JP2017158589A - Method for producing medical cell sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method using a cell culture liquid free from impurity components derived from a production process, which becomes an obstacle to clinical application, in the production of a cell sheet.SOLUTION: A method for producing a cell sheet comprises culturing cells in a density enough to form a cell sheet without substantially proliferating, in a cell culture liquid free from an effective amount of growth factors. There are also provided a cell sheet produced by the method, and a culture medium for use in the method.SELECTED DRAWING: Figure 1B

Description

  The present invention relates to a method for producing a cell sheet without using an effective amount of a growth factor, and a cell sheet produced by such a method, in particular, a cell sheet substantially free of production process-derived impurities.

  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 in order to cope with these, myoblasts were made into sheets, so-called “cell sheets”.

  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).

Thus, the formation of a cell sheet 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. In skeletal myoblasts and the like that tend to differentiate, a steroid agent is usually added to the culture solution of skeletal myoblasts in order to suppress the transition of the proliferated cells to differentiation. If a steroid is not added, skeletal myoblasts will begin to differentiate in about 16 hours of culture, as shown in the experiments described below. When skeletal myoblasts differentiate, they form tubular myotubes, making it impossible to obtain a cell sheet.
Further, selenium may be added to the culture solution for the purpose of antioxidant action.

  However, considering clinical applications, heterogeneous serum components may contain pathogens such as viruses that can infect recipients, and growth factors are usually produced recombinantly using microorganisms. When it remains, it can be an impurity derived from the manufacturing process when it remains, and thus cannot be directly used for transplantation 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. 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.

  Impurities derived from these manufacturing processes are usually removed by washing the cell sheet with a medium that does not contain these substances, but the cell sheet is mechanically extremely fragile and easily destroyed by the water flow during washing. For this reason, it has been extremely difficult to clean impurities from the manufacturing process in the cell sheet to a level that does not cause clinical problems.

Special table 2007-528755 gazette JP 2008-161183 A

  This invention makes it a subject to provide the cell sheet which does not contain the impurity component derived from a manufacturing process which may become an obstacle to clinical application, and its manufacturing method.

For the purpose of promoting the proliferation ability of the cells to be cultured, the cell culture medium contains a growth factor as a component in addition to the heterogeneous serum components as described above. Moreover, a steroid agent is added depending on the cell type, and selenium may be contained depending on the applied basal medium.
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.

In the cell sheet manufacturing process, the present inventors consider that it is not necessary to consider cell proliferation in the cell sheet manufacturing process, and generally consider it as a factor necessary for cell proliferation. When cells were cultured in a non-cell proliferation culture medium substantially free of added growth factors, it was surprisingly found that cell sheets could be produced.
As a result of further research, the use of a non-cell growth system for the culture medium eliminates the need for steroids added to suppress the differentiation of skeletal myoblasts and the need for selenium in the production of cell sheets. In addition, the present inventors have found that sera from recipients can be used instead of heterologous sera, thereby completing the present invention.

More specifically, the present inventors assumed MCDB131 medium (manufactured by Invitrogen) mainly composed of amino acids, vitamins, and electrolytes as a basal medium, and assumed serum derived from recipients instead of heterogeneous serum components. A cell culture solution containing 5-40% human serum (manufactured by Cambrex or from research blood collection) was prepared, and cell sheet formation by human skeletal myoblasts was examined. As a result, it has been found that a cell sheet as a three-dimensional tissue structure having an appropriate strength can be formed and has stable functionality.
The human serum contained here is preferably a serum derived from a recipient from the viewpoint of ensuring high safety, and its concentration is preferably 10 to 20%.

Next, the present inventors used a cell culture solution containing epidermal growth factor and dexamethasone sodium phosphate in MCDB131 medium containing 10-20% human serum, and a cell culture solution not containing these components. When the above cells were seeded to prepare a cell sheet, it was confirmed that the purity of the cell sheet prepared with a cell culture solution containing no epidermal growth factor and dexamethasone sodium phosphate was superior.
This suggests that the non-proliferating cell culture medium also has the effect of suppressing the growth of fibroblasts, which are the same adherent cells but have higher proliferation ability than skeletal myoblasts. became.

Moreover, in the cell culture medium containing epidermal growth factor and dexamethasone sodium phosphate, multinucleation showing differentiation of skeletal myoblasts was observed after 25 hours of culture, whereas epidermal growth factor and dexamethasone sodium phosphate were contained. Multinucleation was not observed in the cell culture medium that did not.
These results support the assumption that it is not necessary to consider cell proliferation in the preparation of the cell sheet described above, and it was therefore clarified that a steroid agent for inhibiting cell differentiation is also unnecessary. .

By the way, the MCDB131 medium used by the present inventors as a basal medium contains a small amount of selenious acid. This component is designated as a poisonous designated item in the “Poisonous and Deleterious Substances Designation Order” (Decree No. 2 of January 4, 1965). For this reason, as with MCDB131 medium, DMEM medium (manufactured by Invitrogen) containing amino acids, vitamins, and electrolytes as main components and containing no selenium component was used as a basal medium, and the obtained cell sheet was obtained. Thus, it was considered that there was no influence of selenium on the production of the cell sheet.
Therefore, in addition to growth factors and steroids, it was suggested that the addition of selenium component is unnecessary in the preparation of cell sheets.
Table 1 shows the composition of MCDB131 medium and DMEM medium.

Therefore, this invention is shown by the following (1)-(18).
(1) A method for producing a cell sheet, comprising culturing cells having a density capable of forming a cell sheet without substantially proliferating in a cell culture solution containing no effective amount of a growth factor.
(2) The production method of (1), wherein the cells form a monolayer cell sheet.
(3) The production method of (1) or (2), wherein the cells are maintained in an undifferentiated state during the culture period.
(4) The production method of (1) to (3), wherein the cell culture solution contains substantially no steroid component.
(5) The production method of (1) to (4), wherein the cell culture solution does not substantially contain a heterogeneous serum component.
(6) The production method of (1) to (5), wherein the cell culture solution contains a homologous serum component.
(7) The production method of (6), wherein the same-type serum component is derived from a recipient.
(8) The production method of (1) to (7), wherein the cell culture solution contains substantially no selenium component.
(9) The production method of (1) to (8), wherein the cell culture solution contains a basal medium containing amino acids and vitamins as components.
(10) The amino acid is at least L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L -The manufacturing method of (9) containing threonine, L-tryptophan, L-tyrosine, and L-valine.
(11) The amino acid concentrations are 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 to 30 mg / L 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: 18.1 104 mg / L, L-valine: The manufacturing method of (10) which is 94-117 mg / L.
(12) The production method of (9), wherein the vitamin preparation contains at least calcium D-pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine, and pyridoxine.
(13) The concentration of the vitamin preparation is D-calcium pantothenate: 4 to 12 mg / L, choline chloride: 4 to 14 mg / L, folic acid: 0.6 to 4 mg / L, i-inositol: 7.2 mg / L, Niacinamide: 4 to 6.1 mg / L, Riboflavin: 0.0038 to 0.4 mg / L, Thiamine: 3.4 to 4 mg / L, Pyridoxine: 2.1 to 4 mg / L, Production of (12) Method.
(14) The manufacturing method of (1)-(13) which does not include the process of removing an impurity derived from a manufacturing process.
(15) A cell sheet produced by the production method of (1) to (14).
(16) A cell sheet used for the treatment of diseases and injuries, which is substantially free of growth factors, steroids and selenium components.
(17) The cell sheet according to (15) or (16), which does not contain a heterogeneous serum component.
(18) A culture medium for cell sheet preparation, which contains a basic medium containing amino acids and vitamins, and allogeneic serum but does not substantially contain growth factors, steroids, and selenium components.

  According to the present invention, in the production of a cell sheet as a three-dimensional tissue structure, since it is possible to culture in a non-cell proliferation system culture solution that does not contain a growth factor that is generally added as a factor necessary for cell proliferation, In addition to avoiding contamination of endotoxin and the like that can be contained in a recombinant growth factor, a steroid agent added as a skeletal myoblast differentiation inhibitor is not necessary. Furthermore, according to the present invention, selenium or a derivative thereof for preventing oxidation is not necessary, and it is possible to use recipient-derived serum instead of heterologous serum. As a result, it is possible to provide a clinically safe cell sheet that does not contain impurities from the manufacturing process.

In addition, an operation such as washing for the purpose of removing impurities derived from the production process in which the produced cell sheet is likely to be damaged is unnecessary, and the cell sheet can be produced more reliably and stably.
Furthermore, since a cell sheet having 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 a treatment of a living body using the cell sheet more flexibly and easily.

It is the figure which showed the conventional cell sheet preparation flow. It was necessary to provide a cleaning process to remove impurities derived from the manufacturing process. It is the figure which showed the cell sheet preparation flow by this invention. By using a cell culture solution that does not contain manufacturing process-derived impurities, the washing operation can be omitted. It is the photograph figure which showed the external appearance of the cell sheet formed using the cell culture solution containing a 20% human serum component (* 200). It is the photograph figure which showed the external appearance of the cell sheet produced with the cell culture solution which consists of MCDB131 culture medium containing 20% human serum (x200). As a comparative control in FIG. 3A, the appearance of a cell sheet prepared with a cell culture medium composed of MCDB131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL dexamethasone sodium phosphate injection is shown. It is the shown photograph figure (x200). It is the photograph figure which showed the cell sheet external appearance property 25 hours after culture | cultivation produced with the cell culture solution containing an epidermal growth factor and a dexamethasone sodium phosphate (* 100). It is the photograph figure which showed the expression of MHC in the cell sheet | seat 16 hours after culture | cultivation produced with the cell culture solution of FIG. 4A (* 100). It is the photograph figure which showed the cell sheet external appearance property 25 hours after culture | cultivation produced with the cell culture solution which does not contain an epidermal growth factor and a dexamethasone sodium phosphate (* 200). It is the photograph figure which showed the external appearance of the cell sheet produced with the cell culture solution which consists of a DMEM culture medium containing 20% human serum (x200). As a comparative control in FIG. 5A, the appearance of a cell sheet prepared with a cell culture medium composed of MCDB131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL dexamethasone sodium phosphate injection is shown. It is the shown photograph figure (x200).

FIG. 1A shows a conventional cell sheet manufacturing flow, and FIG. 1B shows a non-limiting example of the cell sheet manufacturing flow according to the present invention.
The present invention relates to a method for producing a cell sheet, comprising culturing a cell having a density capable of forming a cell sheet without substantially proliferating in a cell culture solution containing no effective amount of a growth factor.
The cells in the present invention include any cells that can form a cell sheet. 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 sheet, such as myoblasts, are preferred. The cells can be derived from any organism capable of being treated with a cell sheet. 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 sheet, the ratio (purity) of the most cells is 65% or more, preferably 70% or more, more preferably at the end of cell sheet production. 75% or more.

In the present invention, the “density capable of forming a cell sheet without substantially proliferating” means a cell density capable of forming a cell sheet when cultured in a culture solution containing no growth factor. 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 were seeded on a plate in order to form a cell sheet ( For example, refer to Patent Document 1), even if cells having such a density are cultured in a culture solution not containing a growth factor, a cell sheet cannot be formed. Therefore, the cell density in the method of the present invention 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 sheet 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 sheet but does not shift 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 the formation of the cell sheet. In this embodiment, the number of cells after forming the cell sheet 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. 100% or less.

  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.

In the present invention, the “cell sheet” refers to a sheet in which cells are connected to each other, and is typically composed of one cell layer, but composed of two or more cell layers. Including. 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, particularly derived from cells constituting the cell sheet. The cells are at least mechanically linked, but may be further functionally, eg, chemically or electrically linked.
The cell sheet of the present invention preferably does not contain a scaffold (support). The scaffold may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of the cell sheet, eg, made of polyvinylidene difluoride (PVDF) However, the cell sheet of the present invention can maintain its physical integrity even without such a scaffold. In addition, the cell sheet of the present invention is preferably composed only of a cell-derived substance constituting the cell sheet and does not contain any other substance.

In the present invention, “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), fiber Including blast growth factor (FGF).
In the present invention, 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 convenience, cell proliferation in the art. The amount usually added for the purpose. 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 sheet is not adversely affected when applied to a living body, and preferably the growth factor is positively added to the culture solution. Means not to be 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.

  The cell culture solution used in the present invention (sometimes simply referred to as “culture solution”) is not particularly limited as long as it can maintain the survival of the cells, but typically, amino acid, vitamins, and electrolyte are the main components. 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 above. 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 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 sheet 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 medium is substantially free of heterologous serum components. Here, the “heterologous serum component” means a serum component derived from an organism of a species different from the recipient. 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. Therefore, “substantially free of heterogeneous serum components” means that the content of these sera in the culture solution does not have an adverse effect when the cell sheet is applied to a living body (for example, the serum albumin content in the cell sheet) Means that these substances are not positively added to the culture solution.

  In one embodiment of the present invention, the cell culture medium contains allogeneic serum components. As used herein, “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. It is preferred that the allogeneic serum component is an autologous serum component, ie, a serum component derived from the recipient. The content of the allogeneic serum component is not particularly limited as long as it enables the formation of a cell sheet, but is preferably 5 to 40%, more preferably 10 to 20%. In the present specification, unless otherwise specified, “%” means “v / v (capacity / capacity)%”.

  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 sheet is not adversely affected when applied to a living body. This means that the substance is not actively added, that is, the culture solution does not contain 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, 10.6-17.4 μg / dL), and the ratio of human serum contained in the medium 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).

The method of the present invention typically includes (1) a step of seeding cells in a culture solution, (2) a step of culturing cells to form a cell sheet, and (3) a step of peeling the cell sheet. . In the conventional method using a culture solution containing a growth factor, when producing a cell sheet to be applied to a living body, after step (3), impurities derived from a production process such as a growth factor, a steroid component, and a heterogeneous serum component are added. The process of removing by washing etc. was essential. However, one embodiment of the method of the present invention does not include a step of removing impurities derived from the manufacturing 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 the cell sheet 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, a cell sheet having a desired size and shape can be quickly obtained without waiting for the cell sheet to grow to a desired size as in the conventional method. Can be obtained. The size and shape of the cell sheet can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by placing a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing cells.
The cell sheet peeling method is not particularly limited as long as the cell sheet can be peeled from the scaffolding substrate while maintaining the sheet structure at least partially. It is cultured on a temperature-responsive culture dish where the sex changes, and peels non-enzymatically due to the temperature change.

The method of the present invention can be positioned as a step of regenerative treatment from cell collection, cell proliferation and cell sheet production to cell sheet application. Therefore, the present invention
(1) a step of isolating desired cells from a tissue or biological fluid collected from a subject,
(2) proliferating the isolated cells;
(3) culturing the proliferated cells at a density capable of forming a cell sheet without substantially proliferating in a cell culture solution containing no effective amount of growth factor to form a cell sheet;
(4) A step of peeling the cell sheet for application to the subject,
And a method for producing a cell sheet for regenerative treatment.

The present invention also relates to a cell sheet produced by the above production method, and further to a cell sheet substantially free from growth factors, steroid agents, and selenium components. In a preferred embodiment, the cell sheet of the present invention is substantially free of heterologous serum components. This cell sheet can be produced by culturing cells in a culture solution substantially free from growth factors, steroid agents, selenium components, and preferably further different serum components to form cell sheets. Here, that the cell sheet is substantially free of growth factors, steroids, selenium components, or heterologous serum components means that the cell sheet does not contain these components at a concentration that adversely affects the recipient. Meaning, such conditions can be satisfied by forming the cell sheet in a culture solution that is substantially free of growth factors, steroids, selenium components, and preferably further heterogeneous sera.
The cell sheet of the present invention can be used for treatment of a target disease or injury. For example, a cell sheet of skeletal myoblasts can be used for heart diseases such as myocardial infarction and dilated cardiomyopathy.

  The present invention also relates to a basal medium containing amino acids and vitamins, and a culture medium for cell sheet preparation, which contains allogeneic serum but substantially does not contain growth factors, steroids, and selenium components. The definition of each term regarding the culture solution of this invention is as the said definition.

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.
1. Examination of allogeneic serum components In the conventional method, a known fetal calf-derived serum or the like was added to the culture medium when preparing a cell sheet for humans. However, these heterogeneous serum components may contain human infectious viruses. Therefore, we examined the possibility of human serum components with low safety risk.

MCDB131 medium and DMEM medium containing 5%, 10%, 20% and 40% human serum (manufactured by Cambrex or research blood collection), respectively, are prepared, and human myoblasts per 3.0 mL of 3.0 × 10 ^{6 to} 3 .1 × 10 ⁶ cells were suspended and seeded on a φ3.5 cm temperature-responsive culture dish (manufactured by Cellseed Co., Ltd.).
After seeding, the cells were cultured under conditions of 37 ° C. and 5% CO ₂ and the state was observed after 40 hours. As a result, cell sheets as three-dimensional tissue structures could be formed in all cultured cells. Moreover, the cell recovery rate after sheet preparation was 83 to 98%, which was substantially the same as the number of seeded cells, and cell proliferation was not substantially observed.

  FIG. 2 shows an external view of cells prepared with a cell culture medium containing human serum. From the figure, it can be seen that the cells are arranged in a paving stone shape and form a cell sheet.

Next, a comparison was made between a cell sheet prepared with a cell culture medium containing human serum and a cell sheet prepared with a cell culture medium containing known fetal bovine serum (manufactured by Invitrogen). Verification was verified.
For comparative verification, cell viability and purity were used as indices.

The cell viability was measured according to the following procedure.
The formed cell sheet was dissociated with trypsin-like proteolytic enzyme (TrypLE Select, manufactured by Invitrogen), and then the same amount of Trypan Blue Stain 0.4% solution (Invitrogen) was added and mixed.
After mixing, 10 μL of the cell suspension was collected before the cells settled, and injected into a hemocytometer (manufactured by Elma). Immediately after the injection, the number of cells observed in the entire 9 mm ² frame of the two chambers of the hemocytometer was measured with an inverted optical microscope (manufactured by Olympus).
After the measurement, the average number of viable and dead cells in the two chambers was obtained, and the ratio of unstained cells to the total number of cells including stained cells was calculated.

Cell purity was measured according to the following procedure.
The formed cell sheet was dissociated with trypsin-like proteolytic enzyme, centrifuged, and the supernatant was discarded.
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, a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed.
After each antibody was mixed, it was immediately reacted in a cool dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis.
For analysis, a flow cytometer (manufactured by Becton Dickinson) was used to measure the ratio of antibody-positive cells contained in the cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed.
After the analysis, the purity was determined from the difference in the ratio of the positive cell ratio of the cells mixed with each antibody.

As a result of the comparative study, there is a difference in cell viability and purity between all cells cultured using 5% to 40% human serum and cells cultured using a known fetal calf serum as a control. Was not recognized. Table 2 shows a list.

2. Examination on the necessity of growth factors and steroid agents In order to confirm the effect of growth factors on cell proliferation, 3.5 × 10 ⁴ human myoblasts (200 cells / cm ² ) were added at 0 to 0.01 μg / mL. Cells seeded in MCDB131 medium containing 20% fetal bovine serum supplemented with a concentration of epidermal growth factor (Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (Daiichi Sankyo Pharmaceutical), and cultured 10 days later The number was counted and the proliferation ability was observed.

As a result, since the cell growth was clearly low in the medium not containing epidermal growth factor, it was found that epidermal growth factor is a necessary component for the culture solution for cell proliferation. Table 3 shows the results.

In contrast, MCDB131 medium containing 20% human serum and MCDB131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL dexamethasone sodium phosphate injection as a control were prepared. Each 2 mL was suspended with 3.0 × 10 ^{6 to} 3.1 × 10 ⁶ human myoblasts and seeded on a φ3.5 cm temperature-responsive culture dish.
After sowing, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ , and the state was observed 40 hours later. As a result, in any cell culture solution, a cell sheet as a three-dimensional tissue structure could be formed.
From this result, it is possible to consider cell proliferation by controlling the number of seeded cells to 3.0 × 10 ⁶ cells, that is, about 3.0 × 10 ⁵ cells / cm ² or more, in a φ3.5 cm temperature-responsive culture dish. It became clear that it became unnecessary.
Note that the number of seeded cells relative to the effective area of the culture dish to be used is one standard, and is not limited to the effective area and the number of cells illustrated here.

FIG. 3A shows an external view of a cell prepared with a cell culture medium composed of MCDB131 medium containing 20% human serum, and FIG. 3B shows 20% fetal calf serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL phosphate. The external view of the cell produced with the cell culture solution which consists of MCDB131 culture medium containing a dexamethasone sodium injection solution is shown.
Since there was no difference in appearance from the appearance of cells, the effect of cells on the elimination of growth factors and steroids was examined. For verification, cell viability and purity were used as indices.

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

Cell purity was measured according to the following procedure.
The formed cell sheet was dissociated with trypsin-like proteolytic enzyme, centrifuged, and the supernatant was discarded.
To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, a negative control antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed.
After each antibody was mixed, it was immediately reacted in a cool dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis.
For analysis, a flow cytometer was used to measure the ratio of antibody-positive cells contained in cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed.
After the analysis, the purity was determined from the difference in the ratio of the positive cell ratio of the cells mixed with each antibody.

As a result of the comparative study, the purity of the cell sheet prepared with the cell culture medium containing the growth factor and the steroid was 63%. On the other hand, the purity of the cell sheet prepared with the cell culture solution containing no growth factor or steroid was 75%, which was a significantly high value. Table 4 shows a list.

  This suggests that the non-proliferating cell culture medium also has the effect of suppressing the growth of fibroblasts, which are the same adherent cells but have higher proliferation ability than skeletal myoblasts. became.

Furthermore, when the cell properties during culture were observed in the process of cell sheet preparation, multinucleation showing differentiation of skeletal myoblasts was observed after 25 hours in the cell culture medium containing epidermal growth factor and dexamethasone sodium phosphate. It was. Therefore, when the cell differentiation state was confirmed using MHC as a label, expression showing differentiation was observed after 16 hours of culture.
On the other hand, in the cell sheet prepared with the cell culture medium not containing epidermal growth factor and dexamethasone sodium phosphate, the cells were not multinucleated even after 25 hours of culture.

  FIG. 4A shows the appearance of the cell sheet after 25 hours of culturing produced in a cell culture medium containing epidermal growth factor and dexamethasone sodium phosphate, and FIG. 4B shows the MHC expression image after 16 hours of culturing the cell sheet. FIG. 4C shows a cell sheet appearance property diagram after 25 hours of culture prepared in a cell culture medium not containing epidermal growth factor and dexamethasone sodium phosphate.

3. Study on the necessity of selenium DMEM medium containing 20% human serum (medium not containing selenium component) and 20% fetal calf serum as a control, 0.01 μg / mL epidermal growth factor, 4 μg / mL dexamethasone sodium phosphate MCDB131 medium containing an injection solution was prepared, and 3.0 × 10 ⁶ human myoblasts were suspended per 2 mL, and seeded on a φ3.5 cm temperature-responsive culture dish.
After sowing, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ , and the state was observed 40 hours later. As a result, in any cell culture solution, a cell sheet as a three-dimensional tissue structure could be formed.

  FIG. 5A shows an external view of a cell prepared with a cell culture medium composed of DMEM medium containing 20% human serum, and FIG. 5B shows 20% fetal calf serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL phosphate. The external view of the cell produced with the cell culture solution which consists of MCDB131 culture medium containing a dexamethasone sodium injection solution is shown.

  Since there was no difference in the appearance of cells in terms of appearance, the effect of cells on the elimination of selenite in addition to growth factors and steroids was examined. For verification, cell viability and purity were used as indices.

The cell viability was measured according to the following procedure.
The formed cell sheet was dissociated with trypsin-like proteolytic enzyme, and the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of the cell suspension was collected before the cells settled and injected into a hemocytometer. Immediately after the injection, the number of cells observed in the entire 9 mm ² frame of the two chambers of the hemocytometer was measured with an inverted optical microscope.

  After the measurement, the average number of viable and dead cells in the two chambers was obtained, and the ratio of unstained cells to the total number of cells including stained cells was calculated.

Cell purity was measured according to the following procedure.
The formed cell sheet was dissociated with trypsin-like proteolytic enzyme, centrifuged, and the supernatant was discarded.

  To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, a negative control antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed.

  After each antibody was mixed, it was immediately reacted in a cool dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis.

For analysis, a flow cytometer was used to measure the ratio of antibody-positive cells contained in cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed.
After the analysis, the purity was determined from the difference in the ratio of the positive cell ratio of the cells mixed with each antibody.

As a result of the comparative study, no effect on the cells with respect to elimination of selenite was observed. Table 5 shows the results.

Claims (5)

  Seeding the cells in a culture vessel at a density at which the cells do not substantially grow, and culturing the cells in a cell culture medium that does not contain an effective amount of growth factor to form a cell sheet. A method for producing a cell sheet having a high purity of skeletal myoblasts, wherein the cells include skeletal myoblasts and fibroblasts, and the density is equal to or higher than a density at which the cells reach confluence. The method, wherein the purity of skeletal myoblasts is higher than the purity of skeletal myoblasts in a cell sheet produced using a cell culture medium containing an effective amount of a growth factor.   The method of claim 1, wherein the cell culture medium comprises allogeneic serum.   The method according to claim 1 or 2, wherein the cells are collected from a living body.   It is the cell sheet manufactured by the method as described in any one of Claims 1-3, Comprising: This cell sheet is peeling from the culture base material, The said cell sheet.   A combination for use in the method of claim 2 or 3 comprising skeletal myoblasts, fibroblasts, allogeneic serum and culture vessels, wherein the number of cells is confluent. Said combination that is a number that results in a density that is at or above the density that the cells do not substantially grow.