JP5436903B2 - Method for producing sheet cell culture - Google Patents

Description

  The present invention provides a method for producing a sheet-shaped cell culture comprising pressurizing cells against a substrate, and a sheet-shaped cell culture produced by such a method, in particular, impurities derived from the production process. The present invention relates to a sheet-shaped cell culture that does not contain.

  In an ischemic heart disease such as angina pectoris and myocardial infarction, sufficient oxygen does not reach the myocardial tissue, and if this state continues for a long time, the myocardial tissue is damaged. Originally, adult cardiomyocytes are poor in self-replicating ability, so once damaged, the myocardium cannot be repaired, or even if it can be repaired, only limited recovery can be expected and eventually heart failure will occur.

Although heart transplantation is an effective method for treating heart failure, a left ventricular assist device is often used 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 distant coronary sclerosis, and the lack of absolute donors. Patient QOL is very limited due to complications such as illness and infection, and device durability, and long-term assistance is difficult.

Under such circumstances, skeletal myoblast transplantation into myocardial tissue is being studied as a new treatment method.
Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. The myocardium and skeletal muscle have many parts that are similar in structure and function, and it is therefore considered that myoblasts derived from skeletal muscle can repair damaged myocardium. Overseas, autologous skeletal myoblast transplantation into the myocardium is being clinically applied.

  Although the mechanism for suppressing the decline in cardiac function due to transplantation of skeletal myoblasts is not clear, transplantation of myoblasts into beating myocardium causes orientation of the cell arrangement when mechanical stretch is applied Appropriate differentiation is considered to be 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 to the infarcted heart causes loss of transplanted cells, tissue damage during injection of the recipient heart, tissue supply efficiency to the recipient heart, occurrence of arrhythmia, infarct Disadvantages, such as difficulty in treatment of the entire region, have been pointed out, and there has been a craving for making myoblasts into a sheet to deal with these.

  On the other hand, an artificial tissue with the property that the organization is progressed more than expected by proliferating the cells under specific culture conditions and is easy to peel off from the culture dish, and is found in parts other than the adult myocardium. There has been provided a cell sheet as a three-dimensional tissue structure applicable to the heart including cells derived therefrom, and a production method thereof (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 supplemented with a serum such as a known bovine fetal serum. It is considered good (see, for example, Patent Document 1).

  In addition, it is known that fetal bovine serum, horse serum, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) 2 are used as cell culture media used for the proliferation of myocardial progenitor cells (patent) Reference 2).

As described above, the formation of a sheet-shaped cell culture is usually carried out by proliferating cells, and in order to promote cell proliferation, growth factors are generally added in addition to heterogeneous serum components. For example, in skeletal myoblasts that tend to differentiate when confluent, it is common to add a steroid to the culture of skeletal myoblasts in order to suppress the transition of the proliferated cells to differentiation. It is. If a steroid is not added, skeletal myoblasts will begin to differentiate in culture for about 16 hours to form tubular myotubes, making it impossible to obtain a sheet-like cell culture.
Further, selenium may be added to the culture solution for the purpose of antioxidant action.

  However, considering clinical applications, non-self serum components such as heterologous serum components may contain pathogens such as viruses that can infect recipients, and growth factors are also usually recombined using microorganisms. In view of the fact that it can be produced, it can become an impurity derived from the production process if it remains, 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. It is incorporated into proteins as selenocysteine and works mainly as selenoprotein, and protects the living body from active oxygen and radicals in cooperation with vitamins E and C. It is considered to be. However, selenium has a very narrow range of appropriate and toxic doses, and symptoms such as nausea, nausea, diarrhea, loss of appetite, headache, immunosuppression, and high-density lipoprotein (HDL) decrease are known as excess symptoms. Yes. Furthermore, selenite is designated as a poisonous designated item in the “Poisonous and Deleterious Substances Designation Order” (Decree No. 2 of January 4, 1965). On the other hand, steroids are known to have side effects such as adrenal cortex dysfunction and Cushing's syndrome, and all of them are preferable components to be removed at the time of transplantation as impurities derived from the production process.

These 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. Due to the destruction, it has been extremely difficult to wash the impurities from the manufacturing process in the cell culture to a level that does not cause clinical problems.
In addition, autologous serum does not have the risks described above due to non-autologous serum, but it imposes physical and time burdens on recipients and medical staff, such as the need to collect blood from the recipient in advance. Depending on the recipient's condition, it may be difficult to obtain.

Moreover, in order to detach such a cell culture from the culture substrate, a mechanical method such as pipetting or scraping, or a normal adherent cell detachment method such as enzyme treatment using trypsin, or the like, the structure of the cell culture may be broken, In the past, cells were cultured in a culture dish coated with a temperature-responsive material, and the cell culture was exfoliated by changing the temperature and changing the hydrophobicity of the material. Patent Document 1). However, such a culture dish is expensive and has problems such as destruction of cell culture structure and cell damage at the time of detachment due to the waiting time until the temperature of the culture dish decreases and the diversity of detachment state by cells. Has not been completely resolved.
Therefore, a method improvement for obtaining a high-quality sheet-like cell culture that is clinically safe has been demanded.

Special table 2007-528755 gazette JP 2008-161183 A JP 2001-128660 A JP 2004-166603 A JP 2004-166604 A JP 2004-283010 A Special Table 2000-516802

Kato et al., Proc Natl Acad Sci U S A. 1988; 85 (24): 9552-6 Ballock and Reddi, J Cell Biol. 1994; 126 (5): 1311-8

  It is an object of the present invention to provide a high-quality cell culture that does not contain impurity components derived from a manufacturing process that can hinder clinical application, and a method for manufacturing the same.

  The inventors of the present invention have continued intensive research to solve the above-mentioned problems. After seeding the cells on the substrate, the cells are cultured after being pressurized against the substrate. In addition, it has been found that a sheet-like cell culture can be obtained using a serum-free medium, and that the obtained cell culture can be easily peeled off from the base material in the form of a sheet. Was completed.

  A technique for cultivating a cell aggregate in a container such as a hollow fiber by filling a hepatocyte with centrifugal force or water pressure, filling in an insert in the container, and in a medium containing various additives, for example, However, these techniques are premised on the use of the whole container without releasing the cells from the container. A technique for preparing a cartilage tissue-like pellet by putting chondrocytes or mesenchymal progenitor cells in a centrifuge tube and culturing with a medium containing various additives in the centrifuge tube is disclosed in, for example, Patent Document 7 Non-Patent Document 1, Non-Patent Document 2, and the like, but there is no teaching about the production of a free sheet-like cell culture. Therefore, the above findings of the present inventors are surprising.

That is, the present invention relates to the following.
(1) (i) seeding cells on a substrate;
(Ii) pressurizing the cell against the substrate;
(Iii) A method for producing a released sheet-shaped cell culture, comprising a step of culturing cells to form a sheet-shaped cell culture, and (iv) a step of releasing the formed culture from a substrate.
(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 growing.
(3) The method according to (1) or (2) above, wherein the cell comprises a myoblast.
(4) The method according to any one of (1) to (3) above, wherein the substrate has a non-cell-adhesive surface.
(5) The method according to any one of (1) to (4), wherein the pressurization is performed by centrifugal force.
(6) The method according to any one of (1) to (5) above, wherein the pressing is performed with a force of 2 to 2000 G.
(7) The method according to any one of (1) to (6) above, wherein the culture is performed in a serum-free medium.
(8) A cell culture produced by the method according to any one of (1) to (7) above.
(9) The cell culture according to (8), which is used for treatment of diseases and injuries.
(10) The cell culture according to (8) or (9), wherein the cell culture is substantially free from impurities from the production process.
(11) A graft containing the cell culture of any of (8) to (10) above.

  According to the present invention, in the production of a sheet-shaped cell culture, it is possible to culture in a serum that is generally added as a factor necessary for cell proliferation or in a non-cell proliferation culture medium that does not contain a growth factor. In addition to avoiding contamination of components and endotoxin that can be contained in growth factors that are usually recombinant products, a steroid agent added as a differentiation inhibitor is not necessary. Further, the present invention eliminates the need for selenium or its derivatives for preventing oxidation. As a result, it is possible to provide a clinically safe cell culture that does not contain impurities from the manufacturing process, and it is not necessary to prepare autologous serum as a substitute for heterologous serum.

In addition, since the cell culture formed on the base material can be released from the base material by a simple operation such as pipetting, the structure of the cell culture is not broken at the time of peeling as in the prior art. Moreover, since a commercially available culture vessel having a cell non-adhesive surface can be used as the substrate, it is not necessary to obtain an expensive temperature-responsive culture dish.
In addition, when cultured in a medium that does not contain serum, growth factors, etc., operations such as washing for the purpose of removing impurities originating from the manufacturing process that may cause damage to the produced cell culture are no longer necessary. A reliable and stable production is possible, and the strength of the obtained cell culture against proteolytic enzymes can be increased.
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 perform treatment of a living body using the cell culture more flexibly and easily.

FIG. 1 is a photograph showing a cell culture cultured in a serum-free medium in a temperature-responsive culture dish (× 200). FIG. 2 is a photograph showing a cell culture cultured in a serum-containing medium after the cells were pressurized by centrifugation on a substrate having a non-cell-adhesive surface. The upper row is the sample that has not been centrifuged, the middle row is the sample centrifuged at 50 G (500 rpm), the lower row is the sample centrifuged at 450 G (1500 rpm), the left row is the sample peeled from the base material, the right row is the expanded state Samples (× 40) are shown. FIG. 3 is a photograph showing a cell culture cultured in a serum-free medium after the cells are pressurized by centrifugation on a substrate having a non-cell-adhesive surface. The upper row is the sample that has not been centrifuged, the middle row is the sample centrifuged at 50 G (500 rpm), the lower row is the sample centrifuged at 450 G (1500 rpm), the left row is the sample peeled from the base material, the right row is the expanded state Samples (× 40) are shown. FIG. 4 is a photographic diagram showing a cell culture that was cultured in a serum-containing medium (right) or a serum-free medium (left) after the cells were pressurized by centrifugation on a substrate having a non-cell-adhesive surface.

The present invention includes (i) a step of seeding a cell on a substrate, (ii) a step of pressurizing the cell against the substrate, (iii) a step of culturing the cell to form a sheet-like cell culture, And (iv) a step of releasing the formed culture from the substrate, and a method for producing a released sheet-shaped cell culture.
The cell in the present invention includes any cell that can form a cell culture, particularly a sheet-shaped cell culture. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, synovial cells, epithelial cells, endothelial cells, and the like. Among these, in the present invention, those that form a monolayer cell culture, such as myoblasts, are preferred. The cells can be derived from any organism capable of being treated with cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like. Further, only one type of cell may be used in the method of the present invention, but 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 most cell ratio (purity) 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 “substrate” is not particularly limited as long as it has a surface or interface capable of pressurizing cells against it. For example, containers of various materials, solid, semi-solid or liquid in containers Includes surface or interface. The container preferably has a structure / material that does not allow permeation of liquid such as a culture solution. The container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. The container may have a solid, semi-solid, or liquid surface or interface therein. As a solid surface, various materials such as plates and containers, as a semi-solid surface, a gel, a soft polymer matrix, etc., as a liquid surface, as a highly viscous liquid medium, as a liquid interface, Interfaces between liquids with different specific gravity, for example, specific gravity liquids for cell separation by density gradient method (sucrose, cesium chloride, cesium sulfate, Percoll ^(R) , Ficoll ^(R) , Nycodenz ^(R) , OptiPrep ^™ , ISolate ^TM , ISOLYMPH, etc.).
The surface or interface may have various shapes, but is preferably flat. Although the area is not particularly limited, typically, 1～200Cm ^2, preferably 2～100Cm ^2, more preferably 3~50cm ^2.

  In a preferred embodiment of the invention, the substrate has a non-cell-adhesive surface or interface. Here, “cell non-adhesive” means that adherent cells do not adhere or are difficult to adhere. For example, it is known that adherent cells are difficult to adhere if the surface or surface to be contacted has a higher hydrophobicity or hydrophilicity to some extent. Therefore, the non-cell-adhesive surface or interface in the present invention preferably has a certain degree of hydrophobicity or hydrophilicity. The degree of hydrophobicity or hydrophilicity of the surface can be expressed by, for example, a water contact angle. In the present invention, the water contact angle of the non-cell-adhesive surface is preferably 70 ° or more or 50 ° or less, particularly 75. It is more than or less than 40 degrees.

  Examples of the non-cell-adhesive material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, poly Examples include acrylamide and polydimethylacrylamide. Moreover, since the commercially available container for floating cell cultures has a cell non-adhesive surface, this can also be used as a base material of this invention. Furthermore, the surface can be modified to make it non-cell-adhesive. Specifically, for example, the surface is coated with hydrophilic or hydrophobic molecules, or the surface is coated with a temperature-responsive material (see JP-A-2-21865) that becomes hydrophobic or hydrophilic due to temperature change. be able to. However, in one embodiment of the present invention, the substrate does not have a surface composed of a temperature responsive material.

  Examples of temperature-responsive materials include (meth) acrylamide compounds, N-alkyl-substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropyl). Acrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, etc.) N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylic) ), (Meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine, etc.), or homopolymers or copolymers of vinyl ether derivatives (eg methyl vinyl ether).

In a preferred embodiment of the invention, the cells are seeded at a density that can form a sheet cell culture without substantial growth. “The density at which a sheet-like cell culture can be formed without substantially growing” means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain growth factors. Refers to cell density. For example, in the case of skeletal myoblasts, in the conventional method using a culture solution 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 (for example, refer to Patent Document 1), even if cells having such a density are cultured in a culture solution containing no growth factor, a sheet-like cell culture cannot be formed. Therefore, the cell density in this embodiment is higher than that in the conventional method using a culture solution containing a growth factor. Specifically, for example, for skeletal myoblasts, such 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 shift to differentiation, but for skeletal myoblasts, for example, 1,000,000 cells / cm ² . A person 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 proliferate, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start to differentiate 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 range of measurement errors. 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 the formation of the cell culture is typically 300% or less, preferably 200% or less, more preferably 150% or less, even more preferably 125% or less, particularly preferably the number of cells at the time of seeding. Is 100% or less.

In the present invention, “pressurizing the substrate” means that the cell is contacted with a substrate of a predetermined size or other cells that are in contact with the substrate directly or via other cells. It means that The type of force is not particularly limited as long as it exhibits the above action, and examples thereof include centrifugal force and pressure.
When a centrifugal force is applied, a container containing a base material and cells can be centrifuged using a centrifuge. The centrifugal force to be applied is typically about 2 to 2000 G, preferably about 5 to 1000 G, more preferably about 10 to 800 G, and particularly preferably 50 to 450 G. By applying a stronger centrifugal force without damaging the cells, the uniformity of the cell culture can be further increased, that is, the voids of the cell culture can be reduced. The centrifugation time is typically 2 to 60 minutes, preferably 3 to 15 minutes, more preferably 3 to 5 minutes, and the centrifugation temperature is typically 2 to 37 ° C, more preferably 4 to 25 ° C. It is.

  The pressure can be applied, for example, by applying a water pressure to the cells toward the permeable substrate. The water pressure can be applied by a syringe or a pump. The loading water pressure is typically 10 to 100 mmHg, preferably 20 to 80 mmHg, more preferably 30 to 50 mmHg, and the loading time is typically 3 to 90 minutes, preferably 5 to 60 minutes, more preferably. Is 5 to 15 minutes, and the load temperature is typically 2 to 37 ° C, more preferably 4 to 25 ° C.

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

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

  In one embodiment of the present invention, cell culture is performed within a predetermined period, preferably within a period in which cells do not shift to differentiation. Thus, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition to cell differentiation can be evaluated by any method known to those skilled 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 within 48 hours, more preferably within 40 hours, and even more preferably within 24 hours.

  The cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes are used. Can be used. In one embodiment of the present invention, the culture solution is based on a basal medium for cell culture. Such basal media include, but are not limited to, for example, DMEM, MEM, F12, DME, RPMI 1640, 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, the composition of MCDB131 and DMEM is shown in Table 1 below.

The basal medium may be used in a standard composition (for example, as it is commercially available), 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 one in which one or more components are added, removed, increased or decreased.

Examples of amino acids contained in the basal medium include, but are not limited to, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited to vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i - inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin _{B 12,} adenine, thymidine and then, as the electrolyte, without limitation, for _{example, CaCl 2, KCl, MgSO 4} , NaCl, NaH ₂ PO ₄ , NaHCO ₃ , Fe (NO ₃ ) ₃ , FeS O ₄ , CuSO ₄ , MnSO ₄ , Na ₂ SiO ₃ , (NH ₄ ) 6 Mo ₇ O ₂₄ , NaVO ₃ , NiCl ₂ , ZnSO _{4 and the} like are included. 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 as follows: 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 preparation contained in the basal medium is as follows: calcium D-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- 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 that they can cause side effects such as anaphylactic shock to the recipient in the clinic, and should be excluded in clinical application. Accordingly, in a preferred embodiment of the invention, the cell culture medium does not contain an effective amount of at least one of these additives. In a more preferred embodiment of the present invention, the cell culture medium is substantially free of at least one of these additives. Furthermore, in a particularly preferred embodiment of the present invention, the cell culture medium is substantially free of additives. Therefore, the cell culture medium may contain only the basal medium.

  In a preferred embodiment of the present invention, the cell culture medium is substantially free of serum components. A cell culture medium substantially free from serum components may be referred to herein as “serum-free medium”. Serum components include heterogeneous serum components and allogeneic serum components, where “heterologous serum components” refers to serum components derived from an organism of a species different from the recipient when the cell culture is used for transplantation. means. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum components. In addition, “same serum component” means a serum component derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to the homologous serum component. Allogeneic serum components include autologous serum components, ie, serum components derived from the recipient. Therefore, “substantially free of serum components” means that the content of these sera in the culture solution does not have an adverse effect when the cell culture is applied to a living body (for example, serum albumin in the cell culture). It means that the content is less than 50 ng), preferably that these substances are not actively added to the culture solution. In this specification, sera other than autologous serum, that is, heterologous serum and non-autologous serum of the same species may be collectively referred to as allogeneic serum or non-autologous serum.

  In one embodiment of the invention, the cell culture fluid does not contain an effective amount of growth factor. As used herein, “growth factor” means any substance that promotes cell proliferation as compared to the case without it, such as epithelial cell growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast, and the like. Cell growth factor (FGF) and the like. In addition, an “effective amount of growth factor” refers to the amount of growth factor that significantly promotes cell proliferation compared to the absence of growth factor, or for the purpose of cell proliferation in the art for convenience. Means the amount usually added. The significance of cell growth promotion can be appropriately evaluated, for example, by 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 known literature. Specifically, the effective amount of EGF in skeletal myoblast culture is, for example, 0.005 μg / mL or more.

  Therefore, “not containing an effective amount of growth factor” means that the concentration of the growth factor in the culture medium of the present invention is less than the effective amount. For example, the concentration of EGF in the culture medium in skeletal myoblast culture is preferably less than 0.005 μg / mL, more preferably less than 0.001 μg / mL. In a preferred embodiment of the present invention, the concentration of the growth factor in the culture solution 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 skeletal myoblast culture is preferably less than 5.5 ng / mL, more preferably less than 1.3 ng / mL, and even more preferably 0.5 ng / mL. Less than mL. In a further preferred embodiment, the culture medium in the present invention is substantially free from growth factors. Here, “substantially free” means that the content of the growth factor in the culture solution is such that the cell culture is not adversely affected when applied to a living body, and preferably the growth factor is positively added to the culture solution. Means that it is not added. Therefore, in this embodiment, the culture solution does not contain a growth factor at a concentration higher than that contained in other components such as serum.

  In one embodiment of the present 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 a living body such as adrenal cortex dysfunction 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 solution is such that the cell culture is not adversely affected when applied to a living body. This means that these compounds are not actively added, that is, the culture solution does not contain a steroid component at a concentration higher than that contained in other components such as serum.

  In one embodiment of the present invention, the cell culture solution is substantially free of a selenium component. 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 selenite. Therefore, “substantially free of selenium component” means that the content of these substances in the culture solution is such that the cell culture is not adversely affected when applied to a living body. This means that these substances are not actively added, that is, the culture solution does not contain a selenium component at a concentration higher than that contained in other components such as serum. Specifically, for example, in the case of humans, the selenium concentration in the culture solution is the normal value in human serum (for example, 10.6 to 17.4 μg / dL), and the ratio of human serum contained in the medium is The value is lower than the multiplied value (that is, if the content of human serum is 10% (v / v), 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 production processes such as growth factors, steroid components, and heterogeneous serum components, which have been conventionally required when preparing a cell culture to be applied to a living body, are removed by washing or the like. A process becomes unnecessary. Therefore, one embodiment of the method of the present invention does not include the step of removing impurities derived from the production process.
Here, “manufacturing process-derived impurities” typically include those listed below, which are derived from each manufacturing process. That is, a substance derived from a cell substrate (for example, host cell-derived protein, host cell-derived DNA), a substance derived from a cell culture medium (for example, inducer, antibiotic, medium component), or a process after cell culture. It is derived from the extraction, separation, processing, and purification steps of a certain target substance (see, for example, Pharmaceutical Examination No. 571).

Cell culture can be performed under conditions usually used in the art. For example, typical culture conditions include culture at 37 ° C. and 5% CO ₂ . The culture period is preferably within 48 hours, more preferably within 40 hours, and even more preferably within 24 hours, from the viewpoint of sufficient formation of a cell culture and prevention of cell differentiation. Culturing can be performed in containers 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 rapidly transferred without waiting for the cell culture to grow to the desired size as in the conventional method. It becomes possible to get between. The size and shape of the cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by installing a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing the cells with, for example.
The release of the cell culture from the base material is not particularly limited as long as the cell culture can be released from the base material serving as a scaffold while at least partially maintaining the sheet structure, but typically pipetting, etc. Do by. It is also possible to culture cells on a temperature-responsive substrate whose surface hydrophilicity changes with temperature, and to release the formed cell culture non-enzymatically due to temperature changes.

The method of the present invention can also be positioned as one step of regenerative treatment from cell collection to cell growth and cell culture preparation to cell culture application. Therefore, the present invention
(I) isolating desired cells from tissue or biological fluid collected from the subject,
(Ii) growing the isolated cells;
(Iii) seeding cells on a substrate;
(Iv) pressurizing the cell against the substrate;
(V) culturing the cell to form a sheet-shaped cell culture, and (vi) releasing the formed culture from the substrate.
And a method for producing a sheet-shaped cell culture for regenerative treatment.

The present invention also provides a cell culture produced by the above production method, and further a cell culture substantially free of allogeneic serum, growth factor, steroid agent and / or selenium component, particularly a sheet-shaped cell culture. About. 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 produced by culturing cells in a culture solution substantially free of allogeneic serum, growth factors, steroids and / or selenium components to form a cell culture. Here, the cell culture is substantially free of allogeneic serum, growth factors, steroids and / or selenium components, so that the cell culture does not contain these components at concentrations that adversely affect the recipient. At least that means that the cell culture can be formed in a culture solution 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 from autologous serum in addition to impurities originating from the manufacturing process. Here, the meaning of “substantially free” is the same as described above.
The cell culture of the present invention can be used for treatment of a target disease or injury. For example, a cell culture using skeletal myoblasts can be used in the form of a graft for heart diseases such as myocardial infarction and dilated cardiomyopathy. Accordingly, another aspect of the present invention relates to a graft comprising the above cell culture.

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

Comparative Example 1 Examination of serum-free medium MCDB131 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), 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sankyo Pharmaceutical Co., Ltd.), human myoblasts 3.0 × 10 ⁶ -3 in 2 mL each. .1 × 10 ⁶ pieces were suspended and seeded on a φ3.5 cm temperature-responsive culture dish (manufactured by Cellseed).
After seeding, the cells were cultured under 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 these results, it has been clarified that serum components are indispensable for the production of sheet-like cell cultures by conventional techniques.

Example 1 Examination in serum-containing medium Human skeletal myoblasts (Lonza, Lot 6F4296) were grown in 20% (v / v) FBS-containing MCDB131 medium (GIBCO) and then 10% (v / v). ) Cryopreserved in liquid nitrogen in MCDB131 medium containing DMSO, 20% (v / v) FBS. The cells were rapidly thawed at 37 ° C., washed twice with HBSS, suspended in MCDB131 medium containing 20% (v / v) FBS, and multiwell ^™ TC plate 48 for suspension cells having a non-cell-adhesive surface. A well (BD, code: 351178) was seeded at a density of 1.2 × 10 ⁶ cells / well. The plate was set in a centrifuge (RL-603, manufactured by TOMY, TS-9 rotor), centrifuged at 50 G (500 rpm) or 450 G (1500 rpm) at 20 ° C. for 5 minutes, and then the cells were incubated at 37 ° C. and 5% CO ₂ . Cultured for 22 hours. After the culture, the cell culture formed into a sheet by pipetting was detached from the plate (FIG. 2, right column). Furthermore, the cell sheet was transferred to a 35 mm Petri dish (manufactured by BD), and the sheet was spread (FIG. 2, left column). As shown in the figures, the samples that were not centrifuged had many large voids in the cell culture and were very fragile and impractical. The formation of sheet-like cell cultures of clinically usable quality was observed. In addition, the uniformity of the cell culture was improved by increasing the rotational speed (increasing the centrifugal acceleration). Furthermore, the sheet-like structure is formed by the adhesive ability of the cells, and when the cells die, such a structure is not formed, but that the sheet-like cell culture is maintained in all the centrifuged samples, This shows that the viability of the cells constituting this is high.
Furthermore, the contact angle of the Multiwell ^™ TC plate 48-well used here with distilled water was measured with a measuring contact angle measuring device GI type (manufactured by Elma), and it was 75 to 80 °.

Example 2 Examination in serum-free medium Human skeletal myoblasts (Lonza, Lot 6F4296) were grown in 20% (v / v) FBS-containing MCDB131 medium (GIBCO) and then 10% (v / v). ) Cryopreserved in liquid nitrogen in MCDB131 medium containing DMSO, 20% (v / v) FBS. Cells are rapidly thawed at 37 ° C., washed twice with HBSS, suspended in DMEM / F12 (GIBCO) without added serum, and multiwell ^™ TC plates for floating cells with a non-cell-adhesive surface 48 wells (BD, code: 351178) were seeded at 1.2 × 10 ⁶ cells / well. Set in a centrifuge (RL-603, manufactured by TOMY, TS-9 rotor), centrifuged at 50 G (500 rpm) or 450 G (1500 rpm) at 20 ° C. for 5 minutes, and then cultured at 37 ° C., 5% CO ₂ for 46 hours. (FIG. 3, upper side). After the culture, the cell culture formed into a sheet by pipetting was released from the plate. Furthermore, the cell sheet was transferred to a 35 mm Petri dish (manufactured by BD), and the sheet was spread (FIG. 3, lower side). As shown in the figures, the samples that were not centrifuged were not practical for all samples that were not practical because many large voids were present in the cell culture. The formation of a quality sheet-like cell culture was observed. In addition, the uniformity of the cell culture was improved by increasing the rotational speed (increasing the centrifugal acceleration). Furthermore, as described above, it was also shown that the cell viability was high in all the centrifuged samples.
Furthermore, the contact angle of the Multiwell ^™ TC plate 48-well used here with distilled water was measured with a measuring contact angle measuring device GI type (manufactured by Elma), and it was 75 to 80 °.

Example 3 Comparative Study of Serum-Free Medium and Serum-Containing Medium Human skeletal myoblasts (Lonza, Lot 6F4296) were grown in 20% (v / v) FBS-containing MCDB131 medium (GIBCO). It was cryopreserved in liquid nitrogen in MCDB131 medium containing% (v / v) DMSO and 20% (v / v) FBS. Cells were rapidly thawed at 37 ° C., washed twice with HBSS, and suspended in MCDB131 medium containing (1) DMEM / F12 (GIBCO) or 20% (v / v) FBS without addition of serum. It became cloudy and was seeded at a rate of 1.2 × 10 ⁶ cells / well in a 48-well multiwell ^™ TC plate (BD, code: 351178) for suspension cells having a non-cell-adhesive surface. It was set in a centrifuge (RL-603, manufactured by TOMY, TS-9 rotor), centrifuged at 450 G (1500 rpm) at 20 ° C. for 5 minutes, and then cultured at 37 ° C. and 5% CO ₂ for 46 hours (FIG. 4). After culture, the cell culture formed into a sheet by pipetting was released from the plate, and the cell culture was transferred to a 35 mm Petri dish (manufactured by BD). Further, remove HBSS from which the cell culture is suspended, add 1.5 mL of a trypsin-like enzyme digestion solution (Triple Select, manufactured by GIBCO), place it on a shaker (TAITEC), and place the cell culture at 100 rpm. Shake.
When the appearance of the cells was confirmed after 15 minutes, the cell culture prepared with the serum-containing medium was scattered (FIG. 4, right side), but the cell culture prepared with the serum-free medium maintained its shape. Cavity (FIG. 4, left side) reveals that the strength is higher.
Furthermore, the contact angle of the Multiwell ^™ TC plate 48-well used here with distilled water was measured with a measuring contact angle measuring device GI type (manufactured by Elma), and it was 75 to 80 °.

Example 4 Centrifugation in Density Gradient Solution Human skeletal myoblasts (Lonza, Lot 6F4296) were grown in MCDB131 medium containing 20% (v / v) FBS and then 10% (v / v) DMSO. And cryopreserved in liquid nitrogen in MCDB131 medium containing 20% (v / v) FBS. The cells are rapidly thawed at 37 ° C., washed twice with HBSS, and suspended in DMEM / F12 (GIBCO) without addition of serum to prepare a cell suspension. Ficoll-Paque PLUS (manufactured by GE Healthcare Bioscience) having a specific gravity of 1.077 to 1.319 g / mL is placed in a centrifuge tube, and the prepared cell suspension is gently layered in a band shape. The centrifuge tube is centrifuged at 400 G for 30 minutes in an ultracentrifuge at 20 ° C., a density gradient is formed by a self-density gradient forming method, and the cells are collected in a predetermined layer. Here, cells are pressed against an interface consisting of a layer of a specific gravity in a density gradient. The cells in the centrifuge tube are cultured as they are at 37 ° C. and 5% CO ₂ for 24 to 48 hours to form a sheet-like cell culture. Alternatively, the band containing the cell suspension in the centrifuge tube is taken out by decantation, washed gently as necessary, and then cultured in a serum-free medium or a serum-containing medium at 37 ° C., 5% CO ₂ for 24 to 48 hours. To form a sheet-like cell culture. Thus, by centrifuging in a density gradient solution, cells can be collected in a predetermined layer and cultured to form a sheet-shaped cell culture.

Claims (11)

(I) seeding cells on a substrate at a density that can form a sheet-like cell culture without substantially growing ;
(Ii) pressurizing the cell against the substrate;
(Iii) culturing the cells without growing them to form a sheet-like cell culture, and (iv) releasing the formed culture from the substrate. Production method. Incubation period in step (iii) is Ru der within 48 hours The method of claim 1.   The method of claim 1 or 2, wherein the cells comprise myoblasts.   The method according to claim 1, wherein the substrate has a non-cell-adhesive surface.   The method according to claim 1, wherein the pressurization is performed by centrifugal force.   The method according to claim 1, wherein the pressing is performed with a force of 2 to 2000 G. The method according to any one of claims 1 to 6, wherein the culture is performed in a serum-containing medium or a serum-free medium.   A cell culture produced by the method according to claim 1.   The cell culture according to claim 8, which is used for treatment of diseases and injuries.   The cell culture according to claim 8 or 9, which is substantially free from impurities from the production process.   A graft comprising the cell culture according to any one of claims 8 to 10.