WO2017018629A1 - Myocyte-mixed sheet using stem cell support, and manufacturing method therefor - Google Patents

Abstract

The present invention relates to a myocyte-mixed sheet using a stem cell support, and a manufacturing method therefor and, more specifically, to a method for manufacturing an in vivo-implantable, myocyte-mixed sheet by using adipose-derived stem cells as a support. According to the present invention, the myocyte-mixed sheet using an adipose-derived stem cell support, can be easily manufactured in a short period of time at low cost and enables stable and safe in vivo implantation of cells, which are difficult to be manufactured in sheets individually, thereby being useful for industrial purposes in addition to clinical application such as local regenerative therapy.

Description

Myocyte mixed sheet using the stem cell support and a manufacturing method thereof

The present invention relates to a myocyte mixed sheet using a stem cell support and a method for producing the same, and more particularly, to a method for producing a mixed sheet with a myocyte transplantable in vivo using adipose stem cells as a support.

Stem cells are undifferentiated cells with self-replicating capacity and the ability to differentiate into any tissue. Undifferentiated stem cells can be differentiated into various cells and tissues by providing appropriate conditions. The regenerative treatment of damaged tissues using the stem cell differentiation ability has been actively studied.

Representatively, multipotency stem cells, such as adipose-derived stem cells (ADSCs), are stem cells that can only differentiate into specific cells of tissues or organs. It has the function of inducing the maintenance and regeneration of. These tissue specific multipotent cells are collectively referred to as adult stem cells (A. Miranville et al., Circulation , 110: 349, 2004). Unlike embryonic stem cells extracted from human embryos, adult stem cells have fewer ethical problems because they are extracted from adult tissues, and have excellent self-renewal ability and have differentiation ability to differentiate into fat, bone, cartilage, vascular endothelium and cardiomyocytes. Have. In addition, it is possible to obtain a larger amount of stem cells than in other tissues, and the extraction process is safe and easy (PA Zuk et al., Tissue Eng. , 7: 211, 2001).

Recently, after the transplantation of stem cells for regeneration of damaged organs and tissues, recovery of damage has been reported (Strauer et al., Circulation 106: 1913, 2002). In order to transplant stem cells, various cell transplantation methods to damaged organs and tissues have been attempted, such as a method of directly injecting a damaged organ or tissue, and a method of injecting into a vein vessel or systemic administration. However, transplanted stem cells have low engraftment and most have been reported to be wash-out. In particular, following observation of the transplanted cells shows that most of them are damaged in the lung or kidney. I have difficulty identifying direct effects or transplanted cells in tissues. In order to overcome this, transplantation is being carried out by increasing the quantity and frequency of stem cells, but there is a problem of continuously transplanting a high concentration of cells, and even if the transplantation is successful, the treatment effect is higher than expected due to poor cell environment. There is a limit to the example.

Therefore, in recent years, the therapy using the cell culture sheet is actively researched. As a representative method using a cell sheet, a culture dish method is proposed in which a surface is smeared with a temperature-responsive polymer. However, culturing the culture plate plated with a temperature-responsive polymer can produce a cell sheet using a large amount of cells, but there is a disadvantage that the sheet can not be recovered depending on the state of the various types of cells and culture. In addition, it is difficult to manufacture different sheets according to the size of the implanted site, and thus the economic burden is increased. Therefore, a cell transplantation technique using a scaffold has been reported to stably transplant various cells including beating cardiomyocytes which are not easy to form into sheets. Typically, attempts using polymeric materials, mixed scaffolds, and biodegradable scaffolds do not guarantee cell viability during the period in which transplanted cells are engrafted, despite the use of biomaterials. There are limitations in terms of side effects.

Accordingly, the present inventors have made diligent efforts to overcome the disadvantages of the existing cell sheet by applying the multi-differentiation advantage of stem cells to the treatment of damaged organs or tissues, to produce a mixed sheet of cardiomyocytes using adipose stem cells as a support It was confirmed that the function of maximizing the proliferation, survival and stable engraftment of the mixed cells and the like, and completed the present invention.

Summary of the Invention

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing a myocyte mix sheet using a stem cell support and a myocyte mix sheet using a stem cell support prepared by the above method.

In order to achieve the above object, the present invention comprises the steps of (a) culturing the adipose stem cells, and further seeding (seeding) the myocytes for transplantation into adipose stem cells to culture together; And (b) provides a method for producing a mixed sheet of fat stem cell support and transplanted muscle cells comprising the step of peeling and recovering the mixed sheet of fat stem cells and transplanted muscle cells cultured in step (a).

The present invention also provides a mixed sheet of adipose stem cell support prepared by the above method and a myocyte for transplantation.

1 is a graph showing the density of fat stem cells per unit area.

Figure 2 is a graph showing a comparison of cytokines secreted by culturing fat and stem cells at low density and high density.

3 is a photograph obtained by seeding 5 × 10 ⁶ fat stem cells, using an optical microscope (scale bar is 100 ㎛).

Figure 4 is a photograph of the sheet formation results using adipose stem cells.

5 is a multi-layered photograph confirmed by hematoxylin-eosin staining after adipocyte stem cell formation (scale bar is 100 μm).

Figure 6 is a photograph of the mouse embryonic stem cells and bone marrow stem cells after CFDA (Carboxyfluorescein diacetate succinimidyl ester) staining, confirming the sheet formation.

7 is a nuclear staining picture of the sheet formation of mouse embryonic stem cells.

8 is a nuclear staining picture of the sheet formation of mouse bone marrow stem cells.

Figure 9 is a result of photographs and engraftment rate confirming the presence of the inside of the heart confirmed over time after transplantation of adipose stem cell sheet and adipose stem cells in the heart induced myocardial infarction.

FIG. 10 is a multi-layered photograph confirming the presence or absence of the adipose stem cell sheet and the adipose stem cell in the heart induced myocardial infarction by the Masson's trichrome staining technique.

11 is a result of the ratio of pulsatile cardiomyocytes (green) induced from adipose stem cells and mouse embryonic stem cells in the process of making a beating heart muscle cell mixture sheet derived from mouse embryonic stem cells using adipose stem cell support (Scale bar is 100 μm).

12 is a photograph obtained by using an optical microscope after the formation of a beating heart muscle cell mixed sheet derived from mouse embryonic stem cells using adipose stem cell support. Inside the dashed line is the region where the pulsating cardiomyocytes are present (scale bar is 100 μm).

Figure 13 is a photograph of the results of the beating heart muscle cell mixture sheet induced in mouse embryonic stem cells using adipose stem cell support.

Figure 14 is a photograph obtained by using an optical microscope after the cardiomyocyte mixed sheet formation of the mouse neonatal using adipose stem cell support.

FIG. 15 is a photograph of cardiomyocyte cells mixed with a mouse neonatal using adipose stem cell support, followed by cardiomyocytes (red) and surrounding adipose stem cells (nucleus; blue) using immunofluorescence staining (scale bar is 100 μm) ).

Figure 16 shows the results of confirming the formation of a variety of myocytes mixed sheet using adipose stem cell support (a: cardiac myoblasts, b: coronary smooth muscle cells, c: myoblasts).

Figure 17 is a photograph of the observation of the nucleus (blue) of the mixed sheet by immunofluorescence staining after the formation of various myocytes (red) mixed sheet using adipose stem cell (green) support (a: cardiac myoblast, b: coronary artery Smooth muscle cells, c: myoblasts).

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, after culturing the adipose stem cells as a support in a general culture plate, and further seeding myocytes to prepare a mixed sheet, it is possible to produce a mixed sheet of pulsating muscle cells and stem cells difficult to produce a multi-layer sheet there was.

Therefore, the present invention in one aspect (a) culturing the adipose stem cells, and further seeding (seeding) the myocytes for transplantation into adipose stem cells to culture together; And (b) peeling and recovering the mixed sheet of the adipose stem cells and the transplanted muscle cells cultured in the step (a).

In another aspect, the present invention relates to a mixed sheet of adipose stem cell support prepared by the above method and a myocyte for transplantation.

In the present invention, the support is preferably adipose tissue-derived stem cells, but is not limited thereto.

In the present invention, the adipose stem cells used as a support is used for adipose stem cells for maximizing the synergistic effect with the mixed cells as much as differentiation into various cells and recovery effects on damaged organs or tissues are reported. It is desirable to. In addition, the present invention uses mouse adipose stem cells, but is not limited thereto. It is possible to use adipose stem cells isolated from all mammals including humans, white papers, pigs and monkeys.

Adipose stem cells as the support have proliferation and differentiation originating in vivo, and the function of the differentiated cells has been verified so that they can engraft and proliferate in damaged organs or tissues after transplantation in the body. In addition, it is characterized by being compatible with the surrounding tissues to minimize the inflammatory response.

The origin of the adipose stem cells is not particularly limited in the present invention, but the adipose tissue is composed of a combination of various surrounding tissues surrounding the adipose tissue, including the mature adipose cells, and refers to all adipose tissue regardless of the position in the body. Typically, the use of pericardial fat, subcutaneous fat, mesenteric fat, gastrointestinal fat and posterior peritoneal adipose tissue is preferred, and in the present invention, the normal adipose tissue is isolated and washed in male mice to clean pure fat using collagenase. Stem cells were recovered and used.

The separated and recovered adipose stem cells may be cultured up to 2 passages in a culture medium, and then their properties may be confirmed using conventional cell phenotypic markers. These markers are preferably, but not limited to, CD13, CD29, CD34, CD44, CD49a, CD90 and Sca-1. In addition, it is possible to assess the titer of stem cells through various cell differentiation. Differentiation induction includes, but is not limited to, fat, bone, cartilage, nerves, vascular endothelium, cardiomyocytes and the like.

In the present invention, in forming adipose stem cells as the support, it is preferable to incubate in 37%, 5% CO ₂ incubator for 36 hours. If the incubation time is less than the above time, there is a problem that the adipose stem cells are not enough to be formed as a support, and are torn, and if the time is longer than this time, the stem cell titer of the adipose stem cells decreases due to the long incubation time, and hypoxia The time is most appropriate because there is a problem that can lead to apoptosis.

In the present invention, the stem cells are preferably cultured by seeding (seeding) at a density of 0.5 ~ 1.0 × 10 ⁶ cells / cm ² , but is not limited thereto.

In general, it is preferable to seed 5 × 10 ⁶ sheets or more of sheet density on a general 6 wells plate. If the number of adipose stem cells is less than the number of cells shown above, there is a problem in that a dense sheet that cannot serve as a support is not formed. On the contrary, if the number of cells in the range is more than two times, the cells are killed relatively. Since the number of cells increases, the possibility of contamination such as turbidity of the culture solution increases, so the density per area is most suitable. In the present invention, the optimum amount of cells per area for seeding for preparing adipose stem cell sheet as a support was derived (FIG. 1).

In the present invention, as a result of confirming the secretion of growth factors, cytokines and hormones according to the cell density in the production of adipose stem cell sheet as a support, the growth factors Adiponectin, MCP-1, SDF-1, VEGFc, HGF, IGF And it was confirmed that TGFβ1 is secreted more from high-density fat stem cells than low-density fat stem cells. In addition, in the extracellular matrix, statistically significant secretion of Collagen I, Laminin-α4, Fibronectin and Vitronectin was confirmed, but their secretion is not necessarily limited thereto. These factors help to form a solid sheet between the adipose stem cells, and act as a paracrine effect (transcrine effect) after transplantation can represent a better effect of transplantation (Fig. 2).

Cell transplantation using stem cells has been tried in various ways for a long time, and one of the stem cell transplantation methods has recently emerged a method using a cell sheet. However, limitations on sheet formation have been reported depending on the characteristics of the cells to be transplanted. For example, there are no limitations in the production of stem cells or cells forming a multi-layer, but pulsating cardiomyocytes or cells that do not form a multilayer have limitations in current sheet making methods. Therefore, the present invention proposes a new mixed sheet manufacturing method that can overcome the limitations of these cell types.

In the present invention, the type and origin of the additional seeding cells are not particularly limited, and include both cells that can be produced in the form of existing sheets and impossible cells. In the present invention, rat-derived cardiomyocytes, human-derived coronary artery smooth muscle cells, and mouse-derived myoblasts are preferred, and beating heart muscle cells are most preferred, but are not limited thereto.

In the present invention, the additional seeding cells are preferably transplanted myocytes which do not form a multilayer or pulsate, and specifically, but not limited to cardiomyocytes, cardiomyocytes, smooth muscle cells or myoblasts, but is not limited thereto. .

In the present invention, the myocytes may be characterized in that seeding 2 × 10 ⁶ beating cardiomyocytes on a 6 wells plate seeded with adipose stem cells used as a support. If the number of additional mixed cells is less than the number of the above-mentioned cells, the engraftment rate of the mixed cells and the proliferation area are narrowed, and the effect is insignificant. On the contrary, if the number of the above-mentioned cells is exceeded, the hypoxic state of the adipose stem cells used as a support is used. And since it is possible to reduce the strength of the support, it is preferable to adjust and use within the above range.

In the present invention, the optimal ratio of forming a beating cardiomyocyte mixed sheet derived from mouse embryonic stem cells into additional mixed cells was derived (FIG. 11).

Therefore, in the present invention, the stem cells and cardiomyocytes are preferably seeded at a ratio of 2: 3, but are not limited thereto.

In the present invention, in the formation of a beating heart muscle cell mixed sheet, it is preferable to incubate in 37%, 5% CO ₂ incubator for 48 hours. If the incubation time is less than this time, there is a problem that the mixing sheet is not made because the time for engraftment and stabilization of additional mixed cells is not sufficient, and if the time is longer than this time, due to excessive proliferation of fat stem cells as a support with a long incubation time. The time is most appropriate because there is a problem that can lead to a decrease in stem cell titer, apoptosis due to hypoxia.

In the present invention, the same ratio of the cells and the culture time conditions were prepared to prepare a myocyte mixed sheet derived from various species and derived using adipose stem cells as a support, and confirmed the successful formation of the mixed sheet (FIGS. 16 and 17).

In the present invention, the peeling of the mixed cell sheet is most preferably one using a scraper, but is not necessarily limited thereto. However, due to the nature of the general dish, there is a problem of dissociation of all the cells using proteolytic enzymes.

In the present invention, the recovered mixed cell sheet is preferably at least 60% of the total culture dish area, but is not limited thereto. The sheet of the present invention is due to the seeding of appropriate cells, and can be configured in a free size to replenish the area of damaged organs or tissues. There is no restriction | limiting in particular about the shape and area of the said support body.

In addition, in order to verify whether the recovered mixed sheet is mixed with cardiomyocytes, it can be confirmed by immunostaining using a cardiac specific marker (FIGS. 14 and 15). When induced from mouse embryonic stem cells to cardiomyocytes, pulsating cardiomyocytes can be identified, which can be evaluated using cardiac specific markers. These markers include, but are not limited to, cardiac troponin T (cTnT), cardiac troponin I (cTnI), α-cardiac actin, α-actinin and myosin heavy chain (MHC). The phenotype of these cells can be assessed as cardiac specific cells.

The myocardial infarction animal model was used in the implantation of the cardiomyocyte cell mixture sheet using the adipose stem cell support of the present invention, but is not limited thereto. In the present invention, a sheet using adipose stem cells as a support rather than suspended fat stem cells was obtained for a long time (Fig. 9). In the case of the conventional cell mixing sheet, the engraftment rate is significantly lowered after about 28 days compared to the 3-4 days after transplantation, and most of the cell mixing sheets are only the extent of confirming the presence of cells after transplantation, and the cell mixing with excellent transplant efficiency is achieved. There is no sheet. However, the cardiomyocyte mixed sheet using the adipose stem cell support of the present invention, as shown in Figure 6, even after 28 days after transplantation can be confirmed an excellent engraftment rate of about 30% compared to the fourth day. It can be seen that it shows a very good engraftment rate compared to known cell mixing sheets or topical injection therapy.

Furthermore, various materials and biomaterials provided as a source of a conventional support are techniques focused on adhesion and transplantation, but a sheet using adipose stem cells according to the present invention as a support can be engrafted in vivo in addition to adhesion. It is an excellent sheet that may require a paracrine effect, as it shows a reduced recovery of fibrinated sites in damaged tissue (FIG. 10). In addition, in the conventional method of culturing specific cells in a culture dish coated with a temperature-responsive polymer and producing a sheet using a biomaterial such as fibrinogen, a culture dish coated with a temperature-responsive polymer is used. There is a problem that it is impossible to incubate for a long time. In addition, since the time and cost and the side effects of the use of non-biological materials have been reported, the new mixed sheet manufacturing method of the present invention can overcome these limitations.

The method for preparing a cardiomyocyte cell mixture sheet using the adipose stem cell support for producing an implantable sheet according to the present invention is easier to handle than a method for preparing a sheet using a temperature-responsive polymer culture dish, a living body, and a chemical material. Do. In addition, it is a method that minimizes the economic burden and minimizes the time to prepare for cell transplantation by using existing built equipment.Excellent cell survival even after transplantation, and uses stem cells, resulting in fewer side effects. Alternatively, the enhanced effect of cell therapy with sheet in tissue can be expected.

More specifically, in the present invention, it was confirmed that the various growth factors of the adipose stem cells present in the living body, by using such stem cells can remove the immune rejection reaction. In addition, in the case of pulsating cardiomyocytes, it is not only difficult to produce a sheet using alone, but also has a disadvantage in that long-term culture is difficult and recovery is not high. However, when the fat stem cell sheet, which is conventionally used according to the present invention, is used as a support, a problem of recovery rate can be solved by mixing cells (pulsating cells such as cardiomyocytes) that are difficult to produce alone as cell sheets. I can solve it. In addition, the admixture sheet with the adipose stem cells has the advantage of being a cell sheet capable of transplanting for a specific tissue by purely mixing the adipose stem cells and various cells without using other chemical treatment or non-biomaterials.

As a result of transplanting the cardiomyocyte mixed sheet using the adipose stem cell support thus prepared for the treatment of damaged tissues, the cell delivery success rate and function recovery were increased as compared with the transplantation of the previously reported single stem cells. In particular, it was confirmed that angiogenesis was increased in the infarct area in the transplantation into the infarcted myocardium, which has a beneficial effect of shortening the treatment time.

Therefore, the present invention not only consumes less time and money, but also mixes adipose stem cells with a variety of cells in a general culture dish to produce a mixed sheet composed of a dense, solid multilayer instead of a single layer. In other words, unlike conventional transplantation methods using chemical and non-biological components, it is a method of manufacturing a mixed sheet of cells that is expected to be developed into a clinical application such as safe and stable local regeneration treatment, which has no side effects and exhibits a stable effect after transplantation. Accordingly, the present invention is a very useful invention in the fields of medicine and biology such as regenerative engineering, cell engineering, tissue engineering and medical engineering.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of Adipose Stem Cell Sheet as Support

Pure adipose stem cells were recovered from the peri testicular adipose tissue. The recovered stem cells were cultured in two passages in a baseal mesdium containing FBS and various growth factors using MesenCult ™ Proloferation kit (Stem Cell Technology), and positive expression of Sca-1, known as stem cell marker, was confirmed. Induction of differentiation into bone, vascular endothelial and cardiomyocytes into four cells was verified as stem cells.

First, in order to determine the presence or absence of sheet formation according to the density of cells per area, the cells were seeded on 6 wells plates at various cell densities.

As a result, an optimum density condition was derived at a cell density of 5 × 10 ⁶ or less (FIG. 1). In addition, it was confirmed that the secretion of a variety of growth factors and extracellular matrix is more increased than the low density of the fat stem cells of high density (Fig. 2).

Then, stem cells were incubated for 48 hours at 37 ° C., 5% CO ₂ incubator to check the morphology of the cells through an optical microscope (FIG. 3), and then peeled off with a scraper to recover the sheets (FIGS. 4 and 5). .

Example 2 Sheet Preparation Using Various Stem Cells

In this example, stem cell sheet formation using mouse stem cells derived from mice and bone marrow stem cells, which are adult stem cells derived from mice, were confirmed.

Each stem cell was labeled with CFDA (Carboxyfluorescein diacetate succinimidyl ester) staining, and then seeded under the cell density conditions established in Example 1 (approximately 5 x 10 ⁶ cells) at 37 ° C., 5% CO ₂ incubator for 48 hours. Incubated at. Mouse embryonic stem cells were cultured by adding FBS and growth factors using GMEM as the baseal mesdium, and mouse bone marrow stem cells were cultured using a culture medium added with FBS to DMEM containing a large amount of glucose.

As a result, embryonic stem cells and bone marrow stem cells were disintegrated by low density prior to sheet formation, and thus could not form a sheet to the support, or tearing easily occurred even when forming a sheet. 6).

Thus, using the CFDA-labeled embryonic stem cells and frozen sections of bone marrow stem cells, the density and the sheet formation of the stained cells were again confirmed. The nuclei of stem cells stained with green by CFDA were stained with DAPI (blue), and then observed by light microscopy at 100, 200 and 400 times.

As a result, it can be seen that mouse-derived embryonic stem cells are divided into fragments without forming a sheet as a support (FIG. 7). In the case of mouse-derived bone marrow stem cells, intermittent formation of a multi-layered cell layer was observed. Existed in several fragments (FIG. 8).

Thus, it was confirmed that embryonic stem cells and bone marrow stem cells cannot form stem cell sheets as a support. Referring to the results of Figure 2, it appears that adipose stem cells can form a sheet as a support by secretion of various growth factors and extracellular matrix.

Example 3: Transplantation Effect of Adipose Stem Cell Sheet

To confirm the transplantation of grafts into the organs and tissues of the adipose stem cell sheets, 6-7 week old rats were prepared to induce myocardial infarction by ligation of the coronary artery, and then successfully induced the infarction of the induced myocardial infarction. The recovered adipose stem cell sheets and suspended adipose stem cells were transplanted. After transplantation, the presence of adipocytes and the effects of cardiac function over time were checked for 28 days.

As a result, it was confirmed that the transplanted fat stem cell sheet was present in the infarct region for a longer time than the floating fat stem cells, and the degree of fibrosis for assessing the infarcted region after sacrifice was also reduced compared to the control group. Could be (FIGS. 9 and 10). In addition, looking at the engraftment (graft) rate after sheet transplantation, it can be seen that about 30% remained compared to the 28th and 4th day (Fig. 9).

In addition, the results of echocardiography measuring cardiac function increased the cardiac function compared to the control group, and the fat in the infarcted heart due to differentiation into neovascularization and increased growth factor secretion through stem cell transplantation. The positive effect of the stem cell sheet was confirmed.

Example 4 Cardiomyocyte Mixture Sheet Induced from Mouse Embryonic Stem Cells Using Adipose Stem Cell Support

After recovering the mouse embryonic stem cells, induced cardiac myocytes beating in a 5% CO ₂ incubator at 37 ° C. for 4.5 days using an inducer, and then in an alpha-MEM medium containing FBS, 2-mercaptoenthanol, and L-glutamine. Incubated. When the induction system is induced with pulsating cardiomyocytes, the expression of enhanced green fluorescent protein (eGFP) appears, and thus successfully obtained pulsating cardiomyocytes derived from mouse embryonic stem cells showing eGFP expression.

Next, 5 × 10 ⁶ mouse fat stem cells were seeded in 6 wells plates and FBS and various growth factors were obtained using a MesenCult ™ Proloferation kit (Stem Cell Technology) in a 37 ° C., 5% CO ₂ incubator for 36 hours. After culturing in a baseal mesdium containing, 2 × 10 ⁶ seeded pulsating cardiomyocytes derived from mouse embryonic stem cells were additionally seeded and cultured for 12 hours at 37 ° C., 5%, using the same culture medium. Incubated in a CO ₂ incubator.

To investigate the optimal ratio of pulsatile cardiomyocytes derived from adipocytes and mouse embryonic stem cells as scaffolds, pulsatile cardiomyocytes derived from mouse embryonic stem cells, cells mixed with or without mixed cell sheet formation at various ratios EGFP expression and population size were compared.

As a result, the ratio of pulsating cardiomyocytes derived from adipose stem cells and mouse embryonic stem cells as a support was found to be an optimal ratio of 2: 3 to form a mixed sheet (FIG. 11).

In addition, as a result of observing mixed cells of pulsating cardiomyocytes using adipose stem cells as a support by optical microscope, it was confirmed that adipose stem cells as a support and cardiomyocytes derived from mouse embryonic stem cells (in dashed lines) coexist and behave. (FIG. 12).

Subsequently, a mixed sheet of pulsating cardiomyocytes derived from mouse embryonic stem cells using adipose stem cell support was peeled off with a scraper to obtain a mixed sheet of pulsating cardiomyocytes derived from mouse embryonic stem cells using adipose stem cell support. (FIG. 13). The pulsating cardiomyocytes could be identified even after peeling of the mixed sheet, and no separation was observed between the pulsating cardiomyocytes and adipose stem cells.

Example 5 Cardiomyocyte Mixture Sheet of Mouse Neonate Using Adipose Stem Cell Support

Cardiac tissues of mice were recovered immediately after delivery (within 48 hours of delivery), and the recovered cardiac tissues were finely pulverized and treated with collagenase for 30 minutes to obtain mouse neonatal cardiomyocytes. Mouse neonatal cardiomyocytes were cultured in two passages, and then stained using antibodies such as cTnT, cTnI, α-cardiac actin, MHC, etc. to confirm the behaviour.

As a result, the beating properties of the acquired mouse neonatal cardiomyocytes were identified.

Next, 5 × 10 ⁶ mouse fat stem cells were seeded on 6 wells plates, and then FBS and various growth factors were obtained using MesenCultTM Proloferation kit (Stem Cell Technology) in 37 ° C., 5% CO ₂ incubator for 36 hours. After culturing in a baseal mesdium containing, and further 2 × 10 ⁶ obtained cardiac muscle cells of the newborn cells were cultured in 37 ℃, 5% CO ₂ incubator for 48 hours using the same culture medium cultured adipose stem cells It was.

As a result, the mixed cells of neonatal cardiomyocytes using adipose stem cell scaffolds were peeled off with a scraper to obtain a neonatal cardiomyocytes mixed sheet using adipose stem cell scaffolds.

In addition, as a result of microscopic observation of the neonatal cardiomyocyte mixed sheet using adipose stem cell support, it was confirmed that the neonatal cardiac myocytes were beating among adipose stem cells used as a support, and immunization using cTnT antibody was performed. The myocardial cell mixed sheets of neonates using adipose stem cell scaffolds were verified by fluorescence staining (FIGS. 14 and 15).

In the preparation of a mixture sheet of adipose stem cells and cardiomyocytes, a culture of adipose stem cells with a baseal mesdium containing FBS and various growth factors using a MesenCult ™ Proloferation kit (Stem Cell Technology) and seeding additional cardiomyocytes The culture was incubated for 2 days. When both cardiomyocytes derived from mouse embryonic stem cells and cardiac muscle cells of mouse newborns were cultured using the existing mouse adipocyte culture medium, no changes in cardiomyocytes were observed.

Example 6 Various Myocyte Mixture Sheet Using Adipose Stem Cell Support

In this embodiment, a myocyte mixture sheet derived from various species and derivatives was prepared using adipose stem cells as a support. Adipose stem cells were used as supporters to form the respective mixed sheets using rat-derived BDIX cardiomyocytes, human-derived coronary smooth muscle cells, or mouse-derived C3H myoblasts.

As a result, as shown in FIG. 16, the rat-derived BDIX cardiomyocytes, the human-derived coronary smooth muscle cells, and the mouse-derived C3H myoblasts formed a mixed sheet with the adipocytes.

Mouse adipose stem cells were then subjected to CFDA (Carboxyfluorescein diacetate succinimidyl ester; green) staining to differentiate cells, seeded 5 × 10 ⁶ in 6 wells plates, and then 37 ° C., 5% CO for 36 hours. Incubated in ₂ incubators. Next, rat-derived BDIX cardiomyocytes, human-derived coronary smooth muscle cells and mouse derived stained with DiI (1,1'-Dioctadecyl-3,3,3 ', 3'-Tetramethylindocarbocyanine Perchlorate; red) Each of the C3H myoblasts was seeded by 2 × 10 ⁶ cells to the cultured adipose stem cells, and cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours using the same culture medium in which the adipose stem cells were cultured. Then, each obtained mixed sheet was subjected to freezing sections to stain the nucleus with DAPI (blue).

The formation of a mixture of fat stem cells and BDIX cardiomyocytes derived from rats as a support was observed by 100 and 400-fold fluorescence microscopy.As a result, fat stem cells (green) and rat-derived BDIX cardiomyocytes (red) as support were observed. Coexistence was confirmed, and it was confirmed that it was a mixed sheet composed of multiple layers (FIG. 17A. The mixed sheet using fat stem cells as a support and BDIX cardiomyocytes derived from rats (rats) was firm without separation between different cells.

In addition, the formation of a mixture sheet of adipose stem cells and human-derived coronary smooth muscle cells using a 200 and 400-fold fluorescence microscope showed that adipose stem cells (green) and human-derived coronary smooth muscle cells (red) coexist as a support. It could be confirmed (Fig. 17B). In addition, the formation of a mixture of fat stem cells and mouse-derived C3H myoblasts as a support was observed by 200 and 400-fold fluorescence microscopy, and it was also confirmed that adipose stem cells (green) and mouse-derived C3H myoblasts (red) coexist as a support. Could be (FIG. 17C).

In the preparation of a mixed sheet of adipose stem cells and various myocytes, cultivation of adipose stem cells with a baseal mesdium containing FBS and various growth factors using the MesenCult ™ Proloferation kit (Stem Cell Technology), followed by additional BDIX-derived BDIX Cardiac myoblasts, human-derived coronary artery smooth muscle cells or mouse-derived C3H myoblasts were seeded and cultured in the same culture for 2 days. When rat-derived BDIX cardiomyocytes, human-derived coronary smooth muscle cells, and mouse-derived C3H myoblasts were all cultured using conventional mouse adipose stem cell culture, no change of cells was observed.

Therefore, the stem cells as a support was unable to confirm the sheet formation using mouse-derived embryos or bone marrow stem cells, but when mixed sheets prepared with adipose stem cells using various muscle cells regardless of species, all the sheets were firmly mixed without separation. It could be confirmed that is formed.

The mixed sheets of myocytes using the adipose stem cell support according to the present invention can be easily manufactured at low cost in a short time, and can be transplanted into the living body in a stable and safe manner, which makes it difficult to produce a sheet alone, and thus, clinical application such as local regenerative therapy. In addition, it is industrially useful.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Method for producing a mixed sheet of fat stem cell support and graft myocytes comprising the following steps: (a) culturing the adipose stem cells, and further seeding the seed muscle cells for transplantation into the adipose stem cells and culturing them together; And (b) peeling and recovering the mixed sheet of the fat stem cells cultured in step (a) and the myocytes for transplantation. The method of claim 1, wherein the transplanted myocytes are cardiomyocytes, cardiomyocytes, smooth muscle cells or myoblasts. The method of claim 2, wherein the cardiomyocytes are pulsating cells. The method of claim 1, wherein the fat stem cells of step (a) are cultured by seeding (seeding) at a density of 0.5 ~ 1.0 × 10 ⁶ cells / cm ² . The method of claim 1, wherein the additional seeding of the step (a) is characterized in that the ratio of the number of cells in the seeding of the adipose stem cells and the transplanted myocytes is 1: 1-2. The method of claim 1, wherein the peeling of the mixed cell sheet of step (b) is characterized in that using a scraper. The method of claim 1, wherein the recovered mixed cell sheet of step (b) is 60% or more of the total culture dish area. A mixed sheet of adipose stem cell support prepared by the method of any one of claims 1 to 7 and a myocyte for transplantation.

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