal method, suction with a pipette or the like, or a conventional liquid disposal method such as decantation can be used. In a preferred embodiment of the present invention, the culture medium may be washed with a serum-free washing solution after serum disposal. The serum-free cleaning solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components adhering to the culture substrate. For example, without limitation, water, physiological saline, and various buffers are not particularly limited. Liquids (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.). As the cleaning method, a conventional culture substrate cleaning method, for example, without limitation, a method of adding a serum-free cleaning solution on the culture substrate, stirring for a predetermined time (for example, 5 to 60 seconds), and then discarding 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 cell growth compared to its absence, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts. Includes cell growth factor (FGF) and the like. In the coating method, disposal method and washing method of the culture substrate using a growth factor, 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 medium may be coated with a steroid agent. Here, the "steroid component" refers to a compound having a steroid nucleus that can adversely affect the living body such as adrenal insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the coating method, disposal method and washing method of the culture medium with a steroid agent, the dilution concentration at the time of incubation is, for example, 0.1 μg / mL to 100 μg / mL, preferably 0.4 μg / mL to 40 μg / mL as dexamethasone. , More preferably, it is basically the same as serum except that it is 1 μg / mL to 10 μg / mL.

The culture medium may be coated with any one of serum, growth factors and steroids and any combination of these, ie serum and growth factors, serum and steroids, serum and growth factors and steroids, Alternatively, it may be coated with a combination of growth factors and steroids. When coating with a plurality of components, these components may be mixed and coated at the same time, 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 a preferred embodiment of the invention, the cells are seeded at a density capable of forming a sheet-like cell culture without substantial proliferation. "The density at which a sheet-shaped cell culture can be formed without substantially growing" means that a sheet-shaped cell culture can be formed when cultured in a non-proliferative culture solution containing no growth factor. It means cell density. For example, in the case of skeletal myoblasts, in the conventional method using a culture medium containing a growth factor, cells having a density of about 6,500 cells / cm ² are seeded on a plate in order to form a sheet-like cell culture. However (see, for example, Patent Document 1), 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 cell density in this embodiment is higher than that in the conventional method using a culture medium containing a growth factor. Specifically, for example, for skeletal myoblasts, the density is typically 300,000 cells / cm ² or more. The upper limit of the cell density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not undergo differentiation, but for skeletal myoblasts, for example, 1,000,000 cells / cm ² . Those skilled in the art can appropriately determine the cell density suitable for the present invention by experiment. During the culture period, the cells may or may not grow, but if they do, they do not grow to the extent that the properties of the cells change. For example, skeletal myoblasts initiate 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 transition to differentiation. In a preferred embodiment of the invention, the cells do not grow beyond the 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 the formation of the cell culture. In this embodiment, the number of cells after cell culture formation is typically 300% or less, preferably 200% or less, more preferably 150% or less, still more preferably 125% or less, particularly preferably 125% or less of the number of cells at the time of seeding. Is 100% or less.

In the present invention, cell culture is carried out 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 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 composed. 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 at least mechanically connecting cells to each other, and examples thereof include an extracellular matrix. The mediators are preferably derived from cells, particularly those derived from the cells that make up the cell culture. The cells are at least mechanically linked, but may be more functional, eg, chemically or electrically linked.

The sheet-like cell culture of the present invention preferably contains no scaffold (support). Scaffolds may be used in the art to attach cells to and / or inside the surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF). Although manufactured membranes and the like are known, the cell culture of the present invention can maintain its physical integrity even in the absence of such a scaffold. In addition, the cell culture of the present invention preferably comprises only cell-derived substances constituting the cell culture, and does not contain any other substances.

In one embodiment of the invention, the cells are cultured within a predetermined period, preferably within a period during which the cells do not undergo differentiation. Therefore, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition to cell differentiation can be assessed by any method known to those of skill in the art. For example, in the case of skeletal myoblasts, MHC expression and cell multinucleation can be used as indicators of differentiation. In a preferred embodiment of the present invention, the culture period is 48 hours or less, more preferably 40 hours or less, still more preferably 24 hours or less.

The cell culture medium used for culturing (sometimes referred to simply as "culture medium" or "medium") is not particularly limited as long as it can maintain the survival of cells, but is typically amino acids, vitamins, and electrolytes. Can be used as the 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, and the like. .. Many of these basal media are commercially available and their compositions are also known. As an example, Table 1 below shows the compositions of MCDB131 and DMEM.

基礎培地は、標準的な組成のまま（例えば、市販されたままの状態で）用いてもよいし、細胞種や細胞条件に応じてその組成を適宜変更してもよい。したがって、本発明に用いる基礎培地は、公知の組成のものに限定されず、１または２以上の成分が追加、除去、増量もしくは減量されたものを含む。

The 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 those having a known composition, and includes those in which one or more components are added, removed, increased or decreased.

The amino acids contained in the basal medium are not limited, for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, and the like. L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited as vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i. -Inositol, niacinamide, riboflavin, thiamine, pyridoxin, biotin, lipoic acid, vitamin B ₁₂ , adenine, thymidin, etc., and the electrolytes are not limited, for example, CaCl ₂ , KCl, _Л4 , NaCl, Includes 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. Is done. In addition to these components, the basal medium may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and 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 to 84 mg / L, L-cystine: 35 to 63 mg / L, L-glutamine: 4.4 to 584 mg / L. , Glycine: 2.3-30 mg / L, L-histidine: 42 mg / L, L-isoleucine: 66-105 mg / L, L-leucine: 105-131 mg / L, L-lysine: 146-182 mg / L, L -Metionine: 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 to 104 mg / L, L-valine: 94 to 117 mg / L.
Further, in one embodiment 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 component, and selenium component. However, these components are impurities derived from the manufacturing process that cannot be denied to be a cause of side effects such as anaphylactic shock to the recipient in clinical practice, and should be excluded in clinical application. Therefore, in a preferred embodiment of the invention, the cell culture medium does not contain an effective amount of at least one of these additives. Also, in a more preferred embodiment of the invention, the cell culture medium is substantially free of at least one of these additives. Moreover, in a particularly preferred embodiment of the invention, the cell culture medium is substantially free of additives. Therefore, the cell culture medium may contain only the basal medium.

In a preferred embodiment of the invention, the cell culture medium is substantially free of serum components. A cell culture medium containing substantially no serum component is sometimes referred to as "serum-free medium" in the present specification. Examples of the serum component include heterologous serum components and allogeneic serum components, where "heterologous serum component" means a serum component derived from an organism of a species different from the recipient when the cell culture is used for transplantation. do. For example, when the recipient is human, serum derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum component. In addition, "homogeneous serum component" means a serum component derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to the allogeneic serum component. Allogeneic serum components include self-serum components, i.e., serum components derived from the recipient. Therefore, "substantially free of serum components" means that the content of these serums in the culture medium does not adversely affect the application of the cell culture to a living body (for example, serum albumin in the cell culture). The content is less than 50 ng), preferably means that these substances are not positively added to the culture broth. In addition, in this specification, serum other than autologous serum, that is, allogeneic serum homologous to heterologous serum may be collectively referred to as allogeneic serum or non-autologous serum.

In one aspect of the invention, the cell culture medium does not contain an effective amount of growth factor. Here, "growth factor" means any substance that promotes cell growth compared to its absence, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts. Includes cell growth factor (FGF) and the like. Further, "effective amount of growth factor" is an amount of growth factor that significantly promotes cell growth as compared with the case without growth factor, or, for convenience, for the purpose of cell growth in the art. Means the amount normally added as. The significance of cell proliferation promotion can be appropriately evaluated by, for example, any statistical method known in the art, for example, t-test, and the usual addition amount is various in the art. It can be known from publicly known documents. Specifically, the effective amount of EGF in culturing skeletal myoblasts is, for example, 0.005 μg / mL or more.

Therefore, "does not contain an effective amount of growth factor" means that the concentration of growth factor in the culture medium in the present invention is less than such an effective amount. For example, the concentration of EGF in the culture medium in culturing skeletal myoblasts is preferably less than 0.005 μg / mL, more preferably less than 0.001 μg / mL. In a preferred embodiment of the invention, the concentration of growth factor in the culture medium is less than the normal concentration in the living body. In such an embodiment, for example, the concentration of EGF in the culture medium in the culture of skeletal myoblasts is preferably less than 5.5 ng / mL, more preferably less than 1.3 ng / mL, still more preferably 0.5 ng / mL. Less than mL. In a more preferred embodiment, the culture medium in the present invention is substantially free of growth factors. Here, "substantially free" means that the content of the growth factor in the culture solution does not adversely affect the application of the cell culture to a living body, preferably, the growth factor is positively added to the culture solution. It means that it is not added. Therefore, in this embodiment, the culture medium does not contain growth factors having a concentration higher than that contained in other components thereof, such as serum.

In one aspect of the invention, the cell culture medium is substantially free of steroid component. Here, the "steroid component" refers to a compound having a steroid nucleus that can adversely affect the living body such as adrenal insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, "substantially free of steroid component" means that the content of these compounds in the culture medium does not adversely affect the application of the cell culture to a living body, preferably the culture medium. It means that these compounds are not positively added to the culture medium, that is, the culture medium does not contain other components thereof, for example, a steroid preparation component having a concentration higher than that contained in serum or the like.

In one aspect of the 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 a living body, such as selenous acid. Therefore, "substantially free of selenium component" means that the content of these substances in the culture medium does not adversely affect the application of the cell culture to a living body, preferably the culture medium. It means that these substances are not positively added, that is, the culture medium does not contain other components thereof, for example, a selenium component having a concentration higher than that contained in serum or the like. Specifically, for example, in the case of humans, the selenium concentration in the culture medium is the ratio of human serum contained in the medium to the normal value (for example, 10.6-17.4 μg / dL) in human serum. 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 growth factors, steroid agent components, and heterologous serum components, which have been conventionally required when producing 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 this manufacturing process.
Here, the "impurities derived from the manufacturing process" 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. Those derived from the extraction, separation, processing, and purification steps of a certain target substance (see, for example, Pharmaceutical Trial No. 571).

The cells can be cultured under the conditions normally used in the art. For example, typical culture 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, still more preferably 24 hours or less, from the viewpoint of sufficient formation of the cell culture and prevention of cell differentiation. Culturing can be carried out in containers of any size and shape. In the method of the present invention, since the cells do not proliferate substantially, the cell culture of the desired size and shape is short-term without waiting for the cell culture to grow to the desired size as in the conventional method. It will be possible to obtain in between. For the size and shape of the cell culture, adjust the size and shape of the cell adhesion surface of the culture vessel, or install a mold of the desired size and shape on the cell adhesion surface of the culture vessel and inside it. It can be arbitrarily 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 from the scaffolding substrate, at least in part, while preserving the sheet structure, eg, a proteolytic enzyme, eg. It can be performed by enzymatic treatment with trypsin or the like and / or mechanical treatment such as pipetting. Further, by culturing cells on a culture substrate whose surface is coated with a stimulus, for example, a material whose physical properties change in response to temperature or light to form a cell culture, a predetermined stimulus is applied. , The formed cell culture can also be released non-enzymatically.

The method of the present invention can also be positioned as one step of regenerative therapy, from cell collection to cell proliferation and cell culture preparation to application of cell culture. Therefore, the present invention
(I) A step of isolating desired cells from a tissue or biological fluid collected from a subject,
(Ii) Step of growing isolated cells,
(Iii) A step of seeding cells on a culture medium coated with serum,
(Iv) a step of culturing cells to form a sheet-shaped cell culture, and (v) a step of exfoliating the formed culture from a substrate,
It also relates to a method for producing a sheet-shaped cell culture for regenerative therapy, which comprises.

The present invention is also a cell culture prepared by the above-mentioned production method, and further, a cell culture substantially free of allogeneic serum, growth factors, steroids and / or selenium components, particularly a sheet-shaped cell culture. Regarding. In a 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 made by culturing cells in a culture medium that is substantially free of allogeneic serum, growth factors, steroids and / or selenium components to form cell cultures. Here, the cell culture is substantially free of allogeneic serum, growth factors, steroids and / or selenium components, that the cell culture does not contain these components at concentrations that adversely affect the recipient. That is, at least, such conditions can be met by forming the cell culture in a culture medium that is substantially free of allogeneic serum, growth factors, steroids and / or selenium components. In a further preferred embodiment, the cell culture of the present invention is substantially free of self-serum as well as impurities derived from the production process. Here, the meaning of "substantially not included" is the same as described above.

In a preferred embodiment of the cell culture of the present invention, the cell culture is reduced in inflammatory cytokines. The inflammatory cytokine is a general term for cytokines produced in association with inflammation, and examples thereof include, without limitation, TNF-α, IL-1, IL-6, and the like. Therefore, "the amount of inflammatory cytokines is reduced" means that the abundance or production amount (secretion amount) of these cytokines is reduced as compared with the case of cell culture formed in a serum-containing medium. Means. Therefore, cytokines may be present in any form of the process from gene transcription to protein secretion, and may be present, for example, in the form of transcripts such as mRNA or in the form of proteins. The degree of reduction is not particularly limited as long as it exceeds the error range, but is, for example, 15% or more, preferably 25% or more, more preferably 50% or more, as compared with the cell culture formed in the serum-containing medium. It is more preferably 60% or more, and particularly preferably 70% or more.

The amount of cytokine can be determined at the genetic level by any known method such as, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, 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, immunohistochemical method, and immuno-cell chemistry method. .. As the sample, a medium impregnated with the cell culture, a part of the cell culture, or the like can be used.

The cell culture of the present invention can be used for the treatment of a target disease or injury or illness. For example, cell cultures of skeletal myoblasts can be used in the form of implants and the like for heart disease, such as myocardial infarction, dilated cardiomyopathy and the like. Accordingly, another aspect of the invention relates to a graft comprising the cell culture.

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

The present invention further relates to a kit for producing the above-mentioned culture medium, which comprises a culture medium and a coating agent selected from the group consisting of serum, growth factors, and steroids. In the kit, the coating is in various storable forms, eg, in a refrigerated, frozen or lyophilized state, in a suitable container, eg, 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 includes media for diluting and / or reconstitution of the coating agent, such as water, physiological saline, various buffers (eg, PBS, HBS, etc.), and various liquid media (eg, PBS, HBS, etc.). For example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.), and coating methods for culture media. It may include a manual containing information about the above, electronic recording medium such as CD, DVD, and the like. The culture medium, coating agent, etc. contained in this kit can be provided preferably in a sterilized state. Examples of the sterilization method include, without limitation, gas sterilization with ethylene oxide gas and the like, sterilization with ultraviolet rays and radiation (for example, γ-rays), and the like.

The present invention also comprises (i) seeding cells on a culture substrate coated with serum, and (ii) culturing cells in a serum-free medium to form a cell culture. It relates to a method for suppressing the production of inflammatory cytokines of a substance.
Each step in this method is basically as described above with respect to the method for producing a sheet-shaped cell culture of the present invention. In the present method, the inflammatory cytokine is a general term for cytokines produced in association with inflammation, and examples thereof include, without limitation, TNF-α, IL-1, IL-6, and the like. Therefore, "suppressing the production of inflammatory cytokines" means reducing the production of these cytokines as compared to the case of forming cell cultures in a serum-containing medium. The degree of reduction is not particularly limited as long as it exceeds the error range, but is, for example, 15% or more, preferably 25% or more, more preferably 50%, as compared with the case where the cell culture is formed in the serum-containing medium. As mentioned above, it is more preferably 60% or more, and particularly preferably 70% or more.

At the gene level, the amount of cytokine produced is, for example, 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. And at the protein level, it can be detected by any known protein detection method such as immunoprecipitation method, Western blotting method, EIA, ELISA, RIA, immunohistochemical method, and immuno-cell chemistry method. can. As the sample, a medium impregnated with the cell culture, a part of the cell culture, or the like can be used.

Hereinafter, the present invention will be further described based on specific examples, but such specific examples are examples of the present invention and do not limit the present invention.

Comparative Example 1 Examination of serum-free medium For MCDB131 medium, IMDM medium (manufactured by GIBCO), CD hybriddome medium (manufactured by GIBCO), medium for breast epithelial cells (manufactured by GIBCO), or medium for nerve stem cells (manufactured by GIBCO) that does not contain serum components. After adding 0.01 μg / mL epithelial growth factor (manufactured by Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sankyo Pharmaceutical Co., Ltd.), human myoblasts 3.0 × 10 ⁶ to 3 in 2 mL each. .1 × 10 ⁶ pieces were suspended and seeded in untreated φ3.5 cm temperature-responsive culture medium (manufactured by Cellseed).
After seeding, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ , and the state was observed 18 hours later. As a result, no sheet-like cell culture was formed in any of the cultured cells.
FIG. 1 shows an external view after 18 hours of cell culture using a serum-free medium. From this result, it was clarified that the serum component is indispensable for the preparation of the sheet-shaped cell culture by the conventional method.

Comparative Example 2 Preparation of sheet-shaped cell culture by a conventional method 20% fetal bovine serum, 0.01 μg / mL epithelial growth factor (manufactured by Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sankyo Pharmaceutical Co., Ltd.) ) Is suspended in 2 mL of MCDB131 medium containing 3.0 × 10 ⁶ to 3.1 × 10 ⁶ human myoblasts, and seeded in untreated φ3.5 cm temperature-responsive culture dishes (manufactured by Cellseed). did. After seeding, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ concentration, and the state was observed 24 hours later. As a result, a sheet-like cell culture was formed (FIG. 2).

Example 1 Preparation of sheet-shaped cell culture using serum-free medium 400 μL of MCDB131 medium (manufactured by Invitrogen) containing 20% bovine fetal-derived serum (manufactured by Invitrogen) in a 24-well temperature-responsive culture dish (manufactured by Cellseed). The cells were added, incubated at 37 ° C. and 5% CO ₂ concentration, and removed by pipetting after 7 hours.
1 mL of Hanks' Balanced Salt Solution (manufactured by Invitrogen) was added to the culture dish after serum removal, and the mixture was slowly stirred and washed for 20 seconds, and then discarded by pipetting. This was repeated twice.
After washing, 400 μL of F12 / DMEM medium (manufactured by Invitrogen) to which no factors such as serum, growth factors, steroids, and selenium were added was added to the culture dish, and 3.0 × 10 ⁶ skeletal myoblasts (human). Origin) was sown.
After seeding, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ concentration, and the state was observed 24 hours later. As a result, a sheet-like cell culture was formed (FIG. 3). The formed sheet-shaped cell culture was compared with the sheet-shaped cell culture prepared by a known method using a medium containing a serum component (Comparative Example 2, FIG. 2), and no inferiority was observed by visual observation. It wasn't there.

Next, the cell viability and the purity of skeletal myoblasts of the prepared sheet-shaped cell cultures were compared.
The measurement of cell viability was performed according to the following procedure.
The formed sheet-like cell culture was dissociated with a trypsin-like proteolytic enzyme, and then the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of the cell suspension was collected before the cells were submerged and injected into the hemocytometer. Immediately after the injection, the number of cells observed in the entire 9 mm ² frame of the two chambers of the hemocytometer was measured with an inverted optical microscope.
After the measurement, the average number of living and dead cells in the two chambers was calculated, and the ratio of unstained cells to the total number of cells including stained cells was calculated.

The measurement of cell purity was performed according to the following procedure.
First, the formed sheet-like cell culture was dissociated with a trypsin-like proteolytic enzyme, and then centrifuged to discard the supernatant.
After adding 0.5% BSA-containing PBS solution and rinsing the cells, an anti-human CD56 antibody (manufactured by Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold with a 0.5% BSA-containing PBS solution was added and mixed.
Immediately after mixing each antibody, the cells were reacted in a cool and dark place for about 1 hour, 0.5% BSA-containing PBS solution was added, the cells were rinsed, and then 0.5% BSA-containing PBS solution was added for analysis.
For the analysis, a flow cytometer (manufactured by Becton Dickinson) was used to measure the proportion of antibody-positive cells contained in the cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and the number of cells was 5,000 to 10,000.
After the analysis, the purity was determined from the difference in the percentage of positive cells mixed with each antibody.
As the results in Table 2 show, there was no difference in the cell viability and skeletal myoblast purity of the sheet-shaped cell cultures of Example 1 and Comparative Example 2.

Example 2 Examination of safety of sheet-shaped cell culture prepared in serum-free medium In order to verify the effect of using serum-free medium, the following comparative test was carried out.
First, MCDB131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor (manufactured by Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sankyo Pharmaceutical Co., Ltd.) was subjected to a 24-well temperature response. 400 μL was added to a sex culture dish, incubation was performed under the conditions of 37 ° C. and 5% CO ₂ concentration, and the mixture was removed by pipetting after 7 hours. I prepared 6 of these.
As a known method, 400 μL of a medium having the same composition as described above was added to three of them, and 6.7 × 10 ⁵ skeletal myoblasts were seeded. After sowing, 200 μL of the above-mentioned medium impregnated with the sheet-like cell culture formed after culturing under the conditions of 37 ° C. and 5% CO ₂ concentration was collected and used as Specimen 1.

For the remaining 3 sheets, as the method of the present invention, 1 mL of Hanks' Balanced Salt Solution solution was added to the temperature-responsive culture dish that had undergone the above-mentioned incubation, and the mixture was slowly stirred and washed for 20 seconds, and then pipetting. Discarded. This stirring and washing was repeated twice. After washing, 400 μL of DMEM / F12 medium containing no bovine fetal serum, epidermal growth factor, or sodium dexamethasone phosphate was added, and 6.7 × 10 ⁵ skeletal myoblasts were seeded. After sowing, 200 μL of the above-mentioned medium impregnated with the sheet-like cell culture formed after culturing under the conditions of 37 ° C. and 5% CO ₂ concentration was collected and used as Specimen 2.

For each sample, the concentration of the cytokine produced in the cell culture process was measured using interleukin-6 (IL-6), which is known as an inflammatory cytokine, as an index. The interleukin-6 concentration was measured by the ELISA (Enzyme-Linked Immunosorbent Assay) method, and Human IL-6 Immunoassay (manufactured by R & D Systems) was used as the measurement kit. The measurement method with this kit is as follows.
100 μL of each sample was applied to each well of the ELISA plate to which 100 μL of RD1W solution was added in advance, and the mixture was reacted for 2 hours. After washing the wells 4 times, 200 μL of Conjugate was added and reacted for 2 hours. After washing the wells 4 times, 200 μL of Substrate Solution was added to develop color, and after 30 minutes, 50 μL of color stop solution was added, and then the absorbance at 450 nm was measured (control wavelength was 540 nm). Interleukin-6 of each sample was measured from the calibration curve. The concentration was calculated.
As the results in Table 3 show, the calculated interleukin-6 concentration for sample 2 was clearly lower than that of sample 1. This result indicates that the method for producing a sheet-shaped cell culture using a serum-free medium according to the present invention has low irritation to cells. Therefore, it has been clarified that the method of the present invention can provide a sheet-like cell culture having extremely low risk of developing inflammation and having high clinical safety.

Claims (3)

( I) After the step of keeping the culture medium coated with serum at 4 ° C. or lower, and (ii) step (i), the cells are placed on the culture medium coated with serum, and the density at which the cells reach confluence. A method for producing a sheet-shaped cell culture, which comprises a step of seeding at a density higher than or higher , and a step of culturing (iii) cells in a serum-free medium to form a sheet-shaped cell culture. The production method according to claim 1, wherein the cells are skeletal myoblasts. The method according to claim 1, wherein the culture medium comprises serum and a number of skeletal myoblasts seeded on the culture medium at a density reaching or above the confluent. , Combination .

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