JP7074908B2 - Method for producing sheet-shaped cell culture - Google Patents

Description

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

The heart carries oxygen throughout the body by its pumping function, and plays an essential role in maintaining life. Therefore, heart disease is the top cause of death, and in ischemic heart disease such as angina pectoris and myocardial infarction, sufficient oxygen does not reach the myocardial tissue, and if this condition continues for a long time, necrosis occurs. Adult cardiomyocytes have poor self-renewal ability, so once necrotic, they do not regenerate and fall into heart failure. When such a condition is reached, the only cure is to wear a left ventricular assist device or eventually receive a heart transplant.
Under these circumstances, research is underway as a new treatment method for heart failure, which is cell transplantation into the heart. So far, the utilization of fetal cardiomyocytes, skeletal myoblasts, ES cells, bone marrow-derived cells and the like has been reported in experiments using animals.

Of these, skeletal myoblasts are present in skeletal muscle tissue, and when the muscle tissue is damaged, they are activated, proliferate, and fuse to regenerate the skeletal muscle tissue. Skeletal myoblasts have many parts similar in structure and function to the myocardium, and instead of cardiomyocytes that proliferate and cannot repair the infarct lesion, the lesion can be physically supplemented and the pumping function of the heart can be improved. From the idea of whether or not there is, it has been expected to be adapted to heart disease. On the other hand, in recent years, despite its effectiveness, the engraftment rate of transplanted cells is low, so the humoral factors produced by the transplanted cells act on myocardial tissue, and their effectiveness is due to the suppression of cell death and angiogenesis. It is also thought that it may show sex. In addition, in the case of cell transplantation using skeletal myoblasts, there is an advantage that it is possible to avoid the problem of immunocompatibility by using the patient's own cells, and clinical trials are being conducted overseas.
However, when the transplanted cells are administered to the tissue in the state of a cell suspension, there are problems such as poor injection efficiency of the transplanted cells, damage due to puncture into the recipient tissue, and difficulty in extensive tissue repair. Points have been pointed out, and in order to overcome this, cell structures using scaffolds and cell sheets in which cells are formed into sheets have been developed.

It is known that skeletal myoblasts can also form cell sheets by growing them in an appropriate culture medium for a certain period of time (Patent Document 1). In the same document, the skeletal myoblast sheet is formed by seeding cells at a density of about 9 × 10 ⁴ cells / cm ² and proliferating them in a culture medium containing a growth factor or the like for a sufficient period of time. However, in such a production method, there is a safety problem that additional factors such as growth factors, steroids, and heterologous sera contained in the culture solution may remain in the product. Further, in such a conventional manufacturing method, there is a problem that the formed cell sheet has poor physical strength and is difficult to handle during a washing operation, a transplant operation, and a transportation. Furthermore, in recent years, the action of humoral factors produced from transplanted cells has attracted attention as the mechanism of action of cell transplantation as a therapeutic method, but from transplanted cells, in addition to factors related to the effectiveness of treatment, inflammatory cytokines Since there is a possibility that factors that have a detrimental effect on the recipient may be produced, there are many factors related to efficacy such as angiogenesis and suppression of cell death for clinical application of cell sheets. It is desirable to obtain a cell sheet that produces and suppresses the production of inflammatory cytokines. Therefore, there has been a demand for the production of a cell sheet having high strength, safety and effectiveness without having the above-mentioned problems of the prior art.

Special Table 2007-528755 Gazette

An object of the present invention is to produce a sheet-like cell culture having excellent strength, safety and effectiveness without any problems of the prior art.

The present inventors are conducting diligent research to solve the above problems, and when skeletal myoblasts are seeded at a density exceeding 1.0 × 10 ⁶ cells / cm ² , the seeded cells are completely sheeted. It was not possible to form a sheet-like cell culture with sufficient strength due to exfoliation before the formation of cells, but as a pretreatment, the culture substrate was coated with serum and then seeded with high-density cells. , Found that it is possible to form sheet-like cell cultures without spontaneous exfoliation. Further, in the case of a lower seeding density of 3.5 × 10 ⁵ pieces / cm ² , by using a culture dish treated with serum coating, as compared with the case of using a culture dish not subjected to such treatment. It was found that the number of cells constituting the sheet increased, that is, the cell recovery rate increased and the strength of the sheet also increased. As a result of further research, the sheet-like cell culture of non-multinucleated skeletal myoblasts showed angiogenesis, etc., as compared with the sheet-like cell culture of multinucleated skeletal myoblasts, which had progressed in differentiation. When the degree of differentiation was evaluated for the sheet-like cell culture prepared by the above-mentioned production method and the fact that the production of inflammatory cytokines was low while the production of factors having an action related to efficacy was high, the progress of differentiation was evaluated. We found that no cells were found and completed the present invention.

Therefore, the present invention is shown in the following (1) to (10).
(1) A method for producing a sheet-shaped cell culture, which comprises a step of seeding cells having a density capable of forming a sheet-shaped cell culture on a culture substrate coated with serum without substantially growing.
(2) The production method according to (1) above, wherein the cell density is 3.5 × ^{105 or more and 3.4 × 10 6 cells} / cm ² or less.
(3) The production method according to (1) or (2) above, further comprising a step of isolating the sheet-shaped cell culture from the culture medium.
(4) The production method according to any one of (1) to (3) above, further comprising a step of washing the isolated sheet-shaped cell culture.
(5) The production method according to any one of (1) to (4) above, wherein the sheet-shaped cell culture contains skeletal myoblasts in which differentiation into myotubes is suppressed.
(6) A sheet-like cell culture produced by the method according to any one of (1) to (5) above.
(7) The sheet-like cell culture according to (6) above, which has sufficient physical strength for a washing operation.
(8) The sheet-like cell culture according to (6) or (7) above, which has physical strength capable of withstanding a transplantation operation.
(9) The sheet-like cell culture according to any one of (6) to (8) above, which is used for treating a disease or injury.
(10) The sheet-like cell culture according to any one of (6) to (9) above, which is substantially free of impurities derived from production.

The production method of the present invention provides a sheet-like cell culture having sufficient physical strength to withstand washing operations such as immersion stirring and transplantation operations such as removal, retention, and transfer, and thus can be obtained by a conventional production method. It is possible to carry out the manufacturing / transplanting operation more easily and reliably than the cell sheet. Further, since the sheet-shaped cell culture can be obtained with a high cell recovery rate by the production method of the present invention, the cells to be used can be effectively used, which contributes to cost reduction and the like. Furthermore, the sheet-shaped cell culture obtained by the production method of the present invention is effective therapeutically because it produces a large amount of factors having an effect related to efficacy such as angiogenesis, while the production of inflammatory cytokines is low. It is extremely safe for recipients and is extremely clinically useful. Therefore, great contributions can be expected in human medicine and veterinary medicine.

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

The present invention relates to a method for producing a sheet-shaped cell culture, which comprises a step of seeding cells having a density capable of forming a sheet-shaped cell culture on a serum-coated culture substrate without substantially growing. ..
In the present invention, the "culture substrate" is not particularly limited as long as the cells can form a sheet-like cell culture on the cells, and for example, a container of various materials, a solid or semi-solid in the container. Including the surface and so on. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide, metals (eg iron, stainless steel, aluminum, copper, brass) and the like. Also, the container preferably has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside thereof. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices.

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimuli, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-Isopropylacrylamide, N-Isopropyl Acrylamide, N-Cyclopropylacrylamide, N-Cyclopropyl Methulamide, N-ethoxyethylacrylamide, N-ethoxyethyl Methulamide, N-Tetrahydrofurfurylacrylamide, N-Tetrahydrofurylmethacrylate It has an N, N-dialkyl-substituted (meth) acrylamide derivative (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), and a cyclic group (such as amide). Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidin, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo-2-propenyl) -morpholin, 1 -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether) or a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether), a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-211865 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted.
The culture medium may have various shapes, but is preferably flat. The area thereof is not particularly limited, but is typically 1 to 200 cm ² , preferably 2 to 100 cm ² , and more preferably 3 to 50 cm ² .

In the present invention, the culture medium is coated (coated or coated) with serum. "Coated with serum" means a state in which serum components are attached to the surface of the culture medium. Such a condition can be obtained without limitation, for example, by incubating the culture medium with serum. Incubation involves contacting the serum with the culture medium. As the serum, heterologous serum and allogeneic serum can be used. Heterologous serum means serum derived from an organism of a different species than the recipient when transplanting a cell culture. For example, when the recipient is human, serum derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to heterologous serum. In addition, "homogeneous serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera, ie sera from the recipient.
Serum for coating the culture medium can be commercially available or prepared by routine from blood collected from the desired organism. Specific examples thereof include a method in which the collected blood is left at room temperature for about 20 to 60 minutes to coagulate, and the collected blood is centrifuged at about 1000 to 1200 × g to collect the supernatant.

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

Serum may be discarded after incubation. As a serum disposal method, suction with a pipette or the like, or a conventional liquid disposal method such as decantation can be used. In one aspect of the present invention, the culture medium may be washed with a serum-free washing solution after serum disposal. The serum-free cleaning solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components adhering to the culture substrate. For example, without limitation, water, physiological saline, and various buffers are not particularly limited. Liquids (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7. , DMEM / F12, etc.). As the cleaning method, a conventional culture substrate cleaning method, for example, without limitation, a method of adding a serum-free cleaning solution on the culture substrate, stirring for a predetermined time (for example, 5 to 60 seconds), and then discarding is used. be able to.

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

In the present invention, the culture medium may be coated with a steroid agent. Here, the "steroid component" refers to a compound having a steroid nucleus that can adversely affect the living body such as adrenal insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the coating method, disposal method and washing method of the culture medium with a steroid agent, the dilution concentration at the time of incubation is, for example, 0.1 μg / mL to 100 μg / mL, preferably 0.4 μg / mL to 40 μg / mL as dexamethasone. , More preferably, it is basically the same as serum except that it is 1 μg / mL to 10 μg / mL.

The culture medium may be coated with any one of serum, growth factors and steroids and any combination of these, ie serum and growth factors, serum and steroids, or serum and growth factors and steroids. You may coat with the combination of. When coating with a plurality of components, these components may be mixed and coated at the same time, or may be coated in separate steps.

The culture substrate may be seeded with cells immediately after being coated with serum or the like, or may be stored after being coated and then seeded with cells. The coated substrate can be stored for a long period of time, for example, by keeping it at 4 ° C. or lower, preferably −20 ° C. or lower, more preferably −80 ° C. or lower.

In the production method of the present invention, cells are seeded at a density capable of forming a sheet-like cell culture without substantially growing. "Density capable of forming a sheet-like cell culture without substantially growing" means that a sheet-like cell culture is formed when cultured in a non-proliferative culture medium containing substantially no growth factor. Means the cell density that can be. For example, in the case of skeletal myoblasts, in the conventional method using a culture medium containing a growth factor, cells having a density of about 6,500 cells / cm ² are seeded on a plate in order to form a sheet-like cell culture. However (see, for example, Patent Document 1), even if cells having such a density are cultured in a culture medium containing no growth factor, a sheet-shaped cell culture cannot be formed. Therefore, the seeding density in the production method of the present invention is higher than that in the conventional method using a culture solution containing a growth factor. Therefore, in the present specification, such seeding density may be simply referred to as "high density". Specifically, for example, the density is typically 300,000 cells / cm ² or more for skeletal myoblasts. The upper limit of the seeding density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not undergo differentiation, but for skeletal myoblasts, it is less than 3.4 × ¹⁰⁶ cells / cm ² .

Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is 3.0 × 10 ⁵ to 3.4 × 10 ⁶ cells / cm ² in one embodiment, and 3.5 × 10 ⁵ to 3 in another embodiment. .4 × 10 ⁶ pieces / cm ² , 1.0 × 10 ⁶ to 3.4 × 10 ⁶ pieces / cm ² in yet another aspect, 3.0 × 10 ⁵ to 1.7 × 10 in yet another aspect. ⁶ pieces / cm ² , 3.5 x 10 ⁵ to 1.7 x 10 ⁶ pieces / cm ² in another aspect, 1.0 x 10 ⁶ to 1.7 x 10 ⁶ pieces / cm ² in yet another aspect. Is. The above range may include both the upper limit and / or the lower limit as long as the upper limit is less than 3.4 × 10 ⁶ pieces / cm ² . Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0 × 10 ⁵ cells / cm ² or more and 3.4 × 10 ⁶ cells / cm less than ² (including the lower limit and not including the upper limit). ), 3.5 × 10 ⁵ pieces / cm ² or more 3.4 × 10 ⁶ pieces / cm less than ² (including the lower limit, not including the upper limit), 1.0 × 10 ⁶ pieces / cm ² or more 3.4 × 10 ⁶ pieces / cm less than ² (including lower limit, not including upper limit), 1.0 x 10 ⁶ pieces / cm ² more than 3.4 x 10 ⁶ pieces / cm less than ² (not including lower limit or upper limit), 1 It may be 0.0 × 10 ⁶ pieces / cm ² or more 1.7 × 10 ⁶ pieces / cm ² or less (the lower limit is not included, but the upper limit is included). Those skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teachings of the present specification.

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

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

The sheet-like cell culture of the present invention preferably contains no scaffold (support). Scaffolds may be used in the art to attach cells to and / or inside the surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF). Although manufactured membranes and the like are known, the cell culture of the present invention can maintain its physical integrity without such a scaffold. In addition, the cell culture of the present invention preferably comprises only cell-derived substances constituting the cell culture, and does not contain any other substances.

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

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

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

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

The amino acids contained in the basal medium are not limited to, for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, and the like. L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited as vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i. -Inositol, niacinamide, riboflavin, thiamine, pyridoxin, biotin, lipoic acid, vitamin B ₁₂ , adenine, thymidin, etc., and the electrolytes are not limited, for example, CaCl ₂ , KCl, _Л4 , NaCl, Includes NaH ₂ PO ₄ , NaHCO ₃ , Fe (NO ₃ ) ₃ , FeSO ₄ , CuSO ₄ , MnSO ₄ , Na ₂ SiO ₃ , (NH ₄ ) 6Mo ₇ O ₂₄ , NaVO ₃ , NiCl ₂ , ZnSO ₄ , etc. Is done. In addition to these components, the basal medium may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, putrescine and the like.

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

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

Examples of serum contained in the cell culture medium include heterologous serum and allogeneic serum, where "heterologous serum" refers to serum 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 human, serum derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to heterologous serum. In addition, "homogeneous serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera, ie sera from the recipient, and allogeneic sera from non-recipient allogeneic individuals. In the present specification, sera other than self-serum, that is, allogeneic sera and allogeneic sera may be collectively referred to as non-self-sera. In the present invention, allogeneic serum is preferable, and autologous serum is more preferable, in order to avoid side effects at the time of transplantation.

In one aspect of the invention, the cell culture medium is substantially free of serum. A cell culture medium containing substantially no serum may be referred to as a "serum-free medium" in the present specification. Here, "substantially free of serum" means that the content of serum in the culture medium does not adversely affect the application of the cell culture to a living body (for example, the content of serum albumin in the cell culture). The amount is less than 50 ng), preferably means that these substances are not positively added to the culture broth. In the present invention, in order to avoid side effects at the time of transplantation, it is preferable that the cell culture medium contains substantially no heterologous serum, and more preferably does not contain substantially non-self serum.

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

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

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

In one aspect of the invention, the cell culture medium is substantially free of selenium components. Here, the "selenium component" includes a selenium molecule and a selenium-containing compound, particularly a selenium-containing compound capable of liberating the selenium molecule in a living body, such as selenous acid. Therefore, "substantially free of selenium component" means that the content of these substances in the culture medium does not adversely affect the application of the cell culture to a living body, preferably the culture medium. It means that these substances are not positively added, that is, the culture medium does not contain other components thereof, for example, a selenium component having a concentration higher than that contained in serum or the like. Specifically, for example, in the case of humans, the selenium concentration in the culture medium is the ratio of human serum contained in the medium to the normal value (for example, 10.6-17.4 μg / dL) in human serum. It is lower than the multiplied value (ie, if the content of human serum is 10%, the selenium concentration is, for example, 1.0 to less than 1.7 μg / dL).

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

Further, since the sheet-shaped cell culture produced by the method of the present invention has physical strength that can withstand the washing operation, the method of the present invention removes impurities and the like derived from the above-mentioned manufacturing process by washing or the like. It may include a step. Therefore, one aspect of the present invention further comprises washing the isolated sheet cell culture. By adding such a step, it is possible to obtain a sheet-shaped cell culture containing substantially no such impurities even by using a cell culture solution substantially containing the impurities derived from the above-mentioned manufacturing process.

The cells can be cultured under the conditions normally used in the art. For example, typical culture conditions include culturing at 37 ° C. and 5% CO ₂ . The culture period is preferably 48 hours or less, more preferably 40 hours or less, still more preferably 24 hours or less, from the viewpoint of sufficient formation of the cell culture and prevention of cell differentiation. Culturing can be carried out in containers of any size and shape. In the method of the present invention, since the cells do not proliferate substantially, the cell culture of the desired size and shape is short-term without waiting for the cell culture to grow to the desired size as in the conventional method. It will be possible to obtain in between. For the size and shape of the cell culture, adjust the size and shape of the cell attachment surface of the culture vessel, or install a mold of the desired size and shape on the cell attachment surface of the culture vessel and inside it. It can be arbitrarily adjusted by culturing the cells in.

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

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

The present invention also relates to a sheet-like cell culture prepared by the above-mentioned production method.
In a preferred embodiment, the sheet-like cell culture of the present invention has sufficient physical strength for washing operations such as immersion stirring and transplantation operations such as removal, retention and transfer. Having sufficient physical strength means that the sheet-like structure of the cell culture is not impaired by the above-mentioned operation, which means that, for example, the obtained sheet-like cell culture is actually subjected to the above-mentioned operation. It can be confirmed by macroscopically or microscopically inspecting that the sheet-like structure is maintained. Here, the washing operation by immersion stirring is typically that a buffer solution in an amount sufficient to immerse the culture in a culture vessel containing a sheet-shaped cell culture is added and the culture vessel is shaken and stirred together. Say. Therefore, sufficient physical strength can be confirmed by applying the same physical stress to the sheet-like cell culture as the washing operation by such immersion stirring. Further, the sheet-shaped cell culture produced by the above-mentioned production method usually has sufficient physical strength.

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

In a preferred embodiment of the cell culture of the present invention, the cell culture is reduced in inflammatory cytokines. Inflammatory cytokines are a general term for cytokines produced by inflammation, and are not limited to, for example, TNF-α, IL-1 (IL-1α, IL-1β, etc.), IL-4, IL-5. , IL-6, IL-13 and the like. Therefore, "reduced inflammatory cytokines" means that the abundance or production (secretion) of these cytokines is reduced compared to differentiated cell cultures. Thus, cytokines may be present in any form of the process from gene transcription to protein secretion, for example in the form of transcripts such as mRNA or in the form of proteins. The degree of reduction is not particularly limited as long as it exceeds the error range, but is, for example, 15% or more, preferably 25% or more, more preferably 50% or more, still more preferably 60, as compared with the differentiated cell culture. % Or more, particularly preferably 70% or more.

The amount of cytokine can be determined at the gene level by any known method such as, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, real-time PCR, in situ hybridization method, in vitro transcription method and the like. It can be detected by a gene expression analysis method, and at the protein level, by any known protein detection method such as immunoprecipitation method, Western blotting method, EIA, ELISA, RIA, immunity tissue chemistry method, and immuno-cell chemistry method. .. As the sample, a medium impregnated with the cell culture, a part of the cell culture, or the like can be used.

In a preferred embodiment of the invention, the cell culture comprises a therapeutically useful bioactive substance. Such a substance is not particularly limited as long as it has some therapeutic usefulness, but includes, for example, substances involved in angiogenesis, suppression of cell death, tissue remodeling, heart development, induction of stem cells, and the like, and more specifically. In particular, without limitation, for example, VEGF, PIGF, angiogenin, angiopoetin, HGF, PDGF (PDGF-AB, PDGF-BB, etc.), BAG-1, BCL-2, MMP (MMP-2, MMP-3, MMP-9, MMP-10, etc.), TIMP, TNNT2, TNNC1, IGF-1, and the like.

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

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

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

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

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

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

The amount of inflammatory cytokines or therapeutically useful bioactive substances can be determined at the genetic level, for example, by Northern blotting, Southern blotting, RNase protection assay, RT-PCR, PCR methods such as real-time PCR, in vitro hybridization methods, etc. Any known gene expression analysis method such as in vitro transcription method, and at the protein level, any known method such as immunoprecipitation method, Western blotting method, EIA, ELISA, RIA, immunity tissue chemistry method, immuno-cell chemistry method, etc. It can be detected by the protein detection method of. As the sample, a medium impregnated with the cell culture, a part of the cell culture, or the like can be used.

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

Example 1 Effect of seeding density / serum coating treatment on the strength of sheet-shaped cell culture A temperature-responsive culture dish (24-well plate, manufactured by Cellseed) was prepared, and a medium containing serum (DMEM) was prepared in some wells. Serum coating treatment by adding 20% human serum (made by Cambrex or derived from research blood sampling, hereinafter referred to as serum-containing medium) to / F12 and incubating at 37 ° C. and 5% CO ² conditions. Was given. Next, skeletal myoblasts (manufactured by Lonza) are suspended in a serum-containing medium and placed in a serum-coated culture dish or an untreated culture dish at 3.5 × ^{105, 1.0 × 10 6 ,} 1.4 ×. The seeds were sown at a density of 10 ⁶ , 1.7 × 10 ⁶ , 3.4 × 10 ⁶ pieces / cm ² . After culturing for 24 hours, the cell culture was peeled off from the temperature-responsive culture dish, and the possibility of sheet formation and its strength were evaluated. As a result, when untreated culture dishes were used, when seeded at a density exceeding 1.0 × 10 ⁶ cells / cm ² , exfoliation occurred before the seeded cells completely formed a sheet. It was not possible to form a sheet-like cell culture with sufficient strength. On the other hand, when a culture dish coated with serum was used, it was possible to form a sheet-like cell culture without spontaneous exfoliation. A sheet-shaped cell culture could not be obtained at a cell seeding density of 3.4 × 10 ⁶ cells / cm ² .

Further, prepare a temperature-responsive culture dish (φ3.5 cm, manufactured by Cellseed) with or without serum coating treatment in the same manner as above, and suspend the skeletal myoblasts in the serum-containing medium to 3.5 × 10. The seeds were sown at a density of ⁵ pieces / cm ² . After culturing for 24 hours, the sheet-like cell culture was peeled off from the temperature-responsive culture dish, and the strength thereof was evaluated based on the degree of damage caused by immersion and stirring. It was possible to obtain an excellent sheet-shaped cell culture. In addition, when the number of cells constituting the exfoliated sheet-shaped cell culture was examined, when a culture dish coated with serum was used, more cells were sheet-shaped than when a non-treated culture dish was used. It was revealed that cell cultures were formed and the cell recovery rate was significantly higher at 96.3% than 69.8% when the untreated culture dish was used (Table 3).

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

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

Example 3 Measurement of humoral factors Skeletal myoblasts were seeded in a temperature-responsive culture dish (φ3.5 cm, manufactured by Cellseed) at a density of 3.5 × 10 ⁵ cells / cm ² , and cultured for 4 days before culturing. Qing was recovered (undifferentiated sheet-like cell culture supernatant). In addition, the skeletal myoblasts seeded in the temperature-responsive culture dish were mixed with 200 ml of DMEM in 100 μl of 100 μg / ml IGF-1 and 2.6 ml of 7.5% BSA solution for 4 days. After culturing to obtain multinucleated cells (differentiated cells), the cells were further cultured for 4 days, and the culture supernatant of the multinucleated cells was collected (multinuclearized sheet-like cell culture supernatant). Measurement of humoral factors contained in culture supernatants of undifferentiated sheet-like cell cultures and multinucleated sheet-like cell cultures using an antibody array (RayBio ^(R) Human Cytokine Antibody Array G Series 2000, manufactured by RayBiotech). Was done. As a result, it was clarified that the polynuclearized sheet-like cell culture produced more inflammatory cytokines such as IL-1α, IL-4, IL-5, and IL-13 than the skeletal myoblast sheet-like cell culture. (Fig. 2). IL-1α is involved in the induction of inflammatory reaction and activation of T cells and B cells, IL-4 and IL-13 induce the activation of B cells and antibody production, and IL-5 is mainly of eosinophils. Although they act on activation, they are all cytokines that are deeply involved in inducing allergic reactions.

In addition, various humoral factors considered as one of the effective mechanisms in stem cell transplantation include PDGF-AB and PDGF-BB in undifferentiated sheet-like cell cultures as compared with multinucleated sheet-like cell cultures. It was revealed that the production of factors having an action related to efficacy was remarkably high (Fig. 3).
Therefore, when the surface of the culture dish is coated with serum, it is possible to form a sheet-like cell culture even if the number of seeded cells is increased, and it is expected that factors that act therapeutically effectively will be produced.

Claims (5)

A method for producing a sheet-like cell culture in which the production of inflammatory cytokines is suppressed.
(i) A step of seeding cells on a culture medium coated with serum at a cell density of 3.5 × 10 ⁵ cells / cm ² or more and 3.4 × 10 ⁶ cells / cm ² or less .
(ii) The step of culturing the seeded cells to form a sheet-like cell culture without substantially proliferating the cells.
The method for producing a sheet-shaped cell culture. The method according to claim 1, wherein the culture period in step (ii) is 48 hours or less. The method according to claim 1 or 2 , wherein the serum is self-serum. A sheet-like cell culture produced by the method according to any one of claims 1 to 3 . The sheet-like cell culture according to claim 4 , which is used for treating a disease or injury.

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