JP7323919B2 - Cell sheet for treatment of ischemic heart disease - Google Patents

Description

The present invention relates to medical cell sheets. More specifically, the present invention relates to a novel medical cell sheet obtained by co-culturing vascular endothelial progenitor cells and myoblasts for use in ischemic heart disease.

In ischemic heart diseases 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. Since adult myocardial cells are inherently poor in self-renewal ability, once damaged, the myocardium cannot be repaired, or even if repaired, only limited recovery can be expected, eventually leading to heart failure.

On the other hand, the present inventors have proposed a method for amplifying vascular endothelial progenitor cells (EPCs) from bone marrow, umbilical cord blood or peripheral blood-derived mononuclear cell fractions under serum-free culture for ischemic diseases, refractory diseases, etc. It was invented and reported for use as a therapeutic agent for ulcer- or diabetes-related diseases (Patent Document 1, Non-Patent Documents 1 and 2). More specifically, the present inventors have found that stem cell factor (SCF), interleukin-6 (IL-6), FMS, and cell-proliferation conditions under which the mononuclear cell fraction can be differentiated and proliferated into a group of cells that contribute to angiogenesis. We have developed a method to culture mononuclear cells in serum-free medium containing five factors: tyrosine kinase-3 ligand (Flt-3 ligand), thrombopoietin (TPO) and vascular endothelial growth factor (VEGF). It was found that this culture system amplifies functional EPCs, guarantees the quality and quantity of EPCs, and enhances the revascularization ability. This culture system is named Quality and Quantity (QQ) culture. Recently, QQ-cultured peripheral blood mononuclear cells have also been studied as a cell therapy for ischemic heart disease (Non-Patent Document 3).

On the other hand, transplantation of skeletal myoblasts into myocardial tissue is known as a therapeutic method for heart failure. Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. Cardiac muscle and skeletal muscle have many similarities in structure and function, so it is believed that skeletal muscle-derived myoblasts can repair damaged myocardium. In addition, damage loss of transplanted cells during transplantation of myoblast suspension, tissue damage during injection of recipient heart, efficiency of tissue supply to recipient heart, occurrence of arrhythmia, difficulty in treating the entire infarcted area, etc. In order to overcome the drawbacks of the method, the use of sheeted myoblasts has been investigated (eg, Non-Patent Documents 4 to 8).

WO2014/051154

Masuda H. et al. Development of serum-free quality and quantity control culture of colony-forming endothelial progenitor cell for vasculogenesis. Stem Cells Transl Med 1 2012: 160-171 Masuda H. et al. Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential. J Am Heart Assoc. 2014 Jun 25;3(3):e000743 doi: 10.1161/JAHA.113.000743. Amankeldi A. Salybekov1, et al. Regeneration-associated cells improve recovery from myocardial infarction through enhanced vasculogenesis, anti-inflammation, and cardiomyogenesis. PLoS One. 2018 Nov 28;13(11):e0203244. doi: 10.1371/journal.pone. 0203244. eCollection 2018 Sawa Y. et ai. Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case. Surg Today 2012; 42: 181-184 Alshammary S. et al. Impact of cardiac stem cell sheet transplantation on myocardial infarction. Surg Today 2013; 43:970-976 Sawa Y. et al. Cell Sheet Technology for Heart Failure, Current Pharmaceutical Biotechnology 2013; 14: 61-66 Sawa Y. et al. Present and Future Perspectives on Cell Sheet-Based Myocardial Regeneration Therapy. BioMed Research International 2013; http://dx.doi.org/10.1155/2013/583912 Keitaro Domae et al., Examination of safety and efficacy of myocardial regeneration treatment using skeletal muscle myoblast sheets in severe heart failure, Organ Biology 2015; 22(2): 64-68 Terrovitis J. et al. Noninvasive Quantification and Optimization of Acute Cell Retention by In Vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem Cell Delivery. Journal of the American College of Cardiology 2009; 54(17): DOI: 10.1016/j.jacc .2009.04.097

A known problem with cell therapy for ischemic heart disease is that the administered cells have a low survival rate and engraftment rate (Non-Patent Document 9). This low survival rate and survival rate are due to the physical outflow of cells, the environment where cells are difficult to engraft, such as ischemia and inflammation, and the fact that the cells being administered are other people's cells. It is also thought that this is due to immunological rejection due to the fact that they are cells of a different type from myocardial cells. In addition, as other problems of cell therapy for ischemic heart disease, the effect of cell therapy is mainly caused by the paracrine effect, and the degree of myocardial differentiation of the administered cells is low, and there is concern that the administered cells may become tumorigenic. There is also concern about arrhythmia due to electrical and tissue isolation of the administered cells.

An object of the present invention is to provide a method for treating ischemic heart disease more effectively than conventional cell therapy.

The present invention provides the following.
[1] a step of preparing vascular endothelial progenitor cells;
preparing myoblasts;
Vascular endothelial progenitor cells and a sheet-forming effective amount of myoblasts are added at a cell number ratio of vascular endothelial progenitor cells to myoblasts (number of vascular endothelial progenitor cells: number of myoblasts) of 1:0.5 to 20. A method for producing a cell sheet, comprising the steps of seeding and co-cultivating to form a cell sheet.
[2] The production method according to 1, further comprising removing at least part of the co-cultured vascular endothelial progenitor cells.
[3] The production method according to 2, wherein the effective amount for sheet formation is 1.0×10 ³ to 1.0×10 ⁵ pieces/cm ² .
[4] The production method according to any one of 1 to 3, wherein the vascular endothelial progenitor cells are derived from peripheral blood mononuclear cells.
[5] The production method according to 4, wherein the peripheral blood mononuclear cells are obtained from a subject to which the cell sheet is transplanted.
[6] The production method according to 4 or 5, wherein the step of preparing vascular endothelial progenitor cells is a step of culturing peripheral blood mononuclear cells using a serum-free medium.
[7] The production method according to any one of 1 to 6, wherein the myoblasts are obtained from a subject to which the cell sheet is to be transplanted.
[8] A cell sheet containing vascular endothelial progenitor cells and myoblasts.
[9] The cell sheet according to 8, for treatment of ischemic heart disease.
[10] The cell sheet according to 8 or 9, wherein at least one of vascular endothelial progenitor cells and myoblasts is obtained from a subject to which the cell sheet is to be transplanted.
[11] The cell sheet according to any one of 8 to 10, which is a regenerative medicine product.
[12] A kit for preparing the cell sheet according to any one of 8 to 11.
[13] Use of vascular endothelial progenitor cells for use in combination with myoblast sheets.

By using the co-cultured cell sheet according to the present invention, ischemic heart disease can be treated more effectively than when using a conventional cell sheet of myoblasts alone. The use of the cell sheet of the present invention is expected to improve left ventricular ejection fraction (LVEF) and reduce remodeling.
Co-culturing of vascular endothelial progenitor cells can promote angiogenesis in the cell sheet-implanted tissue. Inflammation can also be suppressed.
Since cells differentiated to some extent are used in the cell sheet of the present invention, there is little concern about tumorigenesis.
In the embodiment using cells collected from the subject for which the cell sheet is used, immune rejection of the transplanted cells can be suppressed.

In vitro co-culture protocol of QQ cells and mycoblasts cell photo Photograph of cells stained for CD34 Pictures of cells stained with PECAM-1 and CD34 Photograph of Cx43, Tunel-stained cells RT-PCR results for in vitro cultured cells. Each left bar is the result of mycoblast monoculture and each right bar is the result of co-culture. Results of measurements of inflammation-related cytokines on in vitro cultured cells. Each left bar is the result of mycoblast monoculture and each right bar is the result of co-culture. In vivo evaluation protocol In vivo evaluation results

The present invention relates to a method for producing a cell sheet and a cell sheet produced thereby, comprising the following steps:
providing vascular endothelial progenitor cells;
preparing myoblasts;
Vascular endothelial progenitor cells and a sheet-forming effective amount of myoblasts are added at a cell number ratio of vascular endothelial progenitor cells to myoblasts (number of vascular endothelial progenitor cells: number of myoblasts) of 1:0.5 to 20. A step of seeding and co-cultivating to form a cell sheet.

[Preparation of vascular endothelial progenitor cells]
(vascular endothelial progenitor cells)
Vascular endothelial progenitor cells (EPCs) are used in the present invention. The vascular endothelial progenitor cells used in the present invention are not particularly limited as long as they are undifferentiated cells that can become vascular endothelial cells. Depending on the degree of differentiation, vascular endothelial progenitor cells are classified into differentiated EPC colonies (CFU-Large cell-like EC, also called large EPC colonies) mainly containing cells with a diameter of 20 to 50 μm and undifferentiated cells mainly containing cells with a diameter of 20 μm or less. Two types of EPC colonies (CFU-small cell like EC, also referred to as small EPC colonies) can be distinguished by their different sizes. Undifferentiated (small) EPC colonies appearing at an early stage are EPC colonies at an early differentiation stage with excellent proliferation ability, and differentiated (large) EPC colonies appearing at a late stage are late stage EPC colonies with excellent vascular development. It can be said that they are EPC colonies in the differentiation stage (see, for example, Masuda H. et al., Circulation Research, 109: 20-37 (2011)). Any of them can be suitably used in the present invention.

Vascular endothelial progenitor cells are included in mononuclear cells collected from peripheral blood, bone marrow, umbilical cord blood, and the like. In a preferred embodiment of the present invention, as vascular endothelial progenitor cells, QQ cells (QQCs ) (Patent Document 1, Non-Patent Documents 1 and 2) are used. Vascular endothelial progenitor cells contained in such a cell group have almost the same number of undifferentiated EPC colony-forming cells as compared to the case of sorting with CD34/CD133, but the number of differentiated EPC colony-forming cells is significantly increased. are doing. Preferably, the number of undifferentiated EPC colony-forming cells is the same as in the case of selection by CD34/CD133, but the number of differentiated EPC colony-forming cells is more than twice the same. More preferably, the number of undifferentiated EPC colony-forming cells does not change compared to the case of selection with CD34/CD133, but the number of differentiated EPC colony-forming cells is 4 times, more preferably 5 times that. . In the following, the present invention may be described as an example in which the vascular endothelial progenitor cells are QQ cells. It can be applied appropriately and understood.

(mononuclear cells)
QQ cells are derived from mononuclear cells. Mononuclear cells are a general term for cells with round nuclei contained in peripheral blood, bone marrow, umbilical cord blood, etc., and include lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, hematopoietic stem cells, and the like. Mononuclear cells usually contain CD34 and/or CD133 positive cells. Mononuclear cells can be obtained by collecting bone marrow, umbilical cord blood or peripheral blood from an animal and subjecting it to, for example, density gradient centrifugation to extract the fraction. Density gradient centrifugation is not particularly limited as long as a mononuclear cell fraction can be obtained, but Histopaque-1077 (Sigma-Aldrich) is preferably used.

In a particularly preferred embodiment of the present invention, peripheral blood-derived mononuclear cells are used as a source of vascular endothelial progenitor cells.

The animal species from which the vascular endothelial progenitor cells are derived is not particularly limited. More preferably, the cell sheet is obtained from the subject himself/herself to whom the cell sheet is to be transplanted.

(serum-free medium)
In the present invention, when a cell group obtained by culturing mononuclear cells in a serum-free medium without selection by CD34/CD133 is used as the vascular endothelial progenitor cells, preferred examples of serum-free medium used for the culture is stem cell factor (SCF), interleukin 6 (IL-6), FMS-like tyrosine kinase 3 ligand (Flt-3 ligand, or sometimes abbreviated to FL), thrombopoietin (TPO) and vascular endothelial cell growth factor (VEGF) is a serum-free medium containing five factors.

Stem cell factor (SCF) is a 248 amino acid glycoprotein with a molecular weight of approximately 30,000. A soluble type and a membrane-bound type exist due to alternative splicing, and the SCF used in the present invention may be of any type as long as it is useful for culturing EPCs and the like. A soluble form is preferred. Although the origin of SCF is not particularly limited, a recombinant that is expected to be stably supplied is preferred, and a human recombinant is particularly preferred. Human recombinants of SCF are known to be commercially available.

The concentration of SCF in the serum-free medium varies depending on the type of SCF used, and is not particularly limited as long as it is useful for culturing EPCs and the like. is between 50 and 500 ng/mL, more preferably about 100 ng/mL.

Vascular endothelial growth factor (VEGF) is a growth factor that specifically acts on EPCs and is known to be mainly produced in cells surrounding blood vessels. Although alternative splicing produces several different sized VEGF proteins, the VEGF used in the present invention can be of any type as long as it allows colony formation of EPCs. VEGF165 is preferred. Although the origin of VEGF is not particularly limited, recombinants that are expected to be stably supplied are preferred, and human recombinants are particularly preferred.

The concentration of VEGF in the serum-free medium varies depending on the type of VEGF used, but in the case of human recombinant VEGF165, it is preferably 5 to 500 ng/mL, more preferably about 20 to 100 ng/mL, and even more preferably about 20 to 100 ng/mL. 50 ng/mL.

FMS-like tyrosine kinase 3-ligand (FL) is known as a receptor-type tyrosine kinase ligand that plays an important role in the control of early hematopoiesis. Several alternatively spliced products are known and are reported to stimulate proliferation of hematopoietic stem cells. The FL used in the present invention may be any type of FL as long as it is useful for enrichment of EPC and the like.

The concentration of FL in the serum-free medium varies depending on the type of FL used, but in the case of human recombinant Flt-3 ligand, it is preferably 10 to 1000 ng/mL, more preferably 50 to 500 ng/mL. Preferably it is 100 ng/mL.

Thrombopoietin (TPO) is a type of hematopoietic cytokine and is known to act specifically on the process of producing megakaryocytes from hematopoietic stem cells and promote the production of megakaryocytes. The origin of the TPO used in the present invention is not particularly limited, but a recombinant that is expected to be stably supplied is preferred, and a human recombinant is particularly preferred.

The concentration of TPO in the serum-free medium varies depending on the type of TPO used, but in the case of human recombinant TPO, it is preferably 1-500 ng/mL, more preferably 5-100 ng/mL, and still more preferably 20 ng. /mL.

Various factors added to the serum-free medium are preferably unified with factors derived from the same animal species as the animal from which the mononuclear cells were derived. By unifying the origins of mononuclear cells and various factors in this way, a cell culture suitable for allogeneic transplantation such as allogeneic transplantation can be obtained. It is also possible to obtain cell cultures suitable for allogeneic transplantation by using mononuclear cells derived from an individual for whom cell transplantation is intended. Since it is possible to culture a group of cells containing EPCs and the like in an environment that does not contain any components derived from heterologous animals, the resulting cell culture has the advantage of avoiding the risk of infection and rejection during transplantation. .

As the body of the serum-free medium, a basal medium commonly used for culturing animal cells in the art can be used. For example, a serum-free medium known as a medium for growing hematopoietic stem cells can be used. can be done. Examples of basal media used as serum-free media include DMEM, MEM, IMDM and the like.

(Culture conditions)
Conditions for culturing mononuclear cells in a serum-free medium are not particularly limited, and normal conditions for culturing animal cells can be used. For example, it is cultured at 37° C. for 7 days or longer (eg, 10 days or longer) in a 5% CO ₂ environment. The density of mononuclear cells in the serum-free medium is not particularly limited as long as it allows enrichment of EPCs and the like, but is preferably 0.5 to 10×10 ⁵ cells/mL, more preferably 1 to 5 cells/mL. ×10 ⁵ cells/mL, more preferably 3 to 4×10 ⁵ cells/mL.

(Cell group obtained by serum-free culture)
In the present invention, a cell group obtained by culturing mononuclear cells in a serum-free medium containing the factors described above may contain a large number of differentiated EPC colony-forming cells. Cell populations may also include anti-inflammatory macrophages. Anti-inflammatory macrophages are CD206-positive, anti-inflammatory macrophages that contribute to angiogenesis and repair. M2 macrophages are preferred, and CD206-positive M2 macrophages are more preferred.

Vascular endothelial progenitor cell colony-forming ability can be measured using a commercially available kit. As such a kit, for example, a kit manufactured by Stem Cell Technology (Catalog No. H4236) is commercially available. Differentiation or amplification of anti-inflammatory macrophages or inflammatory macrophages is measured, for example, by labeling CD206 for anti-inflammatory macrophages and CCR2 for inflammatory macrophages with antibodies having affinity to them and performing flow cytometric analysis. can.

The resulting cell population may further include anti-inflammatory Th2 cells and regulatory T cells. Therefore, the obtained cell population can be a cell population enriched with anti-inflammatory Th2 cells and regulatory T cells in addition to EPCs and anti-inflammatory macrophages.

The resulting cell population preferably contains differentiated EPC colony-forming cells and anti-inflammatory macrophages (CD206-positive M2 macrophages). This is because such a group of cells is considered to be extremely effective in that it can not only generate blood vessels but also suppress inflammation at an inflamed site such as an ulcer.

[Preparation of myoblasts]
Myoblasts are used in the present invention. Since myoblasts are cells that have differentiated to some extent, it can be said that the risk of tumorigenesis is less. The origin of myoblasts is not particularly limited, but skeletal myoblasts of skeletal muscle tissue are preferable from the viewpoint that they exist abundantly in vivo and can be collected by a relatively easy operation. . The site to be collected is not particularly limited, and can be, for example, the vastus medialis muscle of the thigh.

When myoblasts are collected from a living body, they are preferably collected from a subject to whom the cell sheet is to be transplanted, from the viewpoint that immune rejection is less likely to occur in the subject.

The amount to be collected depends on the size ^and number of cells required for the desired cell sheet. Yes, and relatively many myoblasts may be required. Myoblasts can be cultured and proliferated outside the body, and can be suspended in a preservation solution and cryopreserved. If necessary, an appropriate amount can be ensured by collecting and culturing multiple times.

[Co-culture]
The cell sheet of the present invention is produced by co-culturing vascular endothelial progenitor cells and myoblasts. Co-culture is initiated by seeding vascular endothelial progenitor cells and myoblasts on the same cultureware surface at the start of culture.

(sowing)
The method of seeding vascular endothelial progenitor cells and myoblasts for co-culture is as long as a myoblast sheet is formed while at least the vascular endothelial progenitor cells and myoblasts coexist on the same substrate surface. Although not particularly limited, in a preferred embodiment, an effective amount of myoblasts for sheet formation is first seeded on the surface of the cultureware, and then vascular endothelial progenitor cells are seeded in a predetermined ratio to the myoblasts. be.

In co-culturing, myoblasts are seeded in an amount that allows myoblasts to form a sheet-like culture after several days to several weeks of culture, that is, in an effective amount for sheet formation. The effective amount for sheet formation is, for example, 1.0×10 ³ to 1.0×10 ⁵ cells/cm ² , preferably 2.0×10 ³ , in order to obtain a sheet by culturing for about one week or less. 5.0×10 ⁴ pieces/cm ² , more preferably 2.0×10 ³ to 2.0×10 ⁴ pieces/cm ² . Alternatively, in order to obtain a sheet without substantially proliferating myoblasts, the effective amount for sheet formation is 0.4×10 ⁶ to 2.5×10 6 cells/cm ² , preferably 0.5×10 ⁶ cells/cm 2 . It is 5×10 ⁶ to 2.1×10 ⁶ pieces/cm ² , more preferably 0.6×10 ⁶ to 1.7×10 ⁶ pieces/cm ² . The seeding amount of myoblasts can also be determined in consideration of the required size and number of cell sheets to be formed.

The seeding amount of vascular endothelial progenitor cells in co-culture can be appropriately calculated based on the amount of myoblasts to be seeded and the ratio of vascular endothelial progenitor cells to myoblasts described below.

(Ratio of vascular endothelial progenitor cells to myoblasts)
The ratio of vascular endothelial progenitor cells and myoblasts at the start of co-culture is not particularly limited as long as sheet formation is possible. number: number of myoblasts) can be 1:0.5-20. Preferably, the ratio of vascular endothelial progenitor cell number to myoblast number is 1:0.2 to 5, more preferably 1:0.3 to 3, still more preferably 1:0.5 to 2. . Co-culture in such a range can be expected to increase the expression of angiogenesis-related factors, and is considered to be advantageous for anti-inflammatory effects and cell survival. If the ratio of vascular endothelial progenitor cells is too low, a sufficient effect to enhance the function of co-cultured myoblasts cannot be expected. Blast cells do not form a cell sheet, which is undesirable. Regarding the present invention, when referring to the ratio of cells, it is a value based on the number of cells unless otherwise specified.

(Culture equipment)
The cultureware for co-cultivation is not particularly limited, but from the viewpoint that the formed cell sheet can be easily peeled off from the equipment, a polymer whose hydration force changes in the temperature range of 0 to 80°C is used. It is preferable to use a cell cultureware with a coating on the surface. That is, cells are cultured in a temperature range where the hydration power of the polymer is weak on a cell cultureware whose surface is coated with a polymer whose hydration power changes in the temperature range of 0 to 80°C. The temperature is preferably around 37° C., which is generally suitable for culturing animal cells. A temperature-responsive polymer may be either a homopolymer or a copolymer. Examples of such polymers include polymers described in JP-A-2-211865. Specific examples include polymers of monomers such as (meth)acrylamide compounds, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, and vinyl ether derivatives, and in the case of copolymers, these It can be a product obtained by polymerizing any two or more of the above. Furthermore, copolymers with monomers other than the above monomers, graft or copolymerization between polymers, or mixtures of polymers and copolymers may be used. Moreover, it may be crosslinked to the extent that it does not impair the original properties of the polymer. At that time, since animal cells are cultured thereon, it is preferable that the exfoliation can be performed in the range of 5°C to 50°C. Propyl acrylamide (lower critical solution temperature of homopolymer 21 ° C.), poly-Nn-propyl methacrylamide (27 ° C.), poly-N-isopropyl acrylamide (32 ° C.), poly-N-isopropyl methacrylamide ( 43° C.), poly-N-cyclopropylacrylamide (45° C.), poly-N-ethoxyethylacrylamide (about 35° C.), poly-N-ethoxyethyl methacrylamide (about 45° C.), poly-N - Tetrahydrofurfuryl acrylamide (about 28°C), poly-N-tetrahydrofurfuryl methacrylamide (about 35°C), poly-N,N-ethylmethylacrylamide (about 56°C), poly-N,N-diethyl Acrylamide (at 32°C) can be mentioned. Temperature-responsive cell cultureware is commercially available, and for example, UpCell (registered trademark) (CellSeed, cat# CS3003, CS3004, CS3005, CS3006, CS3007) can be used for co-culture.

(Removal of vascular endothelial progenitor cells)
In a preferred embodiment, after the vascular endothelial progenitor cells and myoblasts have been co-cultured for a period of time, at least a portion of the co-cultured vascular endothelial progenitor cells are removed. Removal of at least a portion of vascular endothelial progenitor cells can be performed in conjunction with medium replacement. That is, when the culture supernatant is removed from the incubator, floating vascular endothelial progenitor cells can be easily removed together with the supernatant. Removal of at least a portion of the vascular endothelial progenitor cells may be performed at any point during the culture period. For example, 2 to 5 days after co-cultivation, the medium is exchanged, fresh medium is added, and the culture can be continued for the remaining period with at least part of the intravascular progenitor cells removed. .

Vascular endothelial progenitor cells may be partially or wholly removed. If some vascular endothelial progenitor cells are removed and some vascular endothelial progenitor cells remain on the myoblast sheet, it is expected to contribute to improved angiogenesis and blood flow in the affected area when the sheet is transplanted. can. From this point of view, it is preferable that not all of the vascular endothelial progenitor cells are removed, but some of them remain on the myoblast sheet. Of the vascular endothelial progenitor cells used for co-culture, for example, about 20% of the vascular endothelial progenitor cells may be left on the cell sheet, about 10% of the vascular endothelial progenitor cells may be left, and 5% may be left on the cell sheet. Some vascular endothelial progenitor cells may be left.

(Culture medium)
A conventional medium used for culturing myoblasts can be used for the co-culture. For example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7 and other basal media, if necessary, adding components, etc. can be used with modifications.

The medium used for co-cultivation may be a serum-free medium or a medium supplemented with serum. When serum is used, examples of serum include xenogeneic serum, allogeneic serum (allogeneic and autologous serum). The heterologous serum as used herein means, when the cell sheet is transplanted, serum derived from an organism of a species different from that of the transplanted subject. For example, when the subject is a human, serum derived from bovine or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), and the like, correspond to heterologous serum. Also, allogeneic serum means serum derived from organisms of the same species as the subject. For example, when the subject is human, human serum corresponds to allogeneic serum. Allogeneic serum includes allogeneic serum obtained from an individual of the same species other than the subject, and autologous serum, ie, serum obtained from the subject himself. When a cell sheet is transplanted and serum is used for co-culturing, allogeneic serum is preferable, and autologous serum is more preferable, from the viewpoint of avoiding side effects during transplantation.

When serum is used, its concentration in the concentration medium can be 0.5-20%, eg 5%, 10%, 15%. Regarding the present invention, the concentration of serum in the medium is the concentration (v/v) based on the volume of serum and the volume of the entire medium, unless otherwise specified.

In another preferred embodiment, the medium for co-cultivation is substantially free of serum.

(Incubation temperature, period, etc.)
Co-cultivation can be performed under the same conditions as when myoblasts are cultured for the purpose of sheet formation. Typical culturing conditions include culturing at 37° C. and 5% CO ₂ . The culture period can be continued until a sheet-like structure is formed. This period includes the culture period after at least part of the vascular endothelial progenitor cells have been removed, for example, culture when myoblasts are seeded at 1.0×10 ³ to 1.0×10 ⁵ cells/cm ^{2 .} The period is 2 days to 2 weeks, more particularly 3 days to 10 days, for example around 1 week. The culture period can be shorter if the myoblast seeding rate is higher.

(Recovery of cell sheet)
A cell sheet formed through co-culture of vascular endothelial progenitor cells and myoblasts can be peeled off from the surface of the cultureware and collected. The method for collecting the cell sheet is not particularly limited, but when a temperature-responsive cultureware is used, the cell sheet can be collected by changing the temperature to an appropriate temperature. As another example, dilute protease is used. method, a method using a special protease, a method using a chelating agent (for example, EDTA), and a method of physically peeling off using a scraper or the like. The obtained cell sheets can be layered as necessary to form a three-dimensional structure.

[Cell sheet]
The present invention provides a cell sheet containing vascular endothelial progenitor cells and myoblasts obtained by the production method described above.

Since the cell sheet of the present invention is produced by co-culturing vascular endothelial progenitor cells and myoblasts, it typically has a form in which small vascular endothelial cells accompany a sheet formed of myoblasts. doing A sheet formed only of myoblasts has little morphological difference except that small vascular endothelial cells are attached, and therefore, it is considered that it can be used in the same way as a conventional myoblast sheet.

According to the studies of the present inventors, the obtained cell sheet showed increased expression of IL-10, which is an anti-inflammatory/immunoregulatory cytokine, compared to a cell sheet formed only from myoblasts. Along with the increased expression of IL-10, increased expression of some inflammatory cytokines may also be observed.

[Uses of cell sheets, etc.]
The cell sheet of the present invention can be used for treating ischemic heart disease. Ischemic heart disease is a disease caused by tissue damage such as degeneration, atrophy, and fibrosis of cells due to decreased blood flow in the myocardial tissue. Ischemia is roughly classified into obstructive ischemia, compression ischemia, convulsive ischemia, and compensatory ischemia, depending on the cause. Treatment includes treatment of disease, inhibition of progression of disease, prevention of disease, and reduction of risk of developing disease.

The cell sheet of the present invention is also expected to be effective against cardiomyopathy other than ischemic heart disease. Cardiomyopathy is a disease in which cardiac function is reduced due to an abnormality of the heart muscle. Cardiomyopathy includes dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy.

When the cell sheet of the present invention is used for transplantation, at least one of the following effects can be expected:
the loss of transplanted cells is inhibited;
immune rejection of transplanted cells is suppressed;
angiogenesis is promoted in the affected area;
Blood flow in the trunk is improved;
inflammation in the affected area is suppressed;
Increased survival rate and engraftment rate of transplanted cells;
increased myocardial differentiation of the transplanted cells;
less concern that the transplanted cells will become tumorigenic;
Electrical and organizational isolation (arrhythmia) of transplanted cells becomes less likely to occur.

The above effects are considered to be achieved by co-culturing vascular endothelial progenitor cells with myoblasts. Accordingly, the present invention also provides methods of using vascular endothelial progenitor cells for use in combination with myoblast sheets.

The present invention also provides kits for producing cell sheets as described above. The kit includes reagents and instruments used for collecting peripheral blood from a subject to which the sheet is to be transplanted; reagents and instruments for obtaining a mononuclear cell fraction from peripheral blood; preparation and culturing of vascular endothelial progenitor cells; Reagents and instruments; instruments for collecting myoblasts, reagents and instruments for cryopreserving myoblasts; culture instruments for co-culturing vascular endothelial progenitor cells and myoblasts; sheets for co-culturing equipment used to collect serum from a subject to be transplanted; equipment used to transport cells collected at a medical institution to a sheet manufacturer.

The cell sheet of the present invention can be used as a regenerative medicine product. In the case of a regenerative medicine product, the cell sheet must comply with the conditions (good manufacturing practice, GMP) that conform to the manufacturing control and quality control regulations for pharmaceuticals and quasi-drugs and the standards for manufacturing control and quality control of regenerative medicine products ( Good Gene, Cellular, and Tissue-based Products Manufacturing Practice, GCTP).

[Preparation of cells]
(QQ cells)
Mononuclear cells were collected from peripheral blood by centrifugation by a conventional method, and 2×10 ⁶ mononuclear cells/6-well Primaria plate (BD Falcon, Franklin Lakes, NJ) wells were seeded. The medium contains five cytokines, recombinant human vascular endothelial growth factor (rhVEGF) (50 ng/ml), rh interleukin-6 (rhIL-6) (20 ng/ml), rh Fms-related tyrosine kinase-3 ligand ( rhFlt-3L) (100 ng/ml), rh thrombopoietin (rhTPO) (20 ng/ml), rh stem cell factor (rhSCF) (100 ng/ml) (all from PeproTech, Rocky Hill, NJ) and antibiotics Cocktail (Invtrogen) added to Stemline II medium (Sigma, St. Lois, Mo.) was used. After culturing at 37°C and 5% CO ₂ for 7 days, QQ cells (QQCs) were obtained.

(myoblast)
Myoblasts (MBCs) were obtained from a commercially available human-derived cell line (Lonza cat# CC2580, Lot# 0000418971). The cells were cultured in MCDB medium supplemented with 15% FBS and subcultured twice to increase the amount to a sufficient amount.

[In vitro study]
(Co-culture)
The protocol is shown in FIG.
QQCs from healthy volunteers were used. 50 mL of blood was collected and cultured for QQCs. The cultured MBCs were detached using 0.25% trypsin-EDTA and seeded in a normal 6-well dish (9.6 cm ² /well) at 1×10 ⁵ /well. On the next day, QQCs and MBCs were seeded in wells so that the cell numbers were QQCs:MBCs = 1:1, 1:3, 1:10, respectively. After 48 hours of co-cultivation, the medium supernatant was aspirated to remove QQCs ((1) in FIG. 1). The medium of MBCs was added and cultured under normoxia and hypoxia for 3 days. Cells were harvested after 3 days using 0.25% trypsin-EDTA ((2) in FIG. 1) and placed in RNAlater (Invitrogen, cat# AM7021) to generate samples for PCR. After 1 week, the supernatant was replaced with 2 ml, collected after 24 hours, and the concentration of inflammation-related cytokines in the medium was measured by ELISA.

(result)
Photographs of cells after culturing are shown in Figures 2-5. In the co-culture group, many small cells are seen on top of normal cells. These QQCs are considered to remain after collection. No other morphological differences are observed.

FIG. 6 shows the results of RT-PCR, and FIG. 7 shows the results of concentration measurement of inflammation-related cytokines. Increased expression of IL-10, an anti-inflammatory cytokine, was observed.

[In vivo study]
(Creation of cell sheet)
Cell sheets were prepared for in vivo studies. A sheet of MBCs alone and a sheet of QQCs coculture were prepared, respectively. MBCs alone sheets were seeded with MBCs at a density of 1×10 ⁴ /cm ² in 12-well temperature-responsive culture dishes Upcell® (CellSeed, cat# CS3003). The co-culture sheet was the most effective 1:1 protocol from the in vitro studies so _far . were co-cultured. After co-cultivation, QQCs were removed and the cells were further cultured for 3 days.

(Creation of myocardial infarction mice)
Eight-week-old male nude mice were used. After the mouse was intubated and respiratory management was performed with an artificial respirator, the heart was exposed by left thoracotomy in the right lateral decubitus position. The left coronary artery was ligated with a 7-0 monofilament thread to create a myocardial infarction. After preparation, each cell sheet was indwelled so as to cover the ischemic site of the left ventricle, the sheet was fixed by closing the pericardium, and the thoracic cavity was closed to complete the operation.

(Evaluation of cardiac function)
The protocol is shown in FIG. Cardiac function was measured using a small animal ultrasound device immediately, 1 week, 2 weeks and 4 weeks after surgery. After 1 and 4 weeks of ultrasound, mouse hearts were excised and half were cryosectioned for PCR analysis and the other half for histochemistry. For PCR analysis, hearts were excised after perfusion with heparinized PBS and permeated with RNAlater. For histochemical use, the cells were fixed with 4% PFA, dehydrated with 10, 15, and 20% sucrose, embedded in OCT compound, stored at -80°C, and frozen.

(result)
FIG. 9 shows the measurement results of cardiac function. Control (CTL) group = 28.0 ± 1.7%, MBCs alone (MBCs only) group = 28.7 ± 1.3%, MBCs + QQCs mixed administration (MBCs + QQCs) immediately after myocardial infarction Group = 28.0 ± 2.3% and there was no significant difference among the three groups. There was no difference between the CTL group and the MBCs group at 1 week after surgery, but the CTL group showed a downward trend in LVEF thereafter, suggesting the progression of remodeling. On the other hand, the LVEF of the MBCs alone group did not decrease thereafter, and showed a mild improvement trend of 33.0±3.6% at 4 weeks. The MBCs + QQCs group showed an increasing trend of 31.6 ± 2.2 at the first week, and after that, it was always higher than the MBCs alone group, and 39.6 ± 1.6% after 4 weeks, an improvement of about 10% compared to immediately after. rice field. In addition, although there was no significant difference in left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) at 4 weeks, the values were lower in the MBCs + QQCs group, suggesting an effect of reducing remodeling.

Claims (12)

providing vascular endothelial progenitor cells;
preparing myoblasts;
Vascular endothelial progenitor cells and an effective amount of myoblasts for sheet formation are mixed at a cell number ratio of vascular endothelial progenitor cells to myoblasts (number of vascular endothelial progenitor cells:number of myoblasts) of 1:0.5 to 20. A method for producing a cell sheet, comprising the steps of seeding and co-cultivating to form a cell sheet. 2. The production method according to claim 1, further comprising removing at least part of the co-cultured vascular endothelial progenitor cells. 3. The production method according to claim 2, wherein the sheet-forming effective amount is 1.0×10 ³ to 1.0×10 ⁵ pieces/cm ² . The production method according to any one of claims 1 to 3, wherein the vascular endothelial progenitor cells are derived from peripheral blood mononuclear cells. 5. The production method according to claim 4, wherein the peripheral blood mononuclear cells are obtained from a subject to which the cell sheet is to be transplanted. The production method according to claim 4 or 5, wherein the step of preparing vascular endothelial progenitor cells is a step of culturing peripheral blood mononuclear cells using a serum-free medium. The production method according to any one of claims 1 to 6, wherein the myoblasts are obtained from a subject to which the cell sheet is to be transplanted. A cell sheet containing vascular endothelial progenitor cells and myoblasts obtained by a production method comprising the following steps:
providing vascular endothelial progenitor cells;
preparing myoblasts;
Vascular endothelial progenitor cells and an effective amount of myoblasts for sheet formation are mixed at a cell number ratio of vascular endothelial progenitor cells to myoblasts (number of vascular endothelial progenitor cells:number of myoblasts) of 1:0.5 to 20. A step of seeding and co-cultivating to form a cell sheet. 9. A cell sheet according to claim 8 for treatment of ischemic heart disease. 10. The cell sheet according to claim 8 or 9, wherein at least one of vascular endothelial progenitor cells and myoblasts is obtained from a subject to which the cell sheet is to be transplanted. The cell sheet according to any one of claims 8 to 10, which is a regenerative medicine product. Use of vascular endothelial progenitor cells in the production of the cell sheet according to any one of claims 8-11.

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