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WO2017018629A1 - Feuille de mélange de myocytes utilisant un support de cellules souches et son procédé de fabrication - Google Patents

Feuille de mélange de myocytes utilisant un support de cellules souches et son procédé de fabrication Download PDF

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Publication number
WO2017018629A1
WO2017018629A1 PCT/KR2016/002428 KR2016002428W WO2017018629A1 WO 2017018629 A1 WO2017018629 A1 WO 2017018629A1 KR 2016002428 W KR2016002428 W KR 2016002428W WO 2017018629 A1 WO2017018629 A1 WO 2017018629A1
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cells
stem cells
sheet
adipose
mixed
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Korean (ko)
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임도선
주형준
김종호
서하림
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

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  • 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.
  • 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).
  • transplant stem cells for regeneration of damaged organs and tissues
  • 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.
  • transplanted stem cells have low engraftment and most have been reported to be wash-out.
  • 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.
  • 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.
  • a culture dish method is proposed in which a surface is smeared with a temperature-responsive polymer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m).
  • 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 ⁇ m).
  • 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.
  • CFDA Carboxyfluorescein diacetate succinimidyl ester
  • 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.
  • FIG. 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 ⁇ m).
  • FIG. 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 ⁇ m).
  • 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 ⁇ m) ).
  • 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).
  • 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.
  • 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).
  • the present invention relates to a mixed sheet of adipose stem cell support prepared by the above method and a myocyte for transplantation.
  • the support is preferably adipose tissue-derived stem cells, but is not limited thereto.
  • 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.
  • 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.
  • 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.
  • adipose stem cells 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.
  • 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.
  • 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.
  • the optimum amount of cells per area for seeding for preparing adipose stem cell sheet as a support was derived (FIG. 1).
  • the growth factors Adiponectin, MCP-1, SDF-1, VEGFc, HGF, IGF
  • TGF ⁇ 1 is secreted more from high-density fat stem cells than low-density fat stem cells.
  • TGF ⁇ 1 is secreted more from high-density fat stem cells than low-density fat stem cells.
  • Collagen I, Laminin- ⁇ 4, 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).
  • stem cells 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • the optimal ratio of forming a beating cardiomyocyte mixed sheet derived from mouse embryonic stem cells into additional mixed cells was derived (FIG. 11).
  • the stem cells and cardiomyocytes are preferably seeded at a ratio of 2: 3, but are not limited thereto.
  • 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.
  • 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).
  • the peeling of the mixed cell sheet is most preferably one using a scraper, but is not necessarily limited thereto.
  • a scraper due to the nature of the general dish, there is a problem of dissociation of all the cells using proteolytic enzymes.
  • 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
  • a cardiac specific marker (FIGS. 14 and 15).
  • 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), ⁇ -cardiac actin, ⁇ -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.
  • a sheet using adipose stem cells as a support rather than suspended fat stem cells was obtained for a long time (Fig. 9).
  • 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.
  • 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.
  • 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).
  • a culture dish coated with a temperature-responsive polymer is used 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.
  • 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.
  • the various growth factors of the adipose stem cells present in the living body can remove the immune rejection reaction.
  • 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.
  • 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.
  • 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.
  • the cell delivery success rate and function recovery were increased as compared with the transplantation of the previously reported single stem cells.
  • angiogenesis was increased in the infarct area in the transplantation into the infarcted myocardium, which has a beneficial effect of shortening the treatment time.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 TM 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.
  • the cells were seeded on 6 wells plates at various cell densities.
  • 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). .
  • mice 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.
  • 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).
  • 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.
  • mouse-derived embryonic stem cells are divided into fragments without forming a sheet as a support (FIG. 7).
  • mouse-derived bone marrow stem cells intermittent formation of a multi-layered cell layer was observed. Existed in several fragments (FIG. 8).
  • adipose stem cells can form a sheet as a support by secretion of various growth factors and extracellular matrix.
  • adipose stem cell sheets 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.
  • 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.
  • 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.
  • eGFP enhanced green fluorescent protein
  • mice fat stem cells were seeded in 6 wells plates and FBS and various growth factors were obtained using a MesenCult TM Proloferation kit (Stem Cell Technology) in a 37 ° C., 5% CO 2 incubator for 36 hours.
  • MesenCult TM Proloferation kit Stem Cell Technology
  • 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.
  • pulsatile cardiomyocytes derived from adipocytes and mouse embryonic stem cells were compared.
  • 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).
  • 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.
  • 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.
  • mice 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, ⁇ -cardiac actin, MHC, etc. to confirm the behaviour.
  • mice 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 °C, 5% CO 2 incubator for 48 hours using the same culture medium cultured adipose stem cells It was.
  • 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.
  • a culture of adipose stem cells with a baseal mesdium containing FBS and various growth factors using a MesenCult TM Proloferation kit (Stem Cell Technology) and seeding additional cardiomyocytes The culture was incubated for 2 days.
  • 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.
  • 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.
  • 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.
  • CFDA Carboxyfluorescein diacetate succinimidyl ester
  • 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).
  • adipose stem cells 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 TM 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.
  • 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.

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Abstract

La présente invention concerne une feuille de mélange de myocytes utilisant un support de cellules souches et son procédé de fabrication, et, plus précisément, un procédé pour fabriquer une feuille de mélange de myocytes, pouvant être implantée in vivo, par utilisation de cellules souches provenant de tissu adipeux comme support. Selon la présente invention, la feuille de mélange de myocytes utilisant un support de cellules souches provenant de tissu adipeux peut être fabriquée facilement en peu de temps et à faible coût, et permet l'implantation in vivo stable et sans danger de cellules, qui sont difficiles à fabriquer individuellement en feuilles, étant ainsi utile à des fins industrielles en plus de l'application clinique, telle qu'une thérapie régénérative locale.
PCT/KR2016/002428 2015-07-29 2016-03-11 Feuille de mélange de myocytes utilisant un support de cellules souches et son procédé de fabrication Ceased WO2017018629A1 (fr)

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KR101586839B1 (ko) * 2015-07-29 2016-01-19 고려대학교 산학협력단 줄기세포 지지체를 이용한 근세포 혼합시트 및 그의 제조방법
US9783777B1 (en) 2016-10-18 2017-10-10 King Saud University Method of making three-dimensional, leaf-based scaffold for three-dimensional cell culture
KR102200341B1 (ko) * 2018-12-21 2021-01-08 고려대학교 산학협력단 지방줄기세포 시트를 함유하는 심장조직 모사 심장이식용 시트 및 이의 제조방법
KR102460408B1 (ko) * 2020-07-23 2022-10-28 포항공과대학교 산학협력단 줄기세포 시트를 제조하는 방법

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008307205A (ja) * 2007-06-14 2008-12-25 Osaka Univ 心筋芽細胞を含むシート
JP2010017128A (ja) * 2008-07-10 2010-01-28 Toyota Central R&D Labs Inc 培養細胞のハンドリング体、その製造方法及びその利用
JP2012235731A (ja) * 2011-05-11 2012-12-06 Tokyo Women's Medical College 骨格筋芽細胞の心筋細胞への分化方法、心筋移植材の製造方法および心筋移植材
JP2014155494A (ja) * 2005-02-28 2014-08-28 Cellseed Inc 培養細胞シート、製造方法及びその利用方法
KR101518651B1 (ko) * 2013-12-31 2015-05-07 동국대학교 산학협력단 골 및 신생혈관 형성능이 향상된 줄기세포 공배양 방법
KR101586839B1 (ko) * 2015-07-29 2016-01-19 고려대학교 산학협력단 줄기세포 지지체를 이용한 근세포 혼합시트 및 그의 제조방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014155494A (ja) * 2005-02-28 2014-08-28 Cellseed Inc 培養細胞シート、製造方法及びその利用方法
JP2008307205A (ja) * 2007-06-14 2008-12-25 Osaka Univ 心筋芽細胞を含むシート
JP2010017128A (ja) * 2008-07-10 2010-01-28 Toyota Central R&D Labs Inc 培養細胞のハンドリング体、その製造方法及びその利用
JP2012235731A (ja) * 2011-05-11 2012-12-06 Tokyo Women's Medical College 骨格筋芽細胞の心筋細胞への分化方法、心筋移植材の製造方法および心筋移植材
KR101518651B1 (ko) * 2013-12-31 2015-05-07 동국대학교 산학협력단 골 및 신생혈관 형성능이 향상된 줄기세포 공배양 방법
KR101586839B1 (ko) * 2015-07-29 2016-01-19 고려대학교 산학협력단 줄기세포 지지체를 이용한 근세포 혼합시트 및 그의 제조방법

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