JP6546219B2 - Method for producing sheet-like cell culture - Google Patents

Description

  The present invention relates to a method for producing a sheet-like cell culture, comprising the step of seeding cells on a serum-coated culture substrate, a sheet-like cell culture produced by the same method, in particular, an impurity derived from the production process A sheet-like cell culture substantially free of the above, a culture substrate coated with serum used in the above method, a method and a kit for producing the culture substrate, and a method of suppressing inflammatory cytokine production in a cell culture About.

  In ischemic heart diseases such as angina pectoris and myocardial infarction, sufficient oxygen can not be distributed to myocardial tissue, and if this condition continues for a long time, myocardial tissue damage occurs. Originally, adult cardiomyocytes have poor self-replication ability, so once damaged, heart muscle can not be repaired, or even if it can be repaired, only limited recovery can be expected and heart failure eventually occurs.

Although heart transplantation is an effective treatment method for heart failure, in many cases, a left ventricular assist artificial heart is attached as a bridge while waiting for transplantation.
However, heart transplantation has serious problems such as the risk of infection associated with immunosuppressive treatment, the appearance of coronary arteriosclerosis at a distant stage, and absolute donor shortage, etc. Also, the current assisted artificial heart is thromboembolic The patient's QOL is extremely limited due to complications such as illness and infection, and the durability of the device, making long-term assistance difficult.

Under these circumstances, skeletal myoblast transplantation into myocardial tissue is being studied as a new therapeutic method.
Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. Myocardium and skeletal muscle have many similarities in structure, function and so on, so it is thought that skeletal muscle-derived myoblasts can also repair damaged myocardium. Overseas, transplantation of autologous skeletal myoblasts into the myocardium is being clinically applied.

  Although the mechanism for suppression of cardiac function depression due to transplantation of skeletal myoblasts is not clear, transplantation of myoblasts into beating cardiac muscle results in orientation of the cell arrangement at the site where mechanical stretch is added. It is believed that proper differentiation is taking place, and also that it may function as a cytokine delivery system such as VEGF (vascular endothelial growth factor).

  However, in the transplantation of myoblast suspension as a single cell to an infarcted heart, damage to the transplanted cells, tissue damage at the time of injection into the recipient heart, tissue supply efficiency to the recipient heart, occurrence of arrhythmia, infarct Problems such as difficulty in treating the entire site have been pointed out, and sheeting of myoblasts has been desired to cope with these.

  On the other hand, by growing cells under specific culture conditions, an organization develops more than expected and an artificial tissue having a property of being easily detached from the culture dish is found, and it is found in parts other than adult myocardium. There has been provided a cell sheet as a three-dimensional tissue structure applicable to the heart including cells derived from it, and a method for producing the same (see Patent Document 1).

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

  In addition, it is known that fetal bovine serum, equine serum, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) 2 are used as a cell culture solution for proliferation of cardiac muscle precursor cells etc. (patented) Reference 2).

Thus, the formation of a sheet-like cell culture is usually performed by growing cells, and addition of growth factors is generally performed in addition to heterologous serum components to promote cell growth. For example, in skeletal myoblasts which tend to differentiate when confluent, it is general to add a steroid to the culture solution of skeletal myoblasts in order to suppress the transition of proliferated cells to differentiation. It is. Without the addition of the steroid agent, skeletal myoblasts begin to differentiate in about 16 hours of culture to form tubular myotubes, making it impossible to obtain a sheet-like cell culture.
Furthermore, addition of selenium may be performed to the culture solution for the purpose of antioxidative action.

  However, considering clinical application, other serum components may contain pathogens such as viruses that can infect the recipient, and growth factors are also usually modified using microorganisms. If it is considered that it will be manufactured, it may become an impurity derived from the manufacturing process if it remains, so it can not be used for transplantation as it is from the viewpoint of safety.

  In addition, selenium is an essential element necessary for the synthesis of antioxidative enzymes for the living body, and is incorporated into proteins as selenocysteine, mainly acting as selenoproteins, and cooperates with vitamins E and C to protect the living body from active oxygen and radicals It is believed that. However, selenium has a very narrow range of appropriate and toxic levels, and symptoms such as nausea, nausea, diarrhea, anorexia, headache, immunosuppression, high density lipoprotein (HDL) reduction, etc. are known as excess disease. There is. Furthermore, selenium acid is designated as a poison designated item in the “Poison and Dramatic Designation Order” (Decree No. 2 of January 4, 1940). On the other hand, as steroids, adrenal cortical dysfunction, Cushing's syndrome and the like are known as side effects thereof, and it is a component which is preferably removed at the time of transplantation as an impurity derived from the production process.

Although these process-derived impurities are usually removed by washing the cell culture with a medium free of these substances, the cell culture is mechanically extremely fragile, and is easily removed by water flow during washing, etc. Because of the disruption, it has been extremely difficult to wash manufacturing process derived impurities in cell cultures to levels that are not clinically disruptive.
Furthermore, although it is possible to use autologous serum that has less adverse effects on the recipient instead of the allogeneic serum, the autologous serum needs to be prepared by collecting blood from the recipient in advance. It imposes a physical and time burden, and depending on the condition of the recipient, it may be difficult to obtain.
Therefore, there has been a need for a methodological improvement for conveniently obtaining a clinically safe, high-quality sheet-like cell culture.

Japanese Patent Application Publication No. 2007-528755 JP, 2008-161183, A

  An object of the present invention is to provide a high-quality cell culture which does not contain a production process-derived impurity component which may be an obstacle to clinical application, and a method for producing the same.

  The inventors of the present invention have conducted intensive studies to solve the above problems, and after the culture substrate is coated with serum, when cells are cultured on this culture substrate, serum-free cell types are also serum-free. It was found that a sheet-like cell culture can be obtained by the medium. Furthermore, it has been found that by culturing cells in such a culture substrate in a serum-free medium, the production of inflammatory cytokines can be significantly reduced as compared to cell cultures cultured in a serum-containing medium, and the present invention has been completed. .

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

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

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

  According to the present invention, since culture in a non-cell growth system containing no serum or growth factor, which is generally added as a factor necessary for cell growth, is possible in the production of sheet-like cell cultures, Contamination with components and endotoxin and the like that may be included in growth factors, which are usually recombinant products, can be avoided, and no steroid agent added as a differentiation inhibitor is required. Furthermore, the present invention eliminates the need for selenium or its derivatives to prevent oxidation. As a result, it is possible to provide a clinically safe cell culture free of manufacturing process-derived impurities, and it is not necessary to prepare autologous serum as a substitute for heterologous serum.

In addition, by enabling culture in a serum-free medium, it is possible to significantly reduce the production of inflammatory cytokines as compared to a cell culture cultured in a serum-containing medium and to further enhance the clinical safety of the cell culture. Is possible.
Furthermore, when cultured in a medium that does not contain serum, growth factors, etc., operations such as washing for the purpose of removing impurities from manufacturing processes that may cause damage to the prepared cell culture become unnecessary, Reliable and stable manufacturing is possible.
Furthermore, according to the method of the present invention, a cell culture of a desired size and shape can be produced in a short period of time, so that treatment of a living body using the cell culture can be performed more flexibly and easily.

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

The present invention comprises: (i) seeding cells on a serum-coated culture substrate; and (ii) culturing the cells to form a sheet-like cell culture. It relates to the manufacturing method of the object.
The cells in the present invention include cell cultures, in particular any cells capable of forming a sheet-like cell culture. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, synoviocytes, epithelial cells, endothelial cells and the like. Among these, in the present invention, those which form a monolayer cell culture, for example, myoblasts are preferred. The cells can be derived from any organism that can be treated by 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 cells may be used in the method of the present invention, two or more types of cells can also be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the proportion (purity) of the largest number of cells is 65% or more, preferably 70% or more, at the end of cell culture production. Preferably it is 75% or more.

  In the present invention, the “culture substrate” is not particularly limited as long as cells can form a cell culture thereon, and, for example, containers of various materials, solid or semi-solid surfaces in containers, etc. including. The container is preferably of a structure / material which does not allow liquid such as culture fluid to permeate. Such materials include, but are not limited to, for example, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl Acrylamide, metals (for example, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also preferably, the container has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles and the like. Also, the container may have a solid or semi-solid surface inside. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semisolid surfaces include gels and soft polymer matrices.

The culture substrate may be coated on the surface with a material whose physical properties change in response to a stimulus such as temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethyl acrylamide, N-n-propyl acrylamide, N-n-propyl methacrylamide, N-isopropyl acrylamide, N-isopropyl methacrylamide, N-cyclopropyl acrylamide, N-cyclopropyl methacrylamide, N-ethoxyethyl acrylamide, N-ethoxyethyl methacrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacryl Amide, etc.), N, N-dialkyl substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethyl methyl acrylamide, N, N-diethyl) (Meth) acrylamide derivatives having cyclic groups (eg, 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 comprising homopolymer or copolymer of vinyl ether derivative (eg methyl vinyl ether), light absorbing polymer having azobenzene group, triphenylmethane leuco hydro Copolymers of vinyl derivatives of oxides with acrylamide monomers, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By applying a predetermined stimulus to these materials, their physical properties, such as hydrophilicity and hydrophobicity, can be changed to promote detachment of the cell culture adhered on the same materials.
The culture substrate may have various shapes, but is preferably flat. Moreover, the area is not particularly limited, but typically 1 to ²⁰⁰ cm ² , preferably ² to 100 cm ² , more preferably 3 to 50 cm ² .

In the present invention, the culture substrate is coated (coated or coated) with serum. The expression "coated with serum" means that the serum component is attached to the surface of the culture substrate. Such conditions can be obtained without limitation, for example, by incubating the culture substrate with serum and then discarding the serum. Incubation involves contacting the serum with the culture substrate. As serum, heterologous serum and allogeneic serum can be used. Heterologous serum, when transplanting a cell culture, refers to serum from a different species of organism than the recipient. For example, when the recipient is a human, serum derived from cows and horses, such as fetal bovine serum (FBS, FCS), calf serum (CS), equine serum (HS), etc. corresponds to different serum. Also, "homologous 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 routine method from blood collected from a desired organism. Specifically, for example, the collected blood is allowed to stand at room temperature for about 20 to 60 minutes to be coagulated, and this is centrifuged at about 1000 to 1200 × g to collect the supernatant.

When incubated on a culture substrate, serum may be used as a stock solution or may be used after dilution. Dilutions can be any medium, including, but not limited to, water, saline, various buffers (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 131, 153, 199 etc.), L15, SkBM, RITC 80-7, DMEM / F 12 etc. can be used. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate, and for example, 0.5 to 90% (v / v), preferably 1 to 60% (v / v), more preferably Is 5 to 40% (v / v).
The incubation time is also not particularly limited as long as the serum component can adhere to the culture substrate, and for example, 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, further preferably 6 to 12 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, a conventional liquid disposal method such as aspiration with a pipette or decantation can be used. In a preferred embodiment of the present invention, the culture substrate may be washed with a serum-free washing solution after serum disposal. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect serum components attached to the culture substrate, and, for example, without limitation, water, physiological saline, various buffers Solutions (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDBs 102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7 , DMEM / F12 etc.). As a washing method, a conventional culture substrate washing method, for example, without limitation, a serum-free washing solution is added to the culture substrate and stirred for a predetermined time (for example, 5 to 60 seconds) and then discarded 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 to in the absence thereof, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts Cell growth factor (FGF) and the like. In the coating method, the discarding method and the washing method of the culture substrate by 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 substrate may be coated with a steroid agent. Here, the "steroid agent component" refers to a compound having a steroid nucleus that can exert adverse effects such as adrenocortical dysfunction, Cushing's syndrome and the like on a living body. Such compounds include, but are not limited to, eg, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. The coating method, the discarding method and the washing method of the culture substrate with a steroid agent are, for example, 0.1 μg / mL to 100 μg / mL, preferably 0.4 μg / mL to 40 μg / mL as the diluted concentration at the time of incubation as dexamethasone It is basically the same as serum except that it is more preferably 1 μg / mL to 10 μg / mL.

  The culture substrate may be coated with any one of serum, growth factor and steroid, or any combination thereof, ie serum and growth factor, serum and steroid, serum and growth factor and steroid, 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 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 cells can be seeded. The coated substrate can be stored for a long time, for example, by keeping the temperature at 4 ° C. or less, preferably −20 ° C. or less, more preferably −80 ° C. or less.

In a preferred embodiment of the invention, the cells are seeded at a density that can form a sheet-like cell culture substantially without growth. The "density capable of forming a sheet-like cell culture substantially without proliferation" means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain a growth factor. Mean cell density. For example, in the case of skeletal myoblasts, in the conventional method using a culture medium containing growth factor, cells are plated on the plate at a density of about 6,500 cells / cm ² to form a sheet-like cell culture. However, (see, for example, Patent Document 1), it is not possible to form a sheet-like cell culture even if cells of such a density are cultured in a growth factor-free medium. Therefore, the cell density in this embodiment is higher than that in the conventional method using a medium containing a growth factor. Specifically, for example, for skeletal myoblasts, such a 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 go into differentiation, and 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 experiments. During the culture period, the cells may or may not proliferate, but even if they do, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts begin to differentiate when confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a cell culture but does not go into differentiation. In a preferred embodiment of the invention, the cells do not grow beyond the range 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 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 the number of cells at the time of seeding Is less than 100%.

In the present invention, cell culture is performed under conditions suitable for the cells of interest to form a sheet-like cell culture.
In the present invention, “sheet-like cell culture” refers to a cell in which cells are linked to each other into a sheet, and typically consists of one cell layer, but it is composed of two or more cell layers. Including those that are configured. The cells may be linked to each other directly and / or via an mediator. The mediator is not particularly limited as long as it is a substance capable of at least mechanically connecting the cells, and examples thereof include extracellular matrix. The mediator is preferably of cell origin, in particular of the cells constituting the cell culture. The cells are at least mechanically linked, but may be further functionally, eg chemically, electrically linked.

  The sheet-like cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within their surfaces and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Membranes and the like are known, but the cell culture of the present invention can maintain its physical integrity without such scaffolds. In addition, the cell culture of the present invention preferably comprises only the substance derived from cells constituting the cell culture, and does not contain any other substance.

  In one embodiment of the present invention, the culture of the cells is performed within a predetermined period of time, preferably within a period of time in which the cells do not go into differentiation. Thus, 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 one skilled in the art. For example, in the case of skeletal myoblasts, expression of MHC or multinucleation of cells can be used as an indicator of differentiation. In a preferred embodiment of the present invention, the culture period is within 48 hours, more preferably within 40 hours, even more preferably within 24 hours.

  The cell culture solution (sometimes simply referred to as "culture solution" or "medium") used for culture is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, 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 a basal medium includes, but is not limited to, for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDBs 102, 104, 107, 131, 153, 199 etc.), L15, SkBM, RITC80-7, etc. . Many of these basal media are commercially available, and their compositions are also known. As an example, the composition of MCDB 131 and DMEM is shown in Table 1 below.

The basal medium may be used as a standard composition (for example, as it is marketed), or its composition may be changed as appropriate depending on the cell type and cell conditions. Accordingly, the basal medium used in the present invention is not limited to those of known composition, but includes those in which one or more components are added, removed, increased or decreased.

The amino acids contained in the basal medium include, but are not limited to, 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 etc. and as electrolytes, without limitation, for example, CaCl ₂ , KCl, MgSO ₄ , NaCl, NaH ₂ PO ₄ , NaHCO ₃ , Fe (NO ₃ ) ₃ , FeS O ₄ , CuSO ₄ , MnSO ₄ , Na ₂ SiO ₃ , (NH ₄ ) 6Mo ₇ O ₂₄ , NaVO ₃ , NiCl ₂ , ZnSO _{4 and the} like are included, respectively. The basal medium may contain, in addition to these components, 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 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 -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 to 104 mg / L, L-valine: 94 to 117 mg / L.
In one embodiment of the present invention, the concentration of the vitamin agent contained in the basal medium is 4 to 12 mg / L of calcium D-pantothenate, 4 to 14 mg / L of choline chloride, and 0.6 to 4 mg / L of folic acid. , I-inositol: 7.2 mg / L, niacinamide: 4 to 6.1 mg / L, riboflavin: 0.0038 to 0.4 mg / L, thiamine: 3.4 to 4 mg / L, pyridoxine: 2.1 to It is 4 mg / L.

  In addition to the above, the cell culture solution may contain one or more additives such as serum, growth factor, steroid component, selenium component and the like. However, these components are impurities in the manufacturing process that can not be denied to cause side effects such as anaphylactic shock to recipients in the clinic, and should be excluded in the 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. Also, 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. Thus, 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. Cell culture media substantially free of serum components may also be referred to herein as "serum free media". Serum components include heterologous serum components and allogeneic serum components, where "heterologous serum component" means serum components derived from an organism of a species different from that of the recipient when the cell culture is used for transplantation. Do. For example, when the recipient is a human, serum derived from bovine or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), equine serum (HS), etc. corresponds to different serum components. Also, "homologous serum component" means a serum component derived from an organism of the same species as the recipient. For example, when the recipient is a human, human serum corresponds to the homologous serum component. The allogeneic serum component comprises an autologous serum component, ie a serum component derived from the recipient. Therefore, "substantially free of serum components" means that the content of these sera in the culture solution does not adversely affect cell cultures when applied to a living body (for example, serum albumin in cell cultures). The content of less than 50 ng) preferably means that these substances are not actively added to the culture solution. In the present specification, serum other than autologous serum, that is, heterogenous serum and allogeneic serum may be collectively referred to as heterogeneous 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 the proliferation of cells as compared to in the absence thereof, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts Cell growth factor (FGF) and the like. Also, an "effective amount of growth factor" refers to an amount of growth factor that significantly promotes cell proliferation as compared to in the absence of the growth factor or, for convenience, to aim for cell proliferation in the art. It means the amount usually added as The significance of cell growth promotion can be appropriately evaluated, for example, by any statistical method known in the art, such as t-test, etc., and the usual addition amount can be varied according to various art. It can be known from known literature. Specifically, the effective amount of EGF in the culture of skeletal myoblasts is, for example, 0.005 μg / mL or more.

  Thus, "containing no effective amount of growth factor" means that the concentration of growth factor in the culture medium in the present invention is less than the effective amount. For example, the concentration of EGF in the culture medium 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 invention, the concentration of growth factor in the culture medium is less than the normal concentration in the organism. 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. It is less than mL. In a further preferred embodiment, the culture medium in the present invention is substantially free of growth factors. Here, the term "substantially free" means that the content of the growth factor in the culture solution is such that it 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. Meaning not added. Thus, in this aspect, the culture medium does not contain other components therein, such as growth factors at concentrations above that contained in serum or the like.

  In one aspect of the invention, the cell culture fluid is substantially free of a steroid agent component. Here, the "steroid agent component" refers to a compound having a steroid nucleus that can exert adverse effects such as adrenocortical dysfunction, Cushing's syndrome and the like on a living body. Such compounds include, but are not limited to, eg, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, the phrase "substantially free of steroid component" means that the content of these compounds in the culture solution is not adversely affected when the cell culture is applied to a living body, preferably, the culture solution Not actively adding these compounds, that is, the culture solution does not contain the steroid component at a higher concentration than that contained in other components such as serum.

  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, in particular, a selenium-containing compound capable of releasing a selenium molecule in vivo, such as selenium acid and the like. Therefore, the phrase "substantially free of selenium component" means that the content of these substances in the culture solution is not adversely affected when the cell culture is applied to a living body, and preferably to the culture solution. It means that these substances are not actively added, that is, the culture solution does not contain the selenium component at a concentration higher than that contained in other components, such as serum. Specifically, for example, in the case of human, the selenium concentration in the culture solution is the normal value (for example, 10.6 to 17.4 μg / dL) in human serum, and the proportion 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, less than 1.0 to 1.7 μg / dL).

In the above preferred embodiment of the present invention, impurities such as growth factors, steroid agent components, different serum components and the like, which have conventionally been required when producing cell cultures to be applied to living organisms, are removed by washing or the like. The process is unnecessary. Therefore, one aspect of the method of the present invention does not include the step of removing this manufacturing process-derived impurity.
Here, the term "manufacturing process derived impurities" typically includes those listed below which are derived from each manufacturing process. That is, those derived from the cell substrate (for example, host cell-derived protein, host cell-derived DNA), those derived from the cell culture solution (for example, inducer, antibiotics, medium components), or steps after cell culture These include those derived from extraction, separation, processing, and purification steps of certain target substances (see, for example, Japanese Patent Laid-Open No. 571).

Culturing of the cells can be performed under conditions that are commonly used in the art. For example, typical culture conditions include culture at 37 ° C., 5% CO ₂ . The culture period is preferably within 48 hours, more preferably within 40 hours, still more preferably within 24 hours, from the viewpoint of sufficient formation of cell cultures and prevention of cell differentiation. Culturing can be performed in vessels of any size and shape. In the method of the present invention, since the cells do not substantially proliferate, the cell culture of the desired size and shape can be short-termed without waiting for the cell culture to grow to the desired size as in the conventional method. It becomes possible to obtain between. The size and shape of the cell culture can be adjusted by adjusting the size and shape of the cell attachment surface of the culture container, or by placing a mold of the desired size and shape on the cell attachment surface of the culture container Can be optionally adjusted, such as by culturing cells.

  The method may further comprise the step of isolating the cell culture from the culture substrate. The isolation of the cell culture from the substrate is not particularly limited as long as the cell culture can be at least partially released from the scaffolding substrate while maintaining the sheet structure, for example, a proteolytic enzyme such as, for example, It can be carried out by enzyme treatment with trypsin or the like and / or mechanical treatment such as pipetting. In addition, the cells are cultured on a culture substrate coated on the surface with a material whose physical properties change in response to a stimulus such as temperature or light to form a cell culture, thereby applying a predetermined stimulus. It is also possible to release the formed cell cultures non-enzymatically.

The method of the present invention can also be regarded as one step of regenerative therapy, from the collection of cells, through the growth of cells and the preparation of cell cultures, to the application of cell cultures. Therefore, the present invention
(I) isolating desired cells from tissue or biological fluid collected from a subject;
(Ii) proliferating the isolated cells,
(Iii) seeding the cells on a serum-coated culture substrate,
(Iv) culturing the cells to form a sheet-like cell culture, and (v) detaching the formed culture from the substrate,
The present invention also relates to a method for producing a sheet-like cell culture for regeneration treatment, comprising

  The present invention also provides a cell culture prepared by the above-mentioned production method, and further, a cell culture substantially free of allogeneic serum, growth factor, steroid agent and / or selenium component, particularly a sheet-like cell culture. About. In a preferred embodiment, the cell culture of the present invention is substantially free of manufacturing process derived impurities comprising the above components. This cell culture can be produced by culturing the cells in a culture medium substantially free of allogeneic serum, growth factors, steroids and / or selenium components to form a cell culture. Here, when the cell culture is substantially free of allogeneic serum, growth factors, steroids and / or selenium components, the cell culture does not contain these components at a concentration that adversely affects the recipient. Although at least meaning, such conditions can be satisfied by performing the formation of the cell culture in a culture medium 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 autologous serum as well as impurities derived from the production process. Here, the meaning of "substantially free" is the same as above.

  In a preferred embodiment of the cell culture of the present invention, the cell culture is reduced in inflammatory cytokines. Inflammatory cytokines are a generic term for cytokines produced with inflammation, including, but not limited to, TNF-α, IL-1, IL-6 and the like. Therefore, "the inflammatory cytokines are reduced" means that the abundance or production (secretion amount) of these cytokines is reduced as compared to the case of cell cultures formed in serum-containing media. Means Therefore, the cytokine may be present in any form in the process from gene transcription to protein secretion, 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, and for example, it is 15% or more, preferably 25% or more, more preferably 50% or more, as compared to a cell culture formed with a serum-containing medium. More preferably, it is 60% or more, particularly preferably 70% or more.

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

  The cell culture of the present invention can be used to treat a subject's disease or injury. For example, cell cultures with skeletal myoblasts can be used in the form of grafts etc. for heart disease, eg myocardial infarction, dilated cardiomyopathy etc. Thus, another aspect of the present invention relates to a graft 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-like cell culture according to the present invention described above. The coating method and the like are as described above for the method for producing a sheet-like cell culture of the present invention.
The invention further relates to a method of producing the above-mentioned culture substrate, which comprises (i) incubating the culture substrate with serum, and (ii) discarding the serum. The specific production procedure is as described above for the method for producing a sheet-like cell culture of the present invention. In one embodiment of the present invention, after step (ii), (iii) a step of washing the culture substrate with a serum-free washing solution may be added.

  The present invention still further relates to a kit for producing the above-mentioned culture substrate, which comprises a culture substrate and a coating agent selected from the group consisting of serum, growth factor and steroids. In the kit, the coating agent is in various storable forms, such as refrigerated, frozen or lyophilised, in suitable containers, such as vials of various materials (glass, plastic etc), ampoules, bottles, It may be stored in a tube or the like. In addition to the above, the kit of the present invention may be a medium for diluting and / or reconstituting the coating agent, such as water, saline, various buffers (eg PBS, HBS etc.), various liquid media (eg For example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 131, 153, 199 etc.), L15, SkBM, RITC80-7, DMEM / F12 etc., and culture substrate coating techniques And an electronic recording medium such as a CD and a DVD. The culture substrate, the coating agent and the like contained in the present 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 light and radiation (eg, γ-ray), and the like.

The invention also includes the steps of (i) seeding the cells on a serum-coated culture substrate, and (ii) culturing the cells in serum-free medium to form a cell culture. The present invention relates to a method of suppressing inflammatory cytokine production of a substance.
Each step in the present method is basically as described above for the method for producing a sheet-like 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 includes, for example, without limitation, TNF-α, IL-1, IL-6 and the like. Thus, "suppressing inflammatory cytokine production" means reducing the production of these cytokines as compared to when cell cultures are formed with serum-containing media. The degree of reduction is not particularly limited as long as it exceeds the range of error, and for example, it is 15% or more, preferably 25% or more, more preferably 50% or more, as compared to the case of forming a cell culture with a serum-containing medium. The content is more preferably 60% or more, particularly preferably 70% or more.

  At the gene level, cytokine production can be performed by any known method such as Northern blotting, Southern blotting, RNase protection assay, RT-PCR, PCR such as real-time PCR, in situ hybridization, in vitro transcription, etc. The gene expression analysis method of the present invention, and at the protein level, may be detected by any known protein detection method such as immunoprecipitation, western blotting, EIA, ELISA, RIA, immunohistochemistry, immunocytochemistry, etc. it can. As a sample, a medium in which a cell culture is impregnated, a part of a 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 are not intended to limit the present invention.

Comparative Example 1 Examination of serum-free medium MCDB 131 medium without serum components, IMDM medium (GIBCO), CD hybridoma medium (GIBCO), mammary epithelial cell medium (GIBCO) or neural stem cell medium (GIBCO) After adding 0.01 μg / mL epidermal growth factor (manufactured by Invitrogen) and 4 μg / mL dexamethasone sodium phosphate injection (Daichi Sanko Pharmaceutical Co., Ltd.), 2 mL each of human myoblasts 3.0 × 10 ^{6 to} 3 1 × 10 ⁶ were suspended and each was seeded on an untreated φ3.5 cm temperature-responsive culture dish (made by Cellseed).
After seeding, the cells were cultured under conditions of 37 ° C., 5% CO ₂ and subjected to state observation 18 hours later. As a result, no sheet-like cell culture was formed in any of the cultured cells.
FIG. 1 shows an appearance view after 18 hours of cell culture using a serum-free medium. From this result, it has become clear that the serum component is indispensable for the preparation of a sheet-like cell culture in the conventional method.

Comparative Example 2 Preparation of a sheet-like cell culture according to a conventional method 20% fetal calf serum, 0.01 μg / mL epidermal growth factor (manufactured by Invitrogen), 4 μg / mL sodium dexamethasone phosphate injection (manufactured by Daiichi Sanko Pharmaceutical Co., Ltd.) 3.) 3.0 × 10 ^{6 to} 3.1 × 10 ⁶ human myoblasts are suspended in 2 mL of MCDB 131 medium containing C. and seeded in untreated φ3.5 cm temperature-responsive culture dishes (manufactured by Cellseed) did. After seeding, the cells were cultured under conditions of 37 ° C. and 5% CO ₂ concentration, and after 24 hours, observation of conditions was carried out. As a result, a sheet-like cell culture was formed (FIG. 2).

Example 1 Preparation of a sheet-like cell culture using a serum-free medium 400 μL of MCDB 131 medium (Invitrogen) containing 20% fetal bovine serum (Invitrogen) in a 24-well temperature-responsive culture dish (Cellseed) The solution was added, incubated at 37 ° C., 5% CO ₂ concentration, and removed by pipetting after 7 hours.
After removing serum, 1 mL of Hanks' Balanced Salt Solution (manufactured by Invitrogen) was added to the culture dish, and after gently washing with stirring for 20 seconds, this was discarded by pipetting. This was repeated twice.
After washing, add 400 μL of F12 / DMEM medium (manufactured by Invitrogen) to which no factor such as serum, growth factor, steroid or selenium was added to the culture dish, and 3.0 × 10 ⁶ skeletal myoblasts (human ) (Seed).
After seeding, culture was performed under conditions of 37 ° C. and 5% CO ₂ concentration, and after 24 hours, observation of conditions was carried out, and as a result, a sheet-like cell culture was formed (FIG. 3). The formed sheet-like cell culture was compared with the sheet-like cell culture (Comparative Example 2, FIG. 2) prepared by a known method using a medium containing serum components, and any fading was observed in visual observation. It was nothing.

Next, cell viability and skeletal myoblast purity of the prepared sheet-like cell cultures were compared.
The measurement of cell viability followed the following procedure.
The formed sheet-like cell culture was dissociated with trypsin-like protease, and the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of cell suspension was collected before cells were allowed to sink and was injected into a hemocytometer. Immediately after 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 light microscope.
After measurement, the average number of viable and dead cells in the two chambers was determined, and the ratio of unstained cells to the total number of cells including stained cells was calculated.

Measurement of cell purity was performed according to the following procedure.
First, the formed sheet-like cell culture was dissociated with a trypsin-like protease and centrifuged to discard the supernatant.
To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody (Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, one to which a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold in a PBS solution containing 0.5% BSA was added and mixed was prepared.
After mixing the respective antibodies, they were reacted immediately in a cool dark place for about 1 hour and 0.5% BSA-containing PBS was added to rinse the cells, and 0.5% BSA-containing PBS was added for analysis.
The analysis used the flow cytometer (made by Becton Dickinson), and measured the ratio of the antibody positive cell contained in the cell which mixed each antibody. In the measurement, the positive rate of the negative control was corrected, and the number of cells was analyzed to 5,000 to 10,000.
After analysis, the purity was determined from the difference in the proportion of positive cells in cells mixed with each antibody.
As the results in Table 2 indicate, no difference was found in the cell viability and skeletal myoblast purity of the sheet-like cell cultures of Example 1 and Comparative Example 2.

Example 2 Study on Safety of Sheet Cell Culture Made in Serum-Free Medium In order to verify the effect using serum-free medium, the following comparative test was performed.
First, MCDB 131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor (Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (Daiichi Sanko Pharmaceutical Co., Ltd.) was used as a 24 well temperature response. 400 μL was added to the culture dishes, incubated at 37 ° C., 5% CO ₂ , and removed by pipetting 7 hours later. Six pieces of this were prepared.
Three of the plates were seeded with 6.7 × 10 ⁵ skeletal myoblasts by adding 400 μL of a medium having the same composition as described above as a known method. After seeding, culture was performed under conditions of 37 ° C., 5% CO ₂ concentration, and after 24 hours, 200 μL of the above-mentioned culture medium impregnated with the sheet-like cell culture formed was collected and used as Sample 1.

In the remaining three plates, 1 mL of Hanks' Balanced Salt Solution solution was added to the temperature-responsive culture dish subjected to the above-mentioned incubation as the method of the present invention, and after gently stirring for 20 seconds, this was washed by pipetting. Discarded. This agitation washing was repeated twice. After washing, 400 μL of DMEM / F12 medium not containing any of fetal bovine serum, epidermal growth factor and dexamethasone sodium phosphate was added to disseminate 6.7 × 10 ⁵ skeletal myoblasts. After seeding, culture was performed under conditions of 37 ° C., 5% CO ₂ concentration, and after 24 hours, 200 μL of the above-mentioned culture medium impregnated with the sheet-like cell culture formed was collected and used as sample 2.

For each sample, the concentration of the same cytokine produced in the cell culture process was measured using interleukin-6 (IL-6) known as an inflammatory cytokine as an index. The measurement of interleukin-6 concentration was performed by ELISA (Enzyme-Linked Immunosorbent Assay) method, and Human IL-6 Immunoassay (manufactured by R & D Systems) was used as a measurement kit. The measurement method with this kit is as follows.
100 μL of each sample was applied to each well of the plate for ELISA to which 100 μL of RD1W solution had been previously added, and allowed to react for 2 hours. After washing the wells 4 times, 200 μL of Conjugate was added and allowed to react for 2 hours. The wells were washed 4 times, and then 200 μL of Substrate Solution was added to develop color, and 30 minutes after that, 50 μL of a color stop solution was added, and then the absorbance at 450 nm was measured (control wavelength is 540 nm). The concentration was calculated.
As the results in Table 3 indicate, the interleukin-6 concentration calculated for sample 2 was clearly lower than for sample 1. The results show that the method for producing a sheet-like cell culture using a serum-free medium according to the present invention gives less stimulation to cells. Therefore, it has been revealed that the method of the present invention can provide a highly safe sheet-like cell culture with a very low risk of developing inflammation.

Claims (3)

(I) maintaining the serum-coated culture substrate at 4 ° C. or lower, and
(Ii) after the step (i), seeding the cells on the serum-coated culture substrate at a density at which the cells reach confluence or higher to form a sheet-like cell culture
A method of producing a sheet-like cell culture, comprising: The culture substrate further comprising a step of coating with serum, the method according to claim 1. The method according to claim 1 or 2 , wherein the serum is autologous serum.