KR102037823B1 - Method for mass preparing of vasculogenic factors using stem cell aggregate, and use thereof - Google Patents

Abstract

The present invention relates to a method for obtaining large quantities of angiogenic factors using stem cell aggregates, and to a composition for preventing and treating ischemic diseases, including the culture medium of the stem cell aggregates. In the case of constructing the aggregates of stem cells through suspension culture in a basal medium that does not contain bovine serum, a poorly stable buffer solution, or an antibiotic component that may increase resistance, the stem cell aggregate culture medium. By obtaining a large amount of angiogenic factors from the transplantation to ischemic tissues and confirming that the engraftment rate in the tissues is significantly increased compared to single cell transplantation, the present invention can be effectively used for the prevention or treatment of ischemic diseases. Said.

Description

Method for mass preparing of vasculogenic factors using stem cell aggregate, and use}

The present invention relates to a method for obtaining large quantities of angiogenic factors using stem cell aggregates and to a method for treating ischemic diseases using the same.

Ischemia is a partial blood deficiency that results in cell damage due to narrowing or contracting of blood vessels or inadequate normal blood vessel formation, resulting in disruption of blood supply. Ischemic diseases are commonly referred to as diseases caused by these ischemic symptoms and include ischemic stroke, ischemic colitis and cardiovascular disease. Ischemic stroke or ischemic heart disease, a disease represented by ischemic disease, causes damage to cerebral nerve cells and heart cells due to ischemia, in which the cerebral artery or coronary artery is closed by blood clots or atherosclerosis, resulting in a decrease in blood flow below the threshold. Apoptosis occurs, leading to brain infarction and myocardial infarction. In addition, interruption of blood supply due to ischemia causes various ischemic diseases such as ischemic heart failure, ischemic enteritis, eye disease, and ischemic leg disease.

Treatment of such ischemic disease is a coronary angioplasty that uses a drug therapy or a stent to expand narrow blood vessels. In the case of the heart, arterial bypass surgery may be performed. However, these treatments do not serve as appropriate treatments if the vessels are too hard, if all the vessels are available for transplantation, or if the recurrence continues due to restenosis. . Therefore, in order to overcome the limitations of the existing treatment methods, a method for improving the neovascularization of ischemic tissues by using a vasculogenic factor to alleviate ischemia is gaining attention as a new treatment strategy.

Therapeutic application of angiogenic factors was first published by Folkman et al. (N. Engl. J. Med. 285, 1182-1186 (1971)). In further studies, animal models of myocardial and lower limb ischemia using recombinant angiogenesis factors such as fibroblast growth factor (FGF) family, endothelial cell growth factor (EGF) and vascular endothelial growth factor (VEGF), etc. It has been confirmed that it is possible to promote and / or enhance the development of the lateral hematogenous pathway in (Circulation 90, II-228-II-234 (1994)). In addition, hepatic parenchymal cell proliferation factor (HGF) acts as an endothelial specific proliferation factor in the same way as VEGF (J. Hypertens. 14, 1067-1072 (1996)) and is useful for the treatment of obstructive atherosclerosis (ASO). Not only can be used (Circulation, Vol. 100, No. 18, No. 1702 (1999), Japanese Circulation Journal Vol. 64, Suppl. I, p478, No. P079 (2000)), but also in myocardial infarction It is well known that HGF gene also works effectively against ischemic recirculation injury (Circulation, Vol. 96, No. 8, No. 3459 (1997), Ann. Thorac. Surg., 67, p1726-1731 ( 1999), Gene Therapy, 7: 417-427 (2000)).

Stem cells are cells capable of differentiating into various cells constituting biological tissues, which collectively refer to undifferentiated cells obtained from each tissue of embryo, fetus and adult. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the cells in which cell division is stopped due to differentiation, proliferation is possible due to cell division (self-renewal). (Proliferation; expansion) is characterized by being able to differentiate into other cells by different environments or different differentiation stimulation, which is characterized by having plasticity (differentiation).

Stem cells are largely derived from embryos and have pluripotency embryonic stem cells (ES cells) with the potential to differentiate into all cells, and multipotency from each tissue. ) Adult stem cells (adult stem cells). The inner cell mass of the blastocyte, the earliest embryonic development, is the part that will form the fetus in the future. Embryonic stem cells formed from the intracellular mass can theoretically differentiate into cells of all tissues that make up an individual. It is a stem cell with potential. That is, embryonic stem cells are undifferentiated cells capable of unlimited proliferation, can differentiate into all cells, and unlike germ stem cells, can make germ cells and can be inherited to the next generation.

Human embryonic stem cells are prepared by isolating and culturing only inner cell mass when forming human blastocysts. Currently, human embryonic stem cells made worldwide are obtained from frozen embryos remaining after infertility. This cell is characterized by its ability to differentiate into all cells, which can be differentiated into any tissue cell, and can also be cultured in immortal and undifferentiated state and also in the preparation of germ cells. And possibly inherited to the next generation (Thomson et al., Science, 282: 1145-1147, 1998; Reubinoff et al., Nat. Biotechnol., 18: 399-404, 2000).

However, various attempts to use human embryonic stem cells, which are capable of differentiating into various cells, as cell therapeutics have yet to fully control the high walls of cancer formation and immune rejection.

Recently, mesenchymal stem cells (MSCs), which have been reported to have an immunomodulatory function, have been proposed as an alternative to overcome these problems. Mesenchymal stem cells (MSCs) first identified in bone marrow by the Friendenstein group (Exp Hematol, 4 (5): 267-274, 1976) are omnipotent and have great potential in regenerative medicine. The MSCs are produced in vivo from several types of mesenchymal lineages, such as osteocytes, chondrocytes, tendon cells, adipocytes, myocytes, and fibroblasts. ) And the like. In addition, under appropriate media conditions, Hung SC et al., Stem Cells, 20 (6): 522-529, 2002; Sanchez-Ramos J et al., Exp Neurol, 164 (2): 247-256, 2000 ), Cardiomyocytes (Kadivar M et al., Biochem Biophys Res Commun, 340 (2): 639-647, 2006), endothelial cells (Reyes M et al., Blood, 98 (9): 2615-2625, 2001) , And hepatocytes (Kang XQ et al., World J Gastroenterol, 11 (47): 7461-7465, 2005). In addition, bone marrow MSCs express class I MHC antigens, but not class II, or express mutually stimulating molecules indicating that MSCs lack immunogenic activity (Klyushnenkova E et al., J Biomed Sci, 12 (1): 47-57). , 2005). Since MSCs exhibit immunosuppressive activity, they can be used as transplant promoters and inhibitors of fetal grafts and fatal grafts and host diseases (Le Blanc K et al., Lancet, 363 (9419): 1439-1441, 2004; El- Badri NS et al., Exp Hematol, 26 (2): 110-116, 1998), from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, human placenta, etc. (Kassis I et al., Bone Marrow Transplant, 37 (10): 967-976, 2006; Wang HS et al., Stem Cells, 22 (7): 1330-1337, 2004; Fukuchi Y et al. , Stem Cells, 22 (5): 649-658, 2004).

However, stem cells have great potential in cell therapy in that they have the potential of differentiation and can induce differentiation from damaged tissues to the cells that are needed.However, at the current developmental level, the survival rate after transplantation in the body is not high. It is hard to find examples of successful and broad success.

In particular, fat, bone marrow or umbilical cord blood-derived stem cell therapy can be used to treat ischemic diseases, and it has been found that blood vessels can be regenerated, but most of the stem cells transplanted to the ischemic site are killed so that the therapeutic effect of the cell therapy is not great. There has been a problem. Rehman J et al. Also reported that adipose derived stem cells secrete factors related to blood vessel regeneration (Circulation 2004; 109 (10): 1292-8). The survival rate of the transplanted cells transplanted into the ischemic region is extremely low, making it difficult to apply the actual clinical practice.

On the other hand, unlike in vivo, to supply the culture medium for artificially cultivating animal cells in vitro, it is sufficient to satisfy the nutrients close to the conditions of the living body based on body fluids such as plasma or lymph fluid and environmental conditions such as pH, temperature, osmotic pressure, etc. Should give. Therefore, the culture medium composition was determined based on the body fluid, and the determined culture medium was selected and serum was added to the cells at an appropriate ratio, and then used for cell culture. In this way, early in the development of the medium around 1950 Morgan (Morgan) and the like based on the fluid composition to make the M199 medium and cultured the fetal cells (primary chick). Subsequent major developments include Eagle's medium used for culturing human and rat fetal cells, Dulbecco's modified eagles medium used for culturing fetal cells in rats, Ham's F-12 and Morore. RPMI-1640 used for this hamster tumor cell culture, etc. are mentioned.

In cell culture, 5-20% of animal serum is added to the medium according to the nutritional requirements of the cells. The serum contains insulin, which promotes cell growth and function, various hormones of the polypeptide system, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum is also used as a buffer to control the osmotic pressure and pH of the culture, and has a viscous action that can protect the cells from proteolysis inhibition and physical shock, making a culture medium by putting a certain concentration of serum in the underlying medium. .

If culture medium containing animal serum (fetal bovine serum) is used to cultivate human stem cells in vitro, fetal bovine serum remains intracellularly, acting as a strong heterologous antigen, and due to the possibility of human infection of cow disease such as mad cow disease. Human transplantation of cells cultured with fetal serum is prohibited in the United States, and serum replacament is being developed to replace it, but many safety issues still arise in human transplants.

The present invention has been made to solve the above-mentioned problems in the prior art, the animal-derived cow serum, low stability buffer solution, an antibiotic-free base medium that does not contain any antibiotic components that can increase resistance (etc.) Stem cell aggregates formed by suspension culture of stem cells in basal medium secrete a large amount of angiogenesis factors that can enhance neovascularization of ischemic tissues, and when the stem cell aggregates are transplanted into ischemic tissues, By revealing a marked increase in tissue engraftment rate, the present invention is intended to suggest that the method of the present invention can not only obtain a large amount of angiogenic factors, but also can be suitably applied to the treatment of ischemic diseases.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a method for obtaining a mass of angiogenesis factors comprising the following steps:

(a) forming stem cell aggregates by floating culture of stem cells in a basal medium without additives; And

(b) obtaining angiogenic factors from the culture medium of the stem cell aggregates.

In one embodiment of the invention, the stem cells are characterized in that selected from the group consisting of mesenchymal stem cells, embryonic stem cells, embryonic germ cells, embryonic tumor cells and induced pluripotent stem cells.

In another embodiment of the present invention, the angiogenesis factor is characterized in that VEGF A (vascular endothelial growth factor A) or HGF (Hepatocyte growth factor).

In another embodiment of the present invention, the basal medium is Eagle's MEM (Eagle's minimum essensial medium), α-MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM (Dulbecco's modification of Eagle's medium) , Mixture of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A, and MCDB or modified medium thereof.

In another embodiment of the invention, the method comprises the steps of (a)

(Iii) culturing the stem cells in a flat plate;

(Ii) separating single cells from the plated stem cells; And

(Iii) forming a cell drop comprising said isolated single cell.

In another embodiment of the present invention, the plate culture is characterized in that the culture in a nutrient medium (rich medium).

In another embodiment of the present invention, the nutrient medium is characterized in that it comprises a serum (serum) or serum replacement (SR).

In another embodiment of the present invention, the cell drop is characterized in that it comprises 500 to 1500 single cells in 10 ~ 100 μl of no addition basal medium.

In another embodiment of the present invention, the cell drop is characterized in that it contains 750 to 1250 single cells in 20 μl of no addition basal medium.

In another embodiment of the invention, the cell drop is characterized in that it contains 1000 single cells in 20 μl of no addition basal medium.

In another embodiment of the present invention, in the suspension culture of the stem cells in the non-added basal medium, the culture time is characterized in that the culture for 24 hours or more.

In another embodiment of the present invention, in the suspension culture of the stem cells in the non-basic culture medium, the culture time is characterized in that the culture for 24 to 72 hours.

In addition, the present invention provides a composition for preventing or treating ischemic disease, including a culture solution of stem cell aggregates having an angiogenic factor as an active ingredient.

In one embodiment of the present invention, the ischemic disease is characterized in that ischemic brain disease or ischemic heart disease.

In another embodiment of the present invention, the ischemic heart disease is selected from the group consisting of coronary artery disease, congestive heart failure, chronic heart failure, cardiomyopathy and myocardial infarction.

In another embodiment, the cardiomyopathy is characterized in that it is selected from the group consisting of ischemic cardiomyopathy, dilated cardiomyopathy, non-large cardiomyopathy, idiopathic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy and valve cardiomyopathy.

According to the method of the present invention, by forming stem cells into spherical aggregates, the angiogenic factors can be excessively secreted into the cell culture medium, and the cell culture medium contains animal-derived components, buffer solutions, antibiotics, indicators, and the like, which have been used in a conventional cell culture medium. Not only is it safe to be clinically applicable, but the cell culture medium contains angiogenesis factors applicable to the treatment of ischemic vascular diseases and stem cell aggregates with increased engraftment rate in tissues. It can be usefully applied to treatment.

FIG. 1 shows the results of observing aggregate formation with an optical microscope (NIKON) after preparing cell drops, culturing at 1, 6, 24, 48, and 72 time periods (magnification: top x100):
a: cell drop making and forms thereof; And
b: As a result of observing the aggregates formed under a microscope.
FIG. 2 shows the cell culture supernatants excluding cells at 1, 6, 24, 48, and 72 time periods from cell drop prepared by culturing in each medium condition, and secreted human vascular endothelial growth factor A (Vascular endothelial) secreted in the supernatant. Growth factor A, VEGF A) and the amount of human hepatocyte growth factor (HGF) were confirmed by ELISA:
a: cell culture cultured cell drop in nutrient medium (hEBM) containing bovine serum and antibiotics; And
b: Cell culture medium in which cell drop was cultured in no addition basal medium (α-MEM).
3 is a tissue photograph of a single cell or a stem cell aggregate of the present invention implanted into cardiac tissue of an ischemic cardiovascular rat model and observed 14 days later (magnification: top x200, bottom x800):
a: single cell transplant tissue; And
b. Stem cell aggregate transplantation tissue of the present invention.

The present inventors conducted a study to develop a method for obtaining a large amount of angiogenic factors secreted from stem cells, and to develop a stem cell therapeutic agent having an increased engraftment rate and an excellent ischemic treatment effect when transplanted into a living body. In the case of constructing the aggregates of stem cells through suspension culture in a basal medium containing no animal-derived bovine serum, a poorly stable buffer solution, or an antibiotic component that may increase resistance, By obtaining a large amount of angiogenic factors from cell aggregate cultures and transplanting them into ischemic tissues, it was found that the engraftment rate in tissues was significantly increased as compared to single cell transplantation, so that the method of the present invention was suitably used for the treatment of ischemic diseases. The present invention has been completed based on this finding.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for obtaining a bulk of angiogenic factors comprising the following steps:

(a) forming stem cell aggregates by floating culture of stem cells in a basal medium without additives; And

(b) obtaining angiogenic factors from the culture medium of the stem cell aggregates.

Recently, as the research on cell therapy using stem cells or other autologous or taga cells for the treatment of refractory and degenerative diseases has been actively conducted, signal substances that exist in vivo and are responsible for the intercellular signaling mechanism have been discovered. Research is also being conducted on the effects. Specifically, studies on the structure and synthesis of neoplastic factors involved in tissue regeneration have been conducted, but most of the neoplastic factors have a three-dimensional complex protein structure, which causes various problems in chemical synthesis. The cost is also very expensive. Therefore, the present inventors have developed a method for obtaining angiogenic factors of active form at a low price while maintaining in vivo activity to efficiently treat ischemic disease.

The development of new blood vessels or angiogenesis is initiated with activation of endothelial cells in the parental blood vessel, but not only stimulates angiogenesis in vivo, but also mitogenically to endothelial cells in vitro. The growth factor, which is shown to act), is referred to herein as an 'angiogenic factor'.

The 'angiogenic factors' are fibroblast growth factor (FGF) family (Science 257, 1401-1403 (1992), Nature 362, 844-846 (1993)), endothelial growth factor (EGF) (J. Surg. Res. 54, 575-583 (1993)), hepatocyte growth factor (HGF) (J. Hypertens. 14, 1067-1072 (1996)) or vascular endothelial growth factor (VEGF).

Hepatocyte growth factor (HGF) is a polymer protein with a molecular weight of 100,000, known as a growth factor for epithelial cells, vascular endothelial cells, cardiomyocytes, and chondrocytes. It turns out. HGF is considered as an essential factor in the formation of tissue organs and regeneration of damaged tissues by inhibiting the expression of TGF-β, which causes apoptosis in chronic injured tissues, and in cardiomyopathy, diabetes mellitus, arteriosclerosis, etc. It is effective as a therapeutic agent for intractable diseases.

Vascular endothelial growth factor (VEGF) (Ferrara, N. et al, Biochem Biophys Res Commun, 161: (2) 851-8 (1989)) is a vascular permeable element (Senger, DR et al., Science, 219: (4587) (983-5 (1983)), a homodimer, has a molecular weight of 34-42 kDa and is a heparin-bound glycoprotein, which has angiogenic elements that promote endothelial mitosis and vascular permeability. Can be improved. VEGF is expressed in a primary structure known as a restricted base sequence, which has homology with the A and B chains of platelet-derived growth factor (PDGF).

According to a preferred embodiment of the present invention, the 'angiogenic factor' is VEGF A (vascular endothelial growth factor A) or HGF (Hepatocyte growth factor).

As used herein, the term 'stem cell' refers to cells capable of differentiating into cells constituting self-renewal and tissues, and to differentiate into specific cells by a specific differentiation stimulus (environment). Has the ability to Stem cells, unlike differentiated cells that cease cell division, are able to self-renewal by cell division, and differentiate into specific cells when differentiation stimulation is applied, and this differentiation is different in different environments or in different ways. It is characterized by the plasticity of differentiation, which can be differentiated into various cells by differentiation stimulation.

Stem cells to which the present invention is applied is not limited, and cells having the characteristics of stem cells, infinite proliferation and differentiation into specific cells are cells to which the present invention can be applied. Stem cells to which the present invention is applied are largely divided into two types: pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG). Embryonic stem cells are derived from the internal cell mass (ICM) of the blastocyst, and embryonic germ cells are derived from primordial germ cells of the 5-10 week old gonadal ridge. Pluripotent stem cells, on the other hand, are found in embryonic, fetal and adult tissues, including adult stem cells. Pluripotent stem cells proliferate indefinitely in vitro and have the ability to differentiate into a variety of cells derived from all three embryonic layers (ectoderm, mesoderm and endoderm).

According to a preferred embodiment of the present invention, the stem cells include mesenchymal stem cells, embryonic stem cells, embryonic germ cells, embryonic tumor cells, or induced pluripotent stem cells, preferably mesenchymal stem cells, but is not limited thereto. It doesn't happen. The term 'induced pluripotent stem cells' is one of pluripotent stem cells artificially derived by inserting certain genes from non-pluripotent cells (eg, somatic cells). Induced pluripotent pluripotent cells are pluripotent stem cells in terms of stem cell gene and protein expression, chromosome methylation, doubling time, embryoid body formation, teratoma formation, viable chimera formation, hybridization and differentiation. For example, embryonic stem cells).

As used herein, the term 'basal medium' is a universal medium used for culturing animal cells, and means a medium containing no animal-derived serum and buffers, antibiotics and indicators. do.

For example, the medium may be Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971) ), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp. Bio.Med., 73: 1) 1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965) ), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), mixtures of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA , et al., Proc. Soc. Exp. Biol. Med. 100: 115 (1959)) and the MCDB series (Ham, RG et al., In Vitro 14:11 (1978)), and modified media thereof. . Detailed description of the medium can be found in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York, which is incorporated herein by reference. Preferably, the medium may be an additive-free α-MEM medium containing no additional ingredients, or any medium conventionally used for culturing animal cells containing no additional ingredients.

It is well known that stem cells can differentiate into different cell types. For example, the stem cells are induced to differentiate into hematopoietic cells, nerve cells, beta cells, heart cells, muscle cells, hepatocytes, chondrocytes, epithelial cells, urinary tract cells and like cells under conditions for cell differentiation. do. Media conditions and methods for the differentiation of stem cells are described in Palacios, et al., PNAS. USA, 92: 7530-7537 (1995), Pedersen, J. Reprod. Fertil. Dev., 6; 543-552 (1994), and Bain et al., Dev. Biol, 168: 342-357 (1995).

As used herein, the term "stem cell aggregate" refers to a spherical cell population in which cells in a stage prior to differentiation into specific cells are formed as a collection of stem cells. It is already known in the art that aggregates may be formed after a predetermined time by floating culture of stem cells in serum-free base medium without adding serum, but aggregates are formed through suspension culture in a non-added base medium, It can be maintained for more than 24 hours as the first revealed in the present invention, therefore, the stem cell aggregate of the present invention is maintained in a suspended culture in a free medium. It is also known in the art that the center of cell aggregates is in a hypoxia state (Cell culture as spheroids: an approach to multicellular resistance. Anticancer Res. 1998 Nov-Dec; 18 (6A): 4147-58 ).

In the present specification, the term 'floating culture' refers to culturing so that cells can be proliferated in a suspended state by using a low adherent petri dish in a concept opposite to a plate culture that induces cell proliferation in an attached form.

The method of the present invention prior to step (a)

(Iii) culturing the stem cells in a flat plate;

(Ii) separating single cells from the plated stem cells; And

(Iii) forming a cell drop comprising said isolated single cell,

After the step (b), it may further include the step of purifying the culture of the cell aggregate, but is not limited thereto.

The plate culture is preferably cultivated in a nutrient medium (rich medium), but is not limited thereto, the term 'nutrition medium' herein is an animal-derived serum and buffer solution in a universal medium used for animal cell culture Means a medium containing additional components such as antibiotics or indicators.

The 'nutrient medium' preferably includes serum (serum) or SR (serum replacement), and may further include any ingredient that can help in vitro proliferation of animal cells.

The cell drop may comprise 500 to 1500 single cells in 10-100 μl free basal medium, and may comprise 750 to 1250 single cells in 20 μl free basal medium, 20 μl free It is most preferred to include 1000 single cells in the basal medium, but is not limited thereto.

In the suspension culture of stem cells in the non-additive basic medium, the culture time is preferably cultured for 24 hours or more, more preferably for 24 hours to 72 hours, but is not limited thereto.

In addition, the present invention provides a composition for preventing or treating ischemic disease, including a culture solution of stem cell aggregates having an angiogenic factor as an active ingredient.

The culture medium of the stem cell aggregates is a cell culture medium obtained by floating culture of stem cell aggregates in a basic medium without additives, and means a culture medium containing cultured stem cell aggregates. Thus, the cell aggregates themselves are transplanted to provide vascular tissue. Not only can the regeneration and recovery be used to treat ischemic diseases, but also contains large amounts of angiogenic factors secreted from cell aggregates, which can be applied more effectively to the treatment of ischemic diseases.

In addition, angiogenic factors secreted from cell aggregates can be collected and applied to the treatment of ischemic diseases, in which case it is safer without the possibility of teratoma or cancer, which consumes less cellular resources and risks stem cell transplantation. It can be a treatment.

The composition for preventing or treating ischemic disease of the present invention refers to a composition comprising a culture solution of stem cell aggregates prepared by the method of the present invention so that the composition can be engrafted by implantation or implantation into a living animal individual, and the culture solution is a stem cell. Aggregates and angiogenic factors secreted therefrom.

The ischemic disease includes, but is not limited to, ischemic cerebrovascular disease or ischemic heart disease, and the ischemic cerebrovascular disease includes, but is not limited to, stroke and cerebrovascular dementia.

In addition, the ischemic heart disease includes, but is not limited to, coronary artery disease, congestive heart failure, chronic heart failure, cardiomyopathy and myocardial infarction, the cardiomyopathy is congenital, valve disease, high blood pressure, coronary artery disease, pericardial disease, etc. Refers to a group of diseases in which abnormalities occur in the heart muscle, such as shortness of breath, chest pain, palpitations, and the like, wherein the cardiomyopathy ischemic cardiomyopathy, dilated cardiomyopathy, non-large cardiomyopathy, idiopathic cardiomyopathy, primary cardiomyopathy, and secondary cardiomyopathy. And valve cardiomyopathy.

The composition for preventing or treating ischemic disease according to the present invention is administered or transplanted into a tissue of a living animal subject, and stem cell aggregates contained in the composition are engrafted into tissue in the subject, or angiogenesis factors contained in the culture medium are neovascular vessels. Ischemic diseases can be treated by improving formation, or by a combinational effect thereof.

The composition of the present invention may include a pharmaceutically acceptable conventional carrier in addition to the active ingredient, and in the case of injection, there may be a preservative, analgesic, solubilizer, or stabilizer, and in the case of topical administration, base, excipient, Lubricants or preservatives;

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oil or aqueous media, and may further comprise dispersants or stabilizers.

The composition of the present invention can be administered parenterally, can be administered intravenously, subcutaneously, intraperitoneally, or topically, and can be applied to tissues in which ischemia is induced to induce apoptosis, and preferably, intracardiac injection (intracardiac). in the form of an injection). Compositions for parenteral administration (e.g. injections) of the compositions according to the invention are infused and dispersed in a pharmaceutically acceptable carrier, e.g., sterile purified water, a buffer of about pH 7, or physiological saline. And, if necessary, include conventional additives such as preservatives, stabilizers and the like.

In addition, the amount of the stem cell aggregate culture solution to be injected in the present invention is not significantly limited, the stem cell aggregate contained in the culture medium may include 10 to 100% compared to the total culture, preferably 20 to 70%, Most preferably 40%, but It is not limited. The dose and stem cell aggregate content of the culture medium may be prescribed in various ways by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response sensitivity. Can be.

As used herein, the term 'prevention' means any action that inhibits or delays ischemic disease by administration of a composition of the present invention.

The term 'treatment' as used herein refers to 1) prevention of the occurrence of a disease or disorder in an animal, preferably a mammal, more preferably a human, which has not yet been diagnosed as having an ischemic disease, 2 A) inhibiting ischemic disease, i.e., inhibiting development, and 3) alleviating ischemic disease. Specifically, when the composition is injected or implanted into a damaged tissue of a subject in need of treatment for an ischemic disease in a therapeutically effective amount, the stem cells of the peripheral central nervous system or peripheral nervous system are differentiated into cells of the corresponding tissue. It can restore tissue function and treat ischemic diseases. Meanwhile, the administration subject may be a mammal including a human.

Terms not otherwise defined herein have the meanings commonly used in the art.

When the composition of the present invention is used for the prevention or treatment of ischemic disease, the composition of the present invention may be used as a monotherapy, but may also be used in combination with other conventional chemotherapy or physical surgery therapy, If performed, the ischemic disease can be treated more effectively.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  1. Formation of Stem Cell Aggregates

1-1. Plate Culture of Stem Cells

To obtain stem cell aggregates of the present invention, bone marrow-derived mesenchymal stem cells (Mesenchymal Stem Cells, MSC) purchased from Lonza were used.

Mesenchymal stem cells (MSCs), often called mesenchymal stromal cells, are present in the bone marrow (BM) but respond to migration signals to maintain connective tissue homeostasis. Is released into the circulation. Recently, mesenchymal stem cells have received widespread attention in the field of cellular and immunotherapy because of their versatility including tissue regeneration, hematopoietic support and immunomodulation (Minguell JJ et al., Exp Biol Med, 226: 507-520 (2001); Caplan AI et al., Trend Mol Med, 7: 259-264 (2001); Tocci A et al., Hematol J., 4: 92-96 (2003)) .

The purchased mesenchymal stem cells were added with 10% fetal bovine serum (GIBCO), 50 IU / ml penicillin and 50 mg / ml streptomycin (Invitrogen) to α-MEM (alpha Minimun Essential Medium, Invitrogen) medium, which is a basal medium. Were plated using α-MEM nutrient medium.

1-2. Isolation of Single Cells from Plated Stem Cells

When the mesenchymal stem cells plated in Example 1-1 filled 80-90% of the culture dish, the nutrient medium was removed and washed with DPBS (Dulbecco's Phosphate Buffered Saline (without calcium and magnesium)). After treatment with 0.25% trypsin / EDTA (GIBCO) for single cell separation, the cells were allowed to stand for 30 seconds in a CO ₂ incubator at 37 ° C. and α containing 10% fetal bovine serum to inactivate 0.25% trypsin / EDTA. After treatment with -MEM culture medium, the cells were precipitated by centrifugation at 1200 rpm for 5 minutes. After centrifugation, the supernatant was removed, and then, the cells not subtracted were added with turbidity, α-MEM medium, which was a non-additive basic medium containing no other additives, and the cells were again precipitated at 1200 rpm for 5 minutes using a centrifuge. The procedure was repeated 1-2 times to completely remove the remaining fetal bovine serum remaining in the cells. Through this, the stem cells adhered to the plate and plated were separated into single cells.

1-3. A cell drop containing a single cell ( drop Formation of

Cells precipitated by centrifugation in Example 1-2 were added and turbidized by addition of α-MEM medium, which is a no addition basal medium, to prepare a cell drop containing 1000 cells / 20 μl per drop.

Comparative controls were 20% KOSR (Knock out serum replacement, Invitrogen), 0.1 mmol / L, β-mercaptoethanol (Sigma), 1% ratio in human Embryonic body medium (hEBM) DMEM / F-12 (Invitrogen) medium. Cell drop containing 1000 cells / 20 μl per drop was prepared using hEBM nutrient medium containing essential amino acid (Invitrogen), 50 IU / ml penicillin and 50 mg / ml streptomycin (Invitrogen).

This results in a drop of cells containing single cells in no addition basal medium (α-MEM) that does not contain serum and other nutrients, and single cells in nutrient medium (hEBM) containing serum substitutes and other nutrients. The included cell drop was constructed.

1-4. Formation of Stem Cell Aggregates

Each cell drop prepared in Examples 1-3 was suspended culture for 1, 6, 24, 48, and 72 hours in a 37 ° C., CO ₂ incubator, and was subjected to optical microscopy (NIKON) for each time zone. ) Was used to observe the formation of cell aggregates in the culture medium.

As a result, it was confirmed that complete spherical cell aggregates were formed and maintained after 24 hours in both the cell culture cultured with the cell drop in α-MEM medium which was the basal medium and the cell culture cultured with the cell drop in hEBM medium which was the nutrient medium. (FIG. 1).

Example  2. Measurement of Angiogenic Factors in Suspension Cultures of Stem Cell Aggregates

Human vascular endothelial growth factor A (Vascularendothelial growth) secreted by recovering the cell culture supernatant excluding the cells from the cell culture medium in which each cell drop prepared in Example 1 was cultured for 1, 6, 24, 48 and 72 hours. factor A, VEGF A) and human hepatocyte growth factor (HGF) were measured.

To this end, ELISA KIT (R & D system) was used, the specific method is as follows. 100 μl of the cell culture supernatant obtained from each medium and the standard solution of each growth factor (VEGF A, HGF) were added to the well coated with the antibody (R & D system) for each growth factor and allowed to stand at room temperature for 2 hours. After the antigen-binding reaction, each well was washed four times with a washing solution, and 200 µl of a solution in which the secondary antibody (R & D system) of each growth factor was added was added thereto, and left at room temperature for 2 hours. After completion of the secondary antibody binding reaction, each well was washed four times with a washing solution, 200 μl of a coloring reagent (R & D system) was added, and the absorbance was measured at 450 nm with an ELISA reader.

As a result, the amount of angiogenic factors in the culture supernatant was included in the nutrient medium as compared to the cultured cell aggregates as compared to the cultured cell aggregates. hHGF was observed to be secreted more than 10 times more (FIG. 2).

Example  3. Observation of graft and engraftment of stem cell aggregates in myocardial tissue

After removing the culture supernatant from the cell culture obtained in Example 2, the obtained cell aggregates were transplanted into the cardiac tissue of the ischemic cardiovascular rat model, and after 14 days, the cardiac tissue was separated and the cell engraftment was observed under a microscope (Carl Zeiss). It was.

Ischemic cardiovascular rat model refers to an animal ischemic cardiovascular disease model created by ligation of the coronary artery of the heart to induce ischemic state. Cell engraftment in each tissue was confirmed in the case (Fig. 3a) and cell aggregates formed through suspension culture for 24 hours in no addition basal medium (Fig. 3b).

To measure the rate of cell engraftment in transplanted tissue, staining with DiI (stained cells are shown in red) for staining of single cells, the conventional transplantation method, and cell aggregates obtained by the method of the present invention, followed by the left ventricle of SD rats ( Transplanted to the left ventricular wall).

Specifically, rats were anesthetized with ether, air containing 2% isoflurane was continuously treated through a ventilator, underwent deep anesthesia (third intercostal), the pericardium was torn with tweezers, and the heart exposed. . Into the exposed heart was injected a syringe with 30G needles and 60 μl of saline containing 1 × 10 ⁵ single cells and cell aggregates. The needle was pushed about 3 mm from the apex through the free wall of the heart and into the planting area. After the implantation, the lung was rapidly removed and returned to the cage after waiting for recovery of autonomic beats. Fourteen days after implantation, the hearts were perfused and frozen to prepare frozen sections, stained with Cytox blue (marked stained nucleus in blue) for nuclear labeling in each tissue section, followed by fluorescence microscopy. (FIG. 3).

As a result, in the case of tissue transplanted with cell aggregates produced by the method of the present invention (FIG. 3B), not only the remaining cells in the tissue are significantly higher than the conventional method of transplanting single cells (FIG. 3A), It was confirmed that the well-fused to the existing tissue, it can be seen that the stem cell aggregate produced by the method of the present invention is very excellent in the engraftment rate in the tissue.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (15)

(a) forming stem cell aggregates by floating culture of stem cells in a basal medium without additives;
(b) culturing the stem cell aggregates in a basal medium without addition;
(c) obtaining a culture supernatant excluding cells from the culture medium of the stem cell aggregate; And
(d) obtaining angiogenic factors from the culture supernatant, the method of mass obtaining angiogenesis factors, comprising
Wherein the basic medium is free of antibiotics and no serum or serum replacement,
The method is characterized in that the secretion of angiogenic factors is increased by using an additive-free basic medium, the risk of antibiotic resistance is lowered.
The method of claim 1, wherein the stem cells are selected from the group consisting of mesenchymal stem cells, embryonic stem cells, embryonic germ cells, embryonic tumor cells and induced pluripotent stem cells.
The method of claim 1, wherein the angiogenic factor is VEGF A (vascular endothelial growth factor A) or HGF (Hepatocyte growth factor).
The method of claim 1, wherein the method is performed before step (a)
(Iii) culturing the stem cells in a flat plate;
(Ii) separating single cells from the plated stem cells;
(Iii) forming a cell drop comprising said isolated single cell.
The method of claim 4, wherein the plate culture is cultured in a nutrient medium.
The method of claim 5, wherein the nutrient medium comprises serum or serum replacement.
The method of claim 4, wherein the cell drop comprises 500 to 1500 single cells in 10-100 μl of no addition basal medium.
The method of claim 4, wherein the cell drop comprises between 750 and 1250 single cells in 20 μl of no addition basal medium.
The method of claim 4, wherein the cell drop comprises 1000 single cells in 20 μl of no addition basal medium.
The method of claim 1, wherein the culturing is carried out for at least 24 hours.
The method of claim 1, wherein the incubation is incubated for 24 hours to 72 hours.
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