WO2012074265A2 - Composition for promoting the stability of stem cells - Google Patents

Abstract

The present invention relates to a composition which can promote the stability of stem cells, and more specifically relates to a composition for promoting the stability of stem cells which contains a mesenchymal stem cell culture and base. The composition for promoting the stability of stem cells according to the present invention has the advantageous effect of promoting the stability of stem cells and maintaining a high survival rate, and, in injectable cell therapeutic agent products that contain the composition for promoting the stability of stem cells according to the present invention, the survival rate of the stem cells comprised in the cell therapeutic agent can be maintained at at least 80% for a long time (10 days), and thus the composition for promoting the stability of stem cells according to the present invention can be used in long-duration transportation of injectable cell therapeutic agent products and, as a consequence of the high survival rates of the stem cells comprised in the cell therapeutic agent, the advantages in cell therapy are outstanding and hence the invention can also be used to advantage in the production of high-quality injectable cell therapeutic agent products.

Description

Composition for enhancing stability of stem cells

The present invention relates to a composition capable of enhancing the stability of stem cells, and more particularly to a composition for enhancing the stability of stem cells containing mesenchymal stem cell culture and base.

In the 21st century, biotechnology aims at human welfare, suggesting the possibility of new solutions to food, environment, and health problems. In particular, the use of stem cells has recently emerged as a new chapter in the treatment of intractable diseases. Previously, organ transplantation and gene therapy have been suggested for the treatment of intractable disease in humans, but its practical use is insufficient due to immune rejection, short supply period, and lack of knowledge of genes.

As interest in stem cell research increased, pluripotent stem cells with the ability to form all organs through proliferation and differentiation were found to be able to fundamentally solve long-term damage as well as most diseases. In addition, many scientists have suggested the possibility of applying stem cells to the treatment of almost all organ regeneration of the human body as well as treatment of Parkinson's disease, various cancers, diabetes and spinal cord injury.

Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells ( multipotent stem cells).

Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8-cells after fertilization of eggs and sperm have these properties. Transplantation can result in one complete individual. Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The inner cell mass inside the blastocyst appears 4-5 days after fertilization. It is called embryonic stem cells and differentiates into a variety of other tissue cells but does not form new life. Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs that contain them.

Multipotent stem cells were first isolated from adult bone marrow (Y. Jiang et al ., Nature , 418: 41, 2002) and subsequently identified in other adult tissues (CM Verfaillie, Trends Cell Biol. , 12: 502, 2002). In other words, bone marrow is the most widely known source of stem cells, but multipotent stem cells have also been identified from skin, blood vessels, muscle and brain (JG Toma et al., Nat. Cell Biol ., 3: 778,2001; M Sampaolesi et al ., Science , 301: 487,2003; Y.Jiang et al., Exp. Hematol. , 30: 896,2002). However, stem cells in adult tissues such as bone marrow are very rare and these cells are difficult to culture without induction of differentiation, making them difficult to culture without specifically screened media. That is, it is very difficult to separate stem cells and preserve them in vitro.

Recently, it has been found that adipose tissue is a new source of multipotent stem cells (B. Cousin et al ., BBRC , 301: 1016,2003; A. Miranville et al., Circulation, 110: 349,2004; S. Gronthos et al., J. Cell Physiol ., 189: 54,2001; MJ Seo et al., BBRC , 328: 258,2005). In other words, it has been reported that the undifferentiated cell population is included in the human adipose tissue obtained by liposuction (liposuction), and it has the ability to differentiate into adipocytes, bone forming cells, myoblasts and chondrocytes in vitro. (PA Zuk et al., Tissue Eng. , 7: 211,2001; AM Rodriguez et al., BBRC , 315: 255,2004). In addition, it has been known through animal model experiments that adipose tissue-derived cells have the ability to promote muscle regeneration and neurovascular differentiation. These adipose tissues have the advantage of being able to be extracted in large quantities, attracting attention as a source of new stem cells to supplement the existing disadvantages.

So far known adipose derived stem cells include human adipose derived adult stem cells capable of differentiation into epithelial cells (M. Brzoska et al ., BBRC , 330: 142,2005), human adipose derived stem stems capable of bone formation and differentiation into adipocytes. Cells (Y. Cao et al ., BBRC , 332: 370,2005), human adipose-derived adult stem cells capable of differentiation into neurons (KM Safford et al., BBRC , 294: 371,2005), capable of differentiating into adipocytes Rat Adipose Stem Cells (R. Ogawa et al., BBRC , 319: 511,2004), Rat Adipose Stem Cells capable of Differentiating to Bone Formation and Cartilage Cells (R. Ogawa et al., BBRC , 313: 871, 2004), human adipose derived stem cells capable of differentiation into chondrocytes (HA Awad et al ., Biomaterials , 25: 3211,2004), and mouse adipose derived stem cells capable of differentiating into neuronal cells (J. Fujimura et al., BBRC , 333). : 116,2005) and adipose derived stem cells capable of differentiating into osteoblasts, chondrocytes, neurons or muscle cells (US Pat. No. 6,777, 231).

Therefore, it is widely known that stem cells that can be obtained in large quantities have the potential to be used in the treatment of a wide variety of disorders, including malignant, metabolic congenital abnormalities, hemochromatosis and immunodeficiency. There is an increasing trend as a cell therapy for injecting cells themselves. Accordingly, as the interest in stem cells increases in the art, it is necessary to provide a stable supply of a plurality of human stem cells over a long distance for various therapeutic purposes, in particular, as a cell therapeutic agent and other medical-related purposes. to be.

To achieve this, it is essential to maintain high stem cell viability even for a long period of 7-10 days. However, at present, there is a problem that the cell viability is significantly lowered if the frozen cells are maintained for a long time. That is, about 40% of the cells are dying in about 10 days, which is a big problem.

Therefore, as a conventional technique for maintaining long-term survival rate, there was a technique for storing human adipose tissue-derived mesenchymal stem cells suspended in physiological saline through refrigerated storage conditions, in which case a survival rate of 70% or more was stored for 48 hours or more. It was hard to express. In order to improve this, when sucrose or albumin is added, survival rate is 70% or more until 48 hours to 72 hours, and it has been confirmed that stability is improved. In addition, there was no significant difference even when the base was used as Hartman-D or PBS instead of physiological saline, and it was confirmed that addition of sucrose and albumin prolonged the stability of stem cells (Korean Patent Application No. 2008 -69063).

In addition, in the case of the technique of freezing and thawing cells, the survival rate at the time of thawing is not comparable to the refrigeration state, and the survival rate may be slightly increased when the excipient is added, but from a biological point of view, long-term damage to cells and stem cells It may have a low activity potential, which may cause problems, and transportation to a frozen state may be more difficult than refrigerated transportation. Thus, a composition for improving stability that maintains the survival rate of stem cells contained in a cell therapy in a refrigerated condition (4 ° C) for a long time stably Development was needed.

In order to achieve the above object, the present inventors have made intensive efforts to develop a stability enhancing composition for maintaining a high survival rate of stem cells, stem cells when the mesenchymal stem cell culture is added as a composition for improving stability, stem It was confirmed that the survival rate of the cells is maintained stably high for 7 to 10 days, to complete the present invention.

Summary of the Invention

An object of the present invention to provide a composition for enhancing the stability of stem cells containing mesenchymal stem cell culture.

It is another object of the present invention to provide a cell therapy injection product containing the composition for enhancing the stability of the stem cells. Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims. Will lose.

In order to achieve the above object, the present invention (a) K-SFM (Keratinocyte-Serum Free Medium) containing mesenchymal stem cells containing N-Acetyl Cysteine (NAC), ascorbic acid, calcium, insulin and hydrocortisone (Hydrocortisone) Culturing for 4-9 days in medium; And (b) mesenchymal stem cell culture obtained by removing the stem cells cultured in the culture medium; And it provides a composition for enhancing the stability of the stem cells containing the base.

The present invention also provides a cell therapy injection product containing the composition for enhancing the stability of the stem cells.

1 is a photograph at 100-fold magnification of cells cultured at 4 ° C. for 3 days.

Figure 2 is a graph showing the viability of the cells according to the concentration of the culture at 4 ℃.

3 is a photograph at 100-fold magnification of cells cultured at 37 ° C. for 3 days.

Figure 4 is a graph showing the viability of the cells according to the concentration of the culture at 37 ℃.

5 is a graph showing the viability of the cells cultured in the cell culture medium and 20% culture concentration at 37 ℃, respectively.

Figure 6 is a graph showing the secretion concentration of procollagen according to the culture time (1 to 9 days) of mesenchymal stem cells.

Detailed Description of the Invention and Preferred Embodiments

As used herein, the term "injection cell therapy or cell therapy" refers to a defect or in the form of parenteral administration, i.e. injection, containing stem cells to treat a defect in a tissue, thereby correcting the defect. It means a pharmaceutical composition that can be.

As used herein, the term "base" refers to a substance added to maintain a form, such as a liquid, in addition to an active ingredient exhibiting pharmacological properties of the pharmaceutical composition, and generally saline as a base for the liquid form. (Saline), Hartmann-D solution, PBS (Phosphate Buffered Saline) is used, but it means a substance that can contain various other compositions.

The term "stem cell" used in the present invention refers to a cell having the ability of self-replicating and differentiating into two or more cells, and "adult stem cell" refers to each organ of the embryo during development. Refers to stem cells appearing in the stage of formation or adulthood.

The term "mesenchymal stem cell" used in the present invention is an undifferentiated stem cell isolated from human or mammalian tissue, and may be derived from various tissues. In particular, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells , Amnion derived mesenchymal stem cells and placental derived mesenchymal stem cells, and techniques for isolating stem cells from each tissue are already known in the art.

As used herein, the term "fat-derived stem cell" is an undifferentiated stem cell isolated from adipose tissue, and the separation method may be as follows. In other words, the suspension containing fat suspended in physiological saline obtained from liposuction, and then treated with trypsin of the stem cell layer attached to the culture vessel, such as a flask and recovered or scraped with a scraper to directly float in a small amount of physiological saline. Adipose-derived mesenchymal stem cells can be separated by a method such as recovery.

The term "culture liquid" used in the present invention means a medium collected after culturing the mesenchymal stem cells in a medium containing a specific component. Eventually, the culture solution contains the cultures (medium components and cell secretions) described below and cultured stem cells.

The term "mesenchymal stem cell culture" used in the present invention refers to the culture of the mesenchymal stem cells in a medium containing a specific component, the medium is collected, and then the cell debris is removed and the remaining broth ). After all, the culture may be said to contain the secretion of the main components and mesenchymal stem cells contained in the medium.

Unless defined otherwise, the terms described herein have been used to mean commonly used terms in the art. In addition, the test method described herein was carried out by a method generally performed in the art, unless otherwise described.

Hereinafter, the present invention will be described in detail.

In one aspect, (a) mesenchymal stem cells in a K-SFM (Keratinocyte-Serum Free Medium) medium containing N-Acetyl Cysteine (NAC), ascorbic acid, calcium, insulin, and hydrocortisone (Hydrocortisone) Incubating for 4-9 days; And (b) mesenchymal stem cell culture obtained by removing the stem cells cultured in the culture medium; And it relates to a composition for enhancing the stability of stem cells containing the base.

According to one embodiment of the present invention, when culturing the mesenchymal stem cells in the K-SFM medium containing the above components, it shows a high productivity for various useful components, particularly preferably in the case of culturing for 4 to 9 days , It was confirmed that the secretion concentration of useful components is high. Most preferably, when cultured for 7 to 9 days, the secretion concentration was about 4 times higher than when cultured for 1 to 2 days. When cultured for 9 days or more, it was confirmed that the concentration of the secreted useful components is reduced, and since the culture medium is a serum-free medium, cells are killed by lack of nutrients during long-term culture, which is obvious to those skilled in the art.

Therefore, in a preferred embodiment of the present invention, mesenchymal stem cells in K-SFM (Keratinocyte-Serum Free Medium) medium containing N-Acetyl Cysteine (NAC), ascorbic acid, calcium, insulin and hydrocortisone (Hydrocortisone) Cultured for 4 to 9 days can be obtained mesenchymal stem cell culture. Most preferably, the mesenchymal stem cell culture may be obtained by culturing for 7 to 9 days.

As a medium used for obtaining the mesenchymal stem cell culture, conventional medium known in the art, which is known to be suitable for stem cell culture, may be used, for example, DMEM, MEM, K-SFM medium, preferably serum free. Medium may be used, and most preferably, K-SFM (Keratinocyte-SFM; Keratinocyte serum free medium) medium may be used.

The medium used for obtaining the mesenchymal stem cell culture of the present invention contains components such as NAC, ascorbic acid, calcium, insulin and hydrocortisone, and thus the mesenchymal stem cells show high productivity for various useful components. Can be. In particular, insulin may be included at a concentration of preferably 1-10 μg / ml.

The medium used for obtaining the mesenchymal stem cell culture may be supplemented with additives known in the art, which promote the proliferation of the undifferentiated phenotype of mesenchymal stem cells while inhibiting differentiation.

In addition, the medium may contain neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. . Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol.

The medium also contains anti-clumping agents, such as those sold by Invitrogen (Cat # 0010057AE), with the aim of preventing the cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.

Among other things, it may be advantageous to use one or more additional additives as follows:

Stem cell factor (SCF, Steel factor), other ligands or antibodies that dimerize c-kit, and other active agents of the same signal transduction pathway

Other tyrosine kinase related receptors such as platelet-Derived Growth Factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and Vascular Endothelial Growth Factor (VEGF) Ligands for

Factors that increase the cyclic AMP concentration, such as forskolin

Factors inducing gp130, such as LIF or Oncostatin-M

Hematopoietic hair growth factors such as thrombopoietin (TPO)

Modified growth factors such as TGFβ1

Other growth factors such as epidermal growth factor (EGF)

Neurotropins, such as CNTF

N-acetyl-L-cysteine (NAC)

Hydrocortisone

Ascrobic Acid

Mesenchymal stem cells for obtaining the mesenchymal stem cell culture of the present invention, for example, can be obtained by the following method.

First, the human adipose tissue obtained from the abdomen by liposuction and the like is separated and washed with PBS, and then the tissue is chopped and digested with DMEM medium containing collagen degrading enzyme, followed by washing with PBS and at 1000 rpm. Centrifuge for 5 minutes. The supernatant is removed and the remaining pellets are washed with PBS and centrifuged at 1000 rpm for 5 minutes. The float was removed using a 100 μm mesh and then washed again with PBS. Incubate in DMEM (10% FBS. 2 mM NAC, 0.2 mM ascorbic acid) medium, and after overnight the cells not attached to the bottom of the culture vessel are washed with PBS, NAC, ascorbic acid, calcium, rEGF, BPE, insulin and By replacing the keratinocytes-SFM medium containing hydrocortisone every two days, the mesenchymal stem cells can be separated and passaged to obtain mesenchymal stem cells. In addition, mesenchymal stem cells can be obtained by methods known in the art. In addition, mesenchymal stem cells can be obtained by methods already known in the art.

Mesenchymal stem cells for obtaining the mesenchymal stem cell culture of the present invention, for example, can be obtained by the following method.

First, the human adipose tissue obtained from the abdomen by liposuction and the like is separated and washed with PBS, and then the tissue is chopped and digested with DMEM medium containing collagen degrading enzyme, followed by washing with PBS and at 1000 rpm. Centrifuge for 5 minutes. The supernatant is removed and the remaining pellets are washed with PBS and centrifuged at 1000 rpm for 5 minutes. The float was removed using a 100 μm mesh and then washed again with PBS. Incubate in DMEM (10% FBS. 2 mM NAC, 0.2 mM ascorbic acid) medium, and after overnight the cells not attached to the bottom of the culture vessel are washed with PBS, NAC, ascorbic acid, calcium, rEGF, BPE, insulin and By replacing the keratinocytes-SFM medium containing hydrocortisone every two days, the mesenchymal stem cells can be separated and passaged to obtain mesenchymal stem cells. In addition, mesenchymal stem cells can be obtained by methods known in the art. In addition, mesenchymal stem cells can be obtained by methods already known in the art.

Mesenchymal stem cell culture of the present invention can be obtained, for example, by the following method. That is, after culturing the mesenchymal stem cells in 90% confluency, incubated in Keratinocyte-SFM medium containing NAC, ascorbic acid, calcium, insulin and Hydrocortisone for 4-9 days, and then the cells were collected to remove the cells. Mesenchymal stem cell culture can be obtained. Most preferably, the mesenchymal stem cell culture can be obtained by culturing for 7 to 9 days to remove the cell debris by collecting the medium, followed by centrifugation and filtering.

In one embodiment of the present invention to include the mesenchymal stem cell culture obtained by the above method in a composition for enhancing stability of stem cells 10 to 50% (v / v), preferably 20% (v / v) In this case, it was confirmed that the survival rate of stem cells was significantly higher than that of the conventional one.

In one example of the present invention, the mesenchymal stem cell culture may be contained in the composition for enhancing stability of the stem cells preferably 10 to 50% (v / v). More preferably, it may be contained in 10 to 30% (v / v), and most preferably in 20% (v / v).

In the present invention, as a base contained in the composition for improving stability of the stem cells, physiological saline, Hartmann-D solution, PBS (Phosphate Buffered Saline), which are generally used, may be used. In addition, any mechanism used in the art may be used.

The mesenchymal stem cell culture obtained by the present invention may contain the following protein components secreted by high concentration of mesenchymal stem cells:

TGF

bFGF

IGF

KGF

HGF

Fibronectin

VEGF

Procollagen

The transforming growth factor-beta (TGF-b) affects the formation of histogenesis, differentiation and proliferation, cell death, etc. in the embryo, and in the adult, functions such as repairing wound tissue and regulating immune cell proliferation. Do it.

IGF-I (Insunlin-like Growth Factor-1) is a 7.6kD, single-chain polypeptide hormone formed of 70 amino acids that forms bone, muscle, and nerve tissue in response to growth hormones. It stimulates the differentiation of cells, thereby regenerating damaged cells and promoting growth.

HGF (hepatocyte growth factor) is composed of a complex group of hormones produced by the body to regulate cell growth and division of skin, bones, blood, and nervous tissues. Cell division, regeneration and cell migration It has been proven to be effective in repairing damaged skin due to scars, burns and peeling. HGFs play important roles in surgical wound healing, such as TGF and Epidermal Growth Factor, by inducing new cell production or by producing new cells with different functions.

VEGF (vascular endothelial growth factor) is a cytokine that increases the permeation of plasma proteins in capillaries and promotes cell division and migration and induces proteolytic enzymes that cause cell reorganization. In addition, by suppressing cell death, the survival of newly formed blood vessels is maintained, and the antigen supply of neurons is suppressed to induce immune regulation to induce cell growth and division. It promotes the migration of vascular cells to promote the development and differentiation of new cells, thereby increasing the composition of the cells.

Procollagen is a protein that makes up the dermal layer of the skin and combines with moisture to keep the skin moist and elastic.

In the present invention, the mesenchymal stem cells are cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, amniotic membrane-derived mesenchyme It may be a mesenchymal stem cell selected from the group consisting of stem cells and placental derived mesenchymal stem cells. Preferably, since the adipose tissue from which the cells are derived is relatively easy to obtain, the production of stem cells may be economical fat-derived mesenchymal stem cells.

In the present invention, the composition for improving the stability of stem cells, if necessary according to the formulation, suspensions, dissolution aids, stabilizers, tonicity agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, painless An agent, a buffer, a sulfur-containing reducing agent, an antioxidant, and the like can be appropriately added.

Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragantmal, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate and the like.

Examples of the solution aid include polyoxyethylene hardened castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

Dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc. are mentioned as a stabilizer.

As an isotonic agent, D-mannitol, sorbitol, etc. are mentioned, for example.

Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hardened castor oil, polyethylene glycol, and the like.

Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetyl homocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione and carbon atoms The thing which has sulfhydryl groups, such as thioalkanoic acid of 1-7, etc. are mentioned.

Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, L-ascorbic acid Chelating agents such as stearate, sodium bisulfite, sodium sulfite, triacyl gallate, propyl gallate or sodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

In another aspect, the present invention provides a cell therapeutic injection product comprising a composition for enhancing stability of the stem cells. The cell therapy injection product may include a compound generally used for cell therapy, and may also include stem cells, which are the main components of the cell therapy, or adult stem cells, which are sub-populations thereof. The stem cells may be cells passaged or subcultured, preferably stem cells derived from autologous tissue, and most preferably mesenchymal stem cells derived from autologous adipose tissue.

Injectables according to the invention can be prepared in the form of filled injections, taking in amounts commonly known in the art, depending on the constitution and type of defect of the patient.

Injectable products according to the present invention can be used by injection in a region or a defect adjacent to the defect to be treated, and defects that can be corrected in this way include wrinkles, stretch marks, scars, skin depression, lip dysfunction, periodontal defects, Soft tissue defects, bone defects, burns, skin ulcers, and the like.

Example

Hereinafter, the present invention will be described in more detail with reference to the following examples and test examples. However, these are provided for easy understanding of the present invention, and the technical scope of the present invention is not limited thereto.

Example 1 Isolation of Mesenchymal Stem Cells from Human Adipose Tissue

Adipose tissue was collected from the abdomen through liposuction and washed with PBS. Then, the tissue was chopped and digested at 37 ° C. for 2 hours using DMEM medium containing collagenase type 1 (1 mg / ml). It was. Next, washed with PBS, centrifuged for 5 minutes at 1000 rpm. The supernatant was removed and the remaining pellets at the bottom were washed with PBS and centrifuged at 1000 rpm for 5 minutes.

The float was removed using a 100 μm mesh and then washed again with PBS. The cells were incubated in DMEM (10% FBS, 2 mM NAC, 0.2 mM ascorbic acid), and after overnight, cells not attached to the bottom of the culture vessel were washed with PBS. The keratinocytes-SFM medium containing 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng / ml rEGF, 50 μg / ml BPE, 5 μg / ml insulin and 74 ng / ml hydrocortisone was then added. Multipotent stem cells were isolated by culturing with replacement every two days.

Example 2: Preparation of Mesenchymal Stem Cell Cultures

Adipose tissue-derived mesenchymal stem cells isolated in Example 1 were cultured at 90% confluency, and then exchanged with keratinocyte-SFM medium containing NAC, ascorbic acid, calcium, insulin and hydrocortisone for 120 hours. .

After incubation, the medium was collected, centrifuged at 1500 rpm for 5 minutes, and cell debris were removed using a 0.22 μm filter to obtain a mesenchymal stem cell culture.

Example 3: Comparative measurement of the survival rate of stem cells according to the culture concentration at 4 ℃

In order to compare the survival rate of stem cells used in the injectable product of a refrigerated cell therapy product which is refrigerated at a temperature of about 4 ° C. until administration through injection, the mesenchymal stem cell culture obtained according to the method of Example 2 An experiment was performed to compare the survival rate of stem cells suspended in an excipient composition including water and the survival rate of stem cells suspended in a general saline solution.

First, stem cells were prepared by the method of Example 1 to prepare adult stem cells, and washed once with PBS. Thereafter, the mesenchymal stem cell culture of Example 2 was mixed with saline (control; saline 100%), 10%, 20%, 30%, 40%, 50% v / v). The divided vial was prepared at a cell concentration of 2 × 10 ⁶ cells / 200 μl per experimental group. This was placed at 4 ° C., the general temperature of the refrigeration agent, and photographed on the third day (FIG. 1), and cell viability was measured at 7 and 10 day intervals.

Cell viability was measured by counting the vial with a finger to release the sunk cells, releasing the cells, pipetting them, diluting 1: 1 with Trypan blue, and Countess ™ Automated Cell from Invitrogen. Survival was measured using a counter.

As a result, as shown in Table 1, when the mesenchymal stem cell cultures were mixed at 20% concentration, cell viability was 86% at 7 days, which was different from that of Saline at 75%, especially on day 10. While the survival rate was still 84%, the control group had a 62% survival rate, and the long-term survival rate was significantly different (Fig. 2). According to the results of the seventh day, the preferred concentration of mesenchymal stem cell culture was found to be 10-50%, more preferably 10-30%, and most preferably 20%.

Table 1

Example 4: Measurement of survival rate of stem cells according to the culture concentration at 37 ℃

In general, since the metabolism of stem cells occurs most actively around 37 ° C., the viability of the cells can be contrasted in a short time, and the mesenchymal stem cells of Example 2 at 37 ° C. under severe conditions. An experiment was performed to compare the survival rate of the stem cells suspended in the composition for enhancing stability of the stem cells including the culture and the survival rate of the stem cells suspended in the saline solution, which is a general base.

First, in order to prepare adult stem cells, the stem cells of Example 1 were prepared and washed once with PBS. Then, the mesenchymal stem cell culture of Example 2 was mixed with saline and divided into a control (saline 100%), 10%, and 20% of 2 x 10 ⁶ cells / 200 μl of cells per experimental group. Vial was prepared by concentration. This was taken at 37 ° C. to take pictures on day 3 (FIG. 3), and cell viability was measured at 7 and 10 day intervals.

To measure the cell viability, use the Invitrogen's Countess ™ Automated Cell Counter to measure the viability using Invitrogen's Countess ™ Automated Cell Counter. It was.

As a result, as shown in Table 2, when the mesenchymal stem cell cultures were mixed at 20% concentration, 59% cell viability was observed at 7 days, which is different from the control of 48%, and still 55% at 10 days. Compared to maintaining the survival rate of the control was 36%, the survival rate was reduced, it was confirmed that the long-term survival rate is significantly different (Fig. 4). According to this result, it was confirmed that the optimum concentration at 37 ° C was 10 to 20%, preferably 20%.

TABLE 2

Example 5: Measurement of survival rate in the composition for enhancing stability of normal medium and stem cells at 37 ° C

In general, in order to compare the survival rate in the serum-free DMEM medium and the culture-containing composition, which is generally used in cell culture, and used as a base for cell therapy for injection, the survival rate was compared by the following method.

First, the stem cells of Example 1 were prepared and washed once with PBS. Thereafter, the mesenchymal stem cell culture of Example 2 was mixed with saline to prepare a 20% (v / v) composition, and the same volume of serum-free DMEM cell culture medium was prepared to 2 x per experimental group. Vials were prepared at a cell concentration of 10 ⁶ cells / 200 μl. Cell viability was measured at 0, 7 and 10 days at 37 degrees Celsius.

Cell viability was measured by releasing the cells by tapping the bottom of the vial with a finger to free the sunk cells, diluting them with Trypan blue 1: 1, and measuring the viability using Invitrogen's Countess ™ Automated Cell Counter. It was.

As a result, as shown in Figure 5 and Table 3, the culture 20% (v / v) composition according to the present invention shows a survival rate of 83% even on day 10, when using a conventional serum-free cell culture medium, 10 In the first day, only 33%, and confirmed that only 39% survives on the 7th day, it was confirmed the superiority of the composition for enhancing the stability of the stem cells according to the present invention.

TABLE 3

Example 6 Analysis of Secretion Levels of Procollagen in Components According to Incubation Time

It is expected that the components contained in the mesenchymal stem cell culture included in the composition for enhancing the stability of the stem cells of the present invention will contribute to increasing the survival rate of the stem cells included in the cell therapy for injection. Therefore, the concentration of protein components such as TGF, bFGF, IGF, KGF, HGF, Fibronectin, VEGF, and Procollagen will vary depending on the culture time of the mesenchymal stem cells. The secretion concentration of the procollagen was confirmed.

Adipose tissue-derived mesenchymal stem cells isolated in Example 1 were incubated in 30 ml of keratinocyte-SFM culture medium containing NAC, ascorbic acid, calcium, insulin and hydrocortisone, and then the medium was collected and centrifuged first. Separation was performed at 1500 rpm for 5 minutes, and secondary cell debris was removed using a 0.22 μm filter to obtain a mesenchymal stem cell culture. The cultures were then analyzed by ELISA to determine the concentration of procollagen. ELISA analysis was performed using the kit of Table 4 below.

Table 4

As a result, as shown in Table 5 and FIG. 6, the concentration of procollagen identified in the culture at each time point started to increase from 3 days, especially after 4 days, to a concentration above 4000ng / ml which is a preferable concentration. It was confirmed that it was included. The highest concentration of 7995.9 ng / ml was confirmed in the culture of the 8th day, and then lowered again. Therefore, it was confirmed that the time for culturing the mesenchymal stem cells is 4 to 9 days, more preferably 7 to 9 days, most preferably 8 days.

Table 5

 As described above, the composition for enhancing the stability of the stem cells according to the present invention has the effect of improving the stability of the stem cells and maintaining a high survival rate, and the cells containing the composition for enhancing the stability of the stem cells according to the present invention. The therapeutic injection product can maintain the survival rate of the stem cells contained in the cell therapy for more than 80% for a long time (10 days), so that it can be used for long-term transportation of the cellular therapy injection product, Depending on the survival rate, the effect of the cell therapy is excellent, it can be usefully used for the production of high-quality cell therapy injection products.

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

Mesenchymal stem cell culture obtained through the following steps; And a composition for enhancing stability of stem cells containing a base: (a) Mesenchymal stem cells are cultured for 4-9 days in K-SFM (Keratinocyte-Serum Free Medium) medium containing N-Acetyl Cysteine (NAC), ascorbic acid, calcium, insulin and Hydrocortisone (Hydrocortisone). step; And (b) removing the stem cells cultured in the culture medium to obtain a mesenchymal stem cell culture. The composition of claim 1, wherein the mesenchymal stem cell culture is prepared by culturing the mesenchymal stem cells for 7 to 9 days. The method of claim 1, wherein the culture is transforming growth factor (TGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibronectin, VEGF (vascular endothelial growth factor) and Procollagen composition for enhancing the stability of the stem cells, characterized in that it contains at least two components selected from the group consisting of. According to claim 1, wherein the composition for enhancing the stability of the stem cells, characterized in that containing 10 to 50% (v / v) of the mesenchymal stem cell culture. The composition for enhancing stability of stem cells according to claim 1, wherein the mesenchymal stem cell culture contains 20% (v / v). According to claim 1, wherein the mesenchymal stem cells are cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells A composition for enhancing the stability of stem cells, characterized in that the mesenchymal stem cells selected from the group consisting of mesenchymal stem cells and placental derived mesenchymal stem cells. The composition of claim 1, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells. The composition of claim 1, wherein the base is one or more selected from the group consisting of saline solution, Hartman-D solution, and PBS (Phosphate Buffered Saline). According to any one of claims 1 to 8, wherein the composition for improving the stability of the stem cells suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, pH adjusters, painless A composition for enhancing stability of stem cells, further comprising at least one selected from the group consisting of an agent, a buffer, a sulfur-reducing agent, and an antioxidant. Cell therapeutic injection product containing a composition for enhancing the stability of the stem cells of any one of claims 1 to 8. A cell therapy injection product containing the composition for enhancing stability of stem cells of claim 9.

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