KR102190231B1 - Immersion Liquids of Tissue and Composition for Cell Culturing Medium Containing Thereof - Google Patents

Abstract

The present invention relates to a tissue-derived immersion solution and a cell culture medium composition comprising the same, and more particularly, to an immersion solution prepared by immersing animal tissue without a cell culture process and a cell culture medium composition containing the same as an active ingredient. About.
The tissue-derived immersion solution of the present invention has advantages in terms of cost and convenience compared to the conventional cell-derived conditioned culture solution, and contains growth factors and cytokines as active ingredients, and improves cell proliferation when applied to various cell groups. And since it was confirmed that the physiological function activity for the therapeutic effect is promoted, it can be usefully used in a cell culture medium composition. In addition, it can be applied as a bovine serum substitute and has stem cell mobilization activity, so it has an effect that can be applied as a cell-free therapeutic agent in the field of regenerative medicine.

Description

Immersion Liquids of Tissue and Composition for Cell Culturing Medium Containing Thereof

The present invention relates to a tissue-derived immersion solution and a cell culture medium composition comprising the same, and more particularly, to an immersion solution prepared by immersing animal tissue without a cell culture process and a cell culture medium composition containing the same as an active ingredient. About.

Stem cells secrete various effective substances (cytokines and growth factors, etc.), and these are known to be the main factors of the therapeutic effect possessed by stem cells, but cell therapy using stem cells does not have a high survival rate after implantation in the body and immune rejection. It is difficult to find an example that has been widely and reliably successful in actual clinical applications. Accordingly, as an alternative to cell therapy, conditioned media of cells are drawing attention. The conditional culture medium for cells (or referred to as a cell culture medium) is a medium that does not contain cells obtained after culturing cells, and contains various components essential for cell growth, such as cytokines and growth factors. The cell culture medium is used to promote the growth of cells or to separate specific components, and in addition, the culture medium itself is applied to the treatment of various diseases.

Recently, several researchers have reported a method of obtaining a high concentration of culture supernatant through cultivation of mesoderm stem cells. In Korean Patent Publication No. 10-2009-0001163 and International Publication No. WO2007-086637, a physical stimulation such as hypoxic culture, ultraviolet irradiation, nutrient deficiency, or mechanical friction is applied to mesenchymal stem cells, and vitamins A, B, C, and Or, it discloses a method of producing several cell growth factors at high concentration by adding D to the culture medium, and an immune disease treatment (Korean Patent Publication No. 10-2018-0072644) and skin regeneration treatment using a stem cell culture medium (Korean Patent Publication) It has been reported that it can be applied to various fields such as No. 10-2010-0114729).

However, in order to prepare a conditioned culture solution, a cell culture process is essential, and there is a disadvantage in that it requires time and cost. In order to solve the above problem, time and cost (for primary culture of cells or purchase and proliferation culture of cells) consumed for existing cell-derived condition culture solutions are reduced, and in the long term, fetal bovine serum that is currently emerging There is a need to prepare a culture solution having a role as a serum (FBS) substitute and a potential for cell-free treatment.

Thus, the present inventors immersion containing growth factors and cytokines that can affect the physiological activity of various cells based on the culture liquid immersed in the frozen sliced flake tissue derived from animal tissue without the process of cell culture The solution was prepared. The tissue-derived immersion solution produced in the present invention contains an effective substance similar to that of the existing cell-derived conditioned culture solution, and culturing other general cells including stem cells using this, not only activates the physiological function of the cell, It was confirmed that cell proliferation was promoted, and the present invention was completed.

An object of the present invention is to provide a tissue-derived immersion liquid and a method for producing the same.

Another object of the present invention is to provide a cell culture medium composition comprising a tissue-derived immersion solution.

Another object of the present invention is to provide a pharmaceutical composition for regeneration or treatment of damaged organs or tissues comprising a tissue-derived immersion solution.

To achieve the above object,

The present invention provides a tissue-derived immersion solution prepared by immersing frozen section tissue derived from animal tissue without a separate cell culture process.

According to a preferred embodiment of the present invention, the tissue may be cartilage tissue.

According to another preferred embodiment of the present invention, the immersion solution may contain a growth factor or cytokine as an active ingredient, specifically, TIMP-2, Decorin, MCP-1 (CCL2), VEGF -A, MIG (CXCL9), IP-10 (CXCL 10) and osteoprotegerin (Osteoprotegerin) may be at least one selected from the group consisting of.

According to another preferred embodiment of the present invention, the immersion liquid is

(a) preparing a tissue, washing it with a washing solution containing antibiotics, and then preparing a 50-200 μm-thick flake using a frozen sectioning machine;

(b) adding the flakes to a solvent and immersing them for 1 to 7 days; can be prepared by a method comprising.

According to another preferred embodiment of the present invention, the solvent in step (b) may be a cell culture medium, an aqueous solvent, or a fat-soluble solvent.

According to another preferred embodiment of the present invention, the cell culture medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and It may be one or more selected from the group consisting of IMDM (Iscove's Modified Dulbecco's Medium).

According to another preferred embodiment of the present invention, the water-soluble solvent may be water or saline.

According to another preferred embodiment of the present invention, the oil-soluble solvent is ethanol (ethanol), methanol (methanol), ethyl acetate (ethyl acetate), butanol (butanol), hexane (hexane), methylene chloride (methylene chloride), 1,3-butylene glycol (1,3-butylene glycol), aglycone (aglycone) and may be one or more selected from the group consisting of dichloromethane (dichloromethane).

According to another preferred embodiment of the present invention, in the step (b), 1 to 5 ml of medium may be added per 100 mg of flakes.

In addition, the present invention provides a cell culture medium composition comprising the tissue-derived immersion solution as an active ingredient.

According to a preferred embodiment of the present invention, the cells may be tissue-derived cells or stem cells.

According to another preferred embodiment of the present invention, the stem cells may be adult stem cells or embryonic stem cells, and the adult stem cells may be mesenchymal stem cells.

In addition, the present invention provides a pharmaceutical composition for regeneration or treatment of damaged organs or tissues comprising the tissue-derived immersion solution as an active ingredient.

In addition, the present invention provides a composition for wound treatment comprising the tissue-derived immersion solution as an active ingredient.

The tissue-derived immersion solution of the present invention has advantages in terms of cost and convenience compared to the conventional cell-derived conditioned culture solution, and contains growth factors and cytokines as active ingredients, and improves cell proliferation when applied to various cell groups. And since it was confirmed that the physiological function activity for the therapeutic effect is promoted, it can be usefully used in a cell culture medium composition. In addition, it can be applied as a bovine serum substitute and has stem cell mobilization activity, so it has an effect that can be applied as a cell-free therapeutic agent in the field of regenerative medicine.

1 is a process for preparing the immersion solution of the present invention. Porcine knee cartilage tissue was collected and frozen to a certain thickness to prepare flakes, and then immersed in a general cell culture solution and immersed in a cell incubator for 1 to 7 days.
Figure 2 is data confirming the growth factors and cytokines contained in the cartilage tissue-derived immersion solution using a multiplex ELISA array assay kit.
3 is data confirming the concentration of glycosaminoglycan (GAG) contained in the cartilage tissue-derived immersion solution. When the cartilage tissue is prepared as a sheet-shaped flake, it is shown that a high concentration of GAG can be contained in the immersion liquid within a relatively quick time compared to the disk (Disc) and the fragment form (Minced).
4 is data obtained by culturing adipose-derived mesenchymal stem cells using a cartilage tissue-derived immersion solution (ACCM). Cells were cultured in 10-100% ACCM without FBS and other external growth factors, and a relatively high degree of cell adhesion was confirmed on the first day of culture compared to serum-free conditions.
5 is data confirming the degree of cell proliferation when adipose-derived mesenchymal stem cells are cultured using a combination of cartilage tissue-derived immersion solution (ACCM) and FBS. It was confirmed that the proliferation rate of adipose-derived stem cells was significantly increased through observation of the microscope and absorbance (FIG. 5A), and as a result of staining the living cells, the actual number of cells was significantly increased (FIG. 5B).
6 is data confirming the degree of cell proliferation when cartilage tissue-derived immersion solution (ACCM) and FBS are used in combination to culture chondrocytes. Through microscopic and absorbance observation, it was confirmed that the proliferation rate of chondrocytes was significantly increased (Fig. 6a), and as a result of performing periodic analysis of chondrocytes, it was confirmed that the ratio of S-phase indicating division was increased compared to the non-containing group. (Fig. 6b).
Figure 7 shows the degree of cell migration of fat-derived mesenchymal stem cells (Fig. 7a), chondrocytes (Fig. 7b), and human umbilical vein vascular endothelial cells (Fig. 7c) using a cartilage-derived immersion solution. ) As the data confirmed through analysis, it was confirmed that the cartilage tissue-derived immersion solution can be applied as a cell chemotaxis factor (inducing cell migration). 7D is data showing the results of an angiogenesis (Tube formation) experiment, and it was confirmed that the vascular structure formation of human umbilical vein vascular endothelial cells was promoted by the cartilage tissue-derived immersion solution.
8 is data confirming the expression levels of collagen II and aggrecan, which are cartilage-related indicators, as a result of inducing cartilage differentiation of synovial mesenchymal stem cells using a cartilage tissue-derived immersion solution (CM).
9 is data confirming the continuous passage culture and proliferation ability of fat-derived mesenchymal stem cells using cartilage tissue-derived immersion solution. The culture medium was cultured by adding 10% FBS and 30% cartilage-derived immersion solution, and subculture was performed up to 7 times.
FIG. 9A is data showing the degree of cell adhesion when cultured for 72 hours in a passage 0 step, and FIG. 9B is a photograph showing cell morphology.
Figure 9c shows the degree of confluency from step 1 to step 7 of subculture. When using cartilage tissue-derived immersion liquid, it was confirmed that confluence of 90% or more was exhibited in all subculture steps.
FIG. 9D is data showing the number of cells and doubling time in each subculture step, and FIG. 9E is data showing the result of calculating the accumulated number of cells during the seven subcultures.
FIG. 10 is data confirming for each subculture whether the ability to differentiate into osteoblasts and adipocytes is maintained when adipose-derived mesenchymal stem cells are subcultured using a cartilage tissue-derived immersion solution. FIG. 10A is data confirmed by staining the degree of differentiation into osteoblasts with Alizarine red S, and FIG. 10B is data confirmed by staining the degree of differentiation into adipocytes with Oil red O.

Hereinafter, the present invention will be described in detail.

Recognizing that it takes time and cost for cell culture to secure the existing cell-derived conditional culture solution, problems with the use of FBS (essentially used for cell culture, but ethical issues in the production process, continuous increase in unit cost, and fetal origin There is a possibility of transmission of diseases such as mad cow disease). Accordingly, in the present invention, as a result of an attempt to develop a culture solution having an advantage in terms of cost and improvement of the above problem, when an immersion solution is produced with an industrialized pig knee cartilage tissue, an effective material having a similar aspect to that of the condition culture solution derived from existing cells At the same time, it was confirmed that it can promote proliferation during cell culture.

Accordingly, in one aspect, the present invention relates to a tissue-derived immersion liquid prepared by immersing a tissue-derived frozen section of animal tissue.

In the present invention, the tissue may be characterized in that the cartilage tissue, preferably the knee cartilage tissue, but it is possible to prepare an immersion solution using various tissues.

In the present invention, the immersion liquid may contain a growth factor or cytokine as an active ingredient, preferably, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG ( CXCL9), IP-10 (CXCL 10), and osteoprotegerin (Osteoprotegerin) may contain one or more selected from the group consisting of as an active ingredient.

In the present invention, the immersion liquid is

(a) preparing a tissue, washing it with a washing solution containing antibiotics, and then preparing a 50-200 μm-thick flake using a frozen sectioning machine;

(b) adding the flakes to a solvent and immersing for 1 to 7 days; characterized in that it is produced by a method comprising.

Specifically, the tissue of step (a) may be used immediately after acquisition or frozen storage as necessary. Antibiotics selected in the washing step with a cleaning solution containing antibiotics (e.g. PBS) are most ideal for having a broad antibiotic effect and low toxicity to the tissues, and can remove microorganisms such as gram-positive bacteria, gram-negative bacteria and fungi. It is washed with physiological saline or a buffer solution such as a phosphate buffer containing antibiotics or antifungal agents, and is usually washed three or more times.

In addition, the manufacturing of flakes using a frozen section machine is a process similar to that of manufacturing a frozen section for tissue observation in general, and a frozen section flake structure can be produced in a thickness of 50 to 200 μm to be suitable for manufacturing an immersion solution. The frozen sectioning machine is a commercially known freeze sectioning machine known in the art, and the CM-1850 model of Laica or the Microtom HM525 model of Thermo Scientific can be used, but is not limited thereto.

In the present invention, the solvent in step (b) may be a cell culture medium, an aqueous solvent, or a fat-soluble solvent. Since the animal tissue-derived frozen section contains various substances, it can be used without various restrictions, such as a cell culture medium, an aqueous solvent, or a fat-soluble solvent, depending on the purpose.

The medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) One characterized by the above, but is not limited thereto, and a solution capable of immersing the tissue may be used without limitation, and may be determined according to the culture conditions of cells and tissues to which the culture solution is to be applied in the future.

The water-soluble solvent may be water or saline, and a conventional solvent capable of obtaining a fraction by extracting the water-soluble component may be used without limitation.

The oil-soluble solvent is a general organic solvent, ethanol, methanol, ethyl acetate, butanol, hexane, methylene chloride, 1,3-butylene glycol It may be one or more selected from the group consisting of (1,3-butylene glycol), aglycone, and dichloromethane, and a conventional organic solvent capable of fractionating by extracting the oil-soluble component can be used without limitation. I can.

The fraction (extract) thus obtained can be prepared in the form of a high-concentration active ingredient powder through methods such as spray drying and freeze-drying, and the active ingredient prepared in such a high concentration can be dissolved in a medium for cell culture and used again. .

In the present invention, in the step (b), 1 to 5 ml of a solvent is added per 100 mg of flakes, but the present invention is not limited thereto.

The immersion period in step (b) can be recovered after 1 to 7 days immersion depending on the amount of flakes used or changes in the immersion liquid, but is not limited to the period. When recovering the immersion liquid, after the first centrifugation Use a pipette so as not to contain suspended solids such as precipitated dead cells or cell debris precipitated after the second centrifugation, and use a pipette to leave a certain amount of excess and then take the immersion liquid. More specifically, after collecting only the solution excluding flakes, first filtering the suspended matter using a cell strainer, and then removing the cell debris and other suspended matter through a centrifuge (300 g/10 min or 2000 g/10 min. Min), through a 0.20 μm syringe filter or filter bottle, unnecessary substances are finally removed according to the standard for cell culture reagents to prepare an immersion solution. When using a 0.20 μm filter for sterilization, you can feel a sense of resistance. In this case, divide the immersion solution into a volume of about 10 ml and use a silage filter in small portions.

1 shows the process of making the immersion solution of the present invention, in a specific embodiment of the present invention, pig cartilage or tissue stored frozen at -80°C immediately after slaughter is collected through a surgical blade, and then used with PBS containing antibiotics. After washing, a 100 μm-thick flake was prepared using a frozen sectioning machine for sample preparation required for pathological tissue observation. After sufficiently washing the sheet-shaped flakes once again, immerse them in a commercial cell culture solution containing 1% concentration of antibiotic-antifungal agent in a cell incubator at 37° C. for 1 to 7 days, and then a cell strainer, centrifugation and a 0.20 μm syringe A cartilage tissue-derived immersion solution was finally prepared using a filter.

The tissue-derived immersion liquid prepared in the present invention contains as active ingredients cytokines and growth factors that can affect the proliferation, differentiation, and physiological activity of cells, similar to the conventional cell-derived conditioned culture solution.

In order to obtain the existing cell-derived conditioned culture solution, it is necessary to purchase a primary culture process and cell line for securing specific cells, which requires skilled technology, cost, and time. In addition, in the case of initial culture from tissue, the acquired tissue cannot be frozen and stored. Since the initial culture process is required immediately after acquisition, it is impossible to store the tissue and use it later. There is a problem that a considerable amount of time and cost are consumed because it must be amplified through successive passages.

However, in the present invention, an immersion solution containing growth factors and cytokines was obtained even with tissues stored for a long period of time frozen, and unlike conventional cells, the cell culture and subculture process itself is omitted, which is advantageous in terms of industrialization.

In a specific embodiment of the present invention, growth factors and cytokines contained in the immersion solution and the chondrocyte-derived culture solution prepared in the present invention were analyzed using a multiplex ELISA array assay kit. As shown in Figure 2 and Table 1, in the immersion solution of the present invention, various cytokines and growth factors such as VEGF, MCP-1, Decorin, and TIMP-2 were contained, which is confirmed in general cell-derived condition culture solution. It was confirmed that it is similar to.

In another specific embodiment of the present invention, in order to find out whether the shape of the tissue affects the preparation of the immersion solution, cartilage tissue is prepared in the form of flakes, discs, and minced to prepare an immersion solution, and then , The concentration of glycosaminoglycan (GAG) contained in the immersion solution was confirmed. As shown in Fig. 3, when the cartilage tissue is manufactured into thin pieces, it is possible to contain a high concentration of a protein sugar (proteoglycan) material in the immersion solution within a relatively quick time compared to the disc (Disc) and fragment form (Minced). Confirmed.

In another aspect, the present invention relates to a cell culture medium composition comprising the tissue-derived immersion liquid of the present invention as an active ingredient.

In the present invention, the cells may be tissue-derived cells or stem cells. The stem cells may be adult stem cells or embryonic stem cells, and the adult stem cells may preferably be mesenchymal stem cells, but are not limited thereto, and may be variously applied to cells cultured using FBS.

In the present invention, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Consisting of Advanced RPMI 1640, F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) It is characterized by one or more selected from the group, but is not limited thereto, and a medium capable of culturing cells may be used without limitation.

In the present invention, it was confirmed whether the tissue-derived immersion solution prepared in the present invention can be used as a substitute for Fetal Bovine Serum (FBS) or a medium for cell culture.

In a specific embodiment of the present invention, adipose-derived mesenchymal stem cells were cultured using 10 to 100% cartilage tissue-derived immersion solution (ACCM) without FBS and other external growth factors, and as shown in FIG. 4, serum-free It was confirmed that the cells were attached at a relatively high ratio compared to the conditions.

In addition, when adipose-derived mesenchymal stem cells or chondrocytes were cultured using a combination of cartilage tissue-derived immersion liquid (ACCM) and FBS, as shown in FIGS. 5 and 6, despite the fact that the FBS content in the culture medium was reduced by half (5%) It was confirmed that a significantly high cell proliferation ability was shown.

In another specific embodiment of the present invention, as shown in Fig. 7, adipose-derived mesenchymal stem cells (Fig. 7a), chondrocytes (Fig. 7b), human umbilical vein vascular endothelial cells ( The degree of cell migration in Fig. 7c) was confirmed. As a result, the cartilage tissue-derived immersion solution will be applied as a chemotactic factor (inducing cell migration) of mesenchymal stem cells, chondrocytes, and human umbilical vein vascular endothelial cells in an environment that simulates in vivo conditions similar to the existing condition culture solution. It was confirmed that it can be. In addition, as shown in Figure 7d, as a result of confirming the degree of angiogenesis (Tube formation), it was confirmed that the vascular structure formation of human umbilical vein vascular endothelial cells was promoted by the cartilage tissue-derived immersion solution.

That is, it was confirmed that the tissue-derived immersion solution of the present invention can be used not only as an FBS replacement agent or a cell culture medium composition, but also as a cell-free-containing therapeutic agent emerging in the field of regenerative medicine in the future.

In another specific embodiment of the present invention, cartilage differentiation of synovial membrane-derived mesenchymal stem cells was induced using a cartilage tissue-derived immersion solution (CM). As shown in Figure 8, it was confirmed that the expression of the cartilage-related indicators of collagen Ⅱ (collagen Ⅱ) and aggrecan (aggrecan) relatively increased.

In addition, in another specific embodiment of the present invention, the ability of continuous passage culture and proliferation of adipose-derived mesenchymal stem cells was confirmed using a cartilage-derived immersion solution. As shown in Figure 9, when using the cartilage tissue-derived immersion liquid, it was confirmed that the confluency state was reached at a higher rate within the same time period, and 90 in all subculture steps from step 1 to step 7 It was confirmed that more than% confluence was shown.

As shown in FIG. 9D, as a result of counting the number of proliferated and cultured cells for each subculture, it was confirmed that the number of amplified MSCs in all subculture steps was greater when cartilage tissue immersion was used, and a shorter doubling time ( doubling time) was confirmed. In addition, as shown in Figure 9e, as a result of calculating the cumulative number of cells through 7 passages, when cultured using a cartilage immersion solution, it was found that about 1.78 times as many cells were obtained as compared to the general 10% FBS supplementation conditions. Confirmed.

In addition, when the adipose-derived mesenchymal stem cells were subcultured using cartilage tissue-derived immersion liquid, it was confirmed for each subculture whether the differentiation ability into osteoblasts and adipocytes was maintained, as shown in FIG. It was confirmed that the differentiation ability was normally maintained even when the immersion solution was supplemented and amplified and cultured.

That is, the tissue-derived immersion solution of the present invention has the advantage of not simply amplifying the number of cells, but also functionally maintaining the differentiation ability of stem cells and obtaining cells with high yield. In addition, since the tissue-derived immersion solution of the present invention has a high cell proliferation ability, it is possible to accumulate growth factors and cytokines, which are effective factors at high concentration through cell culture, so that various growth factors and cytokines can be obtained. There is this.

Further, the present invention relates to a pharmaceutical composition for regeneration or treatment of damaged organs or tissues comprising the tissue-derived immersion solution of the present invention as an active ingredient in another aspect.

In another aspect, the present invention relates to a composition for treating wounds comprising the tissue-derived immersion solution of the present invention as an active ingredient.

As shown in Fig. 7, since it was confirmed that the tissue-derived immersion solution of the present invention is a chemotactic factor (inducing cell migration) of mesenchymal stem cells, chondrocytes, and human umbilical vein vascular endothelial cells, the tissue-derived immersion of the present invention It was confirmed that the solution can be used not only as an FBS replacement agent or a cell culture medium composition, but also as a cell-free-containing therapeutic agent emerging in the field of regenerative medicine in the future.

That is, the tissue-derived immersion solution of the present invention can be used as a composition for mobilizing stem cells, a pharmaceutical composition for regeneration or treatment of organs and/or tissues, and a pharmaceutical composition for wound treatment.

Stem cell mobilization activity promotes the migration of stem cells, in which the stem cells circulate from the bone marrow, organs and/or tissues to the circulating blood; Accumulates in damaged organs and/or tissues from circulating blood; Or it refers to moving inside an organ and/or tissue.

The route of administration of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto. At this time, in order to formulate a formulation for parenteral administration, a hydrogel containing a stem cell mobilization factor is mixed in water with a stabilizer or buffer to prepare a solution or suspension, and this may be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances, which are conventional methods of mixing, granulation. It can be formulated according to the formulation or coating method.

In addition, the dosage of the pharmaceutical composition of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and degree of disease, and based on an adult patient with a body weight of 60 kg, It is generally 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, and may be dividedly administered once a day or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

The pharmaceutical composition of the present invention can be applied to diseases requiring organ and/or tissue regeneration or treatment. Specifically, there are skin diseases, cranial nervous system diseases, cardiovascular diseases, digestive system diseases, respiratory system diseases, urinary system diseases, motor system diseases, vascular vaginal tract, endocrine system diseases, and ophthalmic diseases.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Statistical analysis

The experimental results of the present invention were statistically processed using the GraphPad Prism 7 program (GraphPad Software Inc). The results of each of the three experiments were expressed as mean±standard errors of mean, and were analyzed using a two-way analysis of variance (ANOVA) Tukey's test, and the statistical significance was It was recognized when P<0.05 or less.

Preparation of cartilage tissue-derived immersion solution

1-1: Porcine cartilage tissue preparation

After collecting cartilage tissue from a pig's knee fresh or immediately stored at -80°C, buffered with sterile PBS (Phosphated buffered saline) containing 1% (v/v) antibiotics (Antibiotic-Antimycotic; GIBCO). Blood and various foreign substances were sufficiently removed by washing several times with a solution or sterile physiological saline. Then, it was processed into a shape suitable for frozen sectioning, and then sliced to a thickness of 100 μm using a frozen sectioning machine (CM-1850 from Leica), and then sufficiently washed again.

1-2: immersion of flakes

The weight of the sliced flakes in Example 1-2 was measured and immersed in 10 ml of commercially available cell culture solution (DMEM-F12, GIBCO) per 500 mg.

The tools used in the experiment were kept sterile at a level comparable to that of cell culture. Antibiotic-Antimycotic (GIBCO) was added to the cell culture solution used for immersion at a concentration of 1% (v/v), and other external growth factors were not treated. The immersion solution was prepared by reacting for 1 to 7 days in a cell incubator equipped with conditions of 37°C and 5% CO ₂ .

1-3: Recovery of immersion liquid

Only the supernatant, excluding the flakes of the immersion liquid placed in the incubator for a certain period of time, was recovered using a pipette, and transferred to a new sterilized tube using a cell strainer. Then, after centrifugation at a rate of 300 g for 10 minutes, the supernatant was recovered to remove unnecessary cell groups, and then centrifuged at a rate of 2000 g for 10 minutes, and then the supernatant was recovered to remove the debris of the dead cell group. The recovered supernatant was filtered using a 0.20 µm standard syringe filter or filter bottle to a level to be used for cell culture to finally prepare a cartilage tissue-derived immersion solution. After that, the immersion liquid was stored in a refrigerator at 4°C or at -80°C for a long time.

Component analysis of cartilage tissue-derived immersion solution

In the present invention, growth factors and cytokines contained in the immersion solution prepared in Example 1 were analyzed using an ELISA array assay kit.

Specifically, growth factors and cytokines contained in the cartilage tissue-derived immersion solution obtained and prepared on the 1st, 3rd, 5th and 7th days were multiplexed ELISA array analysis kit (Quantibody® Multiplex ELISA Array; RayBiotech, Porcine cytokine array Q1 ( Using QAP-CYT-1), Q2 (QAP-CYT-2), Q3 (QAP-CYT-3), Q4 (QAP-CYT-4)), analysis was performed according to the manual.

As a control, a culture medium derived from chondrocytes (a culture medium obtained by culturing pig-derived chondrocytes in DMEM-F12 medium) was collected on the third day, and then prepared through a process similar to the preparation of the immersion solution, and then the ELISA array analysis kit manual Analysis was carried out according to.

The results were converted to numerical values using the GenePix®4000B (Molecular Devices) scanner to check the fluorescence intensity for each sample and using the GenePix®Pro Microarray Analysis program (Molecular Devices).

Analysis of growth factors and cytokines contained in immersion solution (unit: pg/ml) Factor Day 1 Day 3 Day 5 Day 7 Chondrocyte CM TIMP-2 8590.86 8601.51 8631.69 8711.90 8528.03 Decorin 3215.54 3173.73 3030.64 2744.57 3318.01 MCP-1 (CCL2) 703.89 771.02 917.83 1075.23 1607.37 VEGF-A 408.10 401.80 544.25 524.59 250.21 MIG (CXCL9) 1279.24 1366.00 2286.37 2494.27 N.D IP-10 (CXCL 10) 8976.14 14499.56 15849.39 18778.77 N.D Osteoprotegerin 56.68 19.05 N.D N.D N.D MIP-1beta (CCL4) N.D N.D N.D N.D 72.35 IL-8 (CXCL8) N.D N.D N.D N.D 22.41

As shown in Table 1 and 2, from the first day of production, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG (CXCL9), IP-10 (CXCL 10) and Osteooff Rotegerin (Osteoprotegerin) was measured. These cytokines and growth factors are factors known to affect the proliferation or migration of various cells.

In particular, TIMP-2, dicholine, MCP-1 (CCL2), and VEGF-A were identified in the same manner as chondrocyte-derived condition culture solutions, and MIG (CXCL9), IP-10 (CXCL 10), and osteoprotegerin were immersion solutions. Only in the case of MIP-1beta (CCL4) and IL-8 (CXCL8) were found only in chondrocyte-derived condition culture.

Preparation of immersion solution according to tissue type

In the present invention, in order to find out whether the shape of the tissue has an effect on the preparation of the immersion solution, cartilage tissue was prepared in various forms, and the immersion solution was prepared in the same manner as in Example 1-3.

The production efficiency of the immersion solution according to the tissue type was confirmed by measuring the concentration of glycosaminoglycan (GAG). The concentration of GAG was quantitatively analyzed using an analysis kit (Blyscan ^TM Sulfated Glycosaminoglycan assay kit; Biocolor). For analysis of each flake shape, a 1 ㎜ thick disk, a 100 ㎛ thick sheet-shaped flake, was cut at random. The immersion liquid was prepared with the same amount of the minced, and the immersion liquid was recovered on days 1, 3, 5, and 7 and repeated three times to measure absorbance.

As a result, as shown in FIG. 3, when the cartilage tissue is manufactured into thin slices, a high concentration of a protein sugar (proteoglycan) material is contained in the immersion solution within a relatively quicker time than that of the disc (Disc) and the fragment form (Minced). I confirmed what I can do.

Cell culture using tissue-derived immersion solution

4-1: Adipose-derived mesenchymal stem cell culture-FBS free

It was confirmed whether the tissue-derived immersion solution prepared in the present invention can be used as a Fetal Bovine Serum (FBS) replacement agent or a medium for cell culture.

Specifically, the addition of FBS or external growth factors was excluded and only the immersion solution was adjusted to various concentrations (10 to 100%) and used as a cell culture solution. DMEM-F12 (GIBCO) was used as the basic medium, and cells were secreted by primary culture of rabbit adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (Passage 2) were added so as to be 10000 per cm ² area of the culture dish, and the medium containing FBS (GIBCO), serum free medium (serum free), and cartilage tissue-derived immersion solution (ACCM) were included in 10 to 100% Each was cultured using the medium, and the degree of adhesion was compared through the CKX-CCSW program (Olympus) by continuously observing through a microscope.

As a result, as shown in FIG. 3, the cartilage tissue-derived immersion solution showed the predominant degree of cell adhesion at all concentrations on the first day of culture compared to the control medium containing FBS and serum free without supplementing external growth factors. It was confirmed to show (Serum free: 26.3%, immersion solution: 52.1%~73.4%).

4-2: Adipose-derived mesenchymal stem cells and chondrocyte culture-FBS combined culture

Rabbit adipose-derived mesenchymal stem cells or pig-derived chondrocytes were cultured using a combination of cartilage tissue-derived immersion solution (ACCM) and FBS, and the two cells were prepared by separating cells from each tissue and then primary culture.

Specifically, the FBS added to the cell culture solution (DMEM-F12, GIBCO) is 10% and 5%, and the cartilage tissue-derived immersion solution is added so that it becomes 30% 10% FBS culture group, 5% FBS culture group, 10 Adipose-derived mesenchymal stem cells and chondrocytes were cultured by dividing into% FBS + 30% ACCM culture group and 5% FBS + 30% ACCM culture group.

Mesenchymal stem cells (Passage 2) and chondrocytes (Passage 3) were added so as to be 5000 per cm ² area of a culture dish, and then cultured using the above four groups of medium, and the degree of cell proliferation was observed under a microscope, and the number of cells was measured. , Absorbance measurement, and cell cycle analysis.

<Measurement of absorbance>

Absorbance measurement was performed using CCK-8 (Dojindo Molecular Technologies) analysis method to measure the absorbance of 450 nm wavelength after 2 hours reaction to the experimental groups on days 3 and 5 of culture, and statistical analysis was performed through 3 repeated experiments.

Confirmation of mesenchymal stem cell proliferation through absorbance measurement Mesenchymal stem cells Day 3 Day 5 10% FBS 1.271 1.868 5% FBS 0.991 1.439 10% FBS + 30% ACCM 1.682 2.719 5% FBS + 30% ACCM 1.384 2.181

Confirmation of chondrocyte proliferation through absorbance measurement Chondrocytes Day 3 Day 5 10% FBS 0.626 0.990 5% FBS 0.494 0.780 10% FBS + 30% ACCM 0.807 1.383 5% FBS + 30% ACCM 0.738 1.126

When the mesenchymal stem cells and chondrocytes were cultured using a cartilage tissue-derived immersion solution, it was confirmed that the cell proliferation ability was significantly higher (5%) despite the fact that the FBS content in the culture medium was reduced by half.

<cell count>

Calcein-AM (Molecular Probes ^TM ) using a flow cytometer (BD Accuri ^TM C6 flow cytometer; BD Biosciences) to confirm that the proliferation rate of cells cultured through the immersion solution was reflected in the increase in the actual number of cells. The number of viable cells stained with was counted.

Specifically, mesenchymal stem cells (Passage 2) were added so as to be 5000 per cm ² area of a culture dish, and then cultured using media of the above four groups, and the attached cells after 3 days of culture were trypsin/EDTA (Trypsin/ EDTA; GE Healthcare).

Thereafter, the cells were suspended in 200 μl of normal medium containing 50 μM calcein-AM fluorescent dye, followed by staining for 20 minutes in a light-shielding state. Then, each 20 μl of single cell suspension was subjected to statistical analysis after repeated experiments 3 times through flow cytometry and BD Accuri ^™ C6 program (BD Biosciences).

As shown in Fig. 5c, it was confirmed that the number of cells in a living state was significantly increased when the immersion solution was included, as in the proliferation rate result through the CCK-8 assay.

<Cell cycle analysis>

For cell cycle analysis, chondrocytes (Passage 3) are cultivated to be 50000 per cm ² area of a culture dish, and when 80-90% of the culture is cultured, serum free cell culture solution not supplemented with growth factors, etc. is used. This makes it starvation.

Thereafter, the 10% FBS culture group, the 1% FBS culture group, and the 1% FBS + 50% ACCM culture group were divided, and medium exchange was performed with the corresponding culture solution, followed by culture for 1 day. Then, the attached cells were recovered using trypsin/EDTA (Trypsin/EDTA; GE Healthcare), suspended in cold 70% ethanol, fixed at 4° C. for 24 hours or more, and washed twice with PBS. Washed cells are suspended in 500 µl of FxCycle ^TM PI/RNase staining solution (Molecular Probes ^TM ) and stained for 30 minutes in a shaded state, and then the single cell suspension is repeated 3 times using a flow cytometer and ModFit LT ^TM (Verity Software House) program. It was analyzed after the experiment.

As shown in FIG. 6B, the S phase ratios of the 10% FBS culture group, 1% FBS culture group, and 1% FBS + 50% ACCM culture group were 32.53%, 18.18% and 29.74%, respectively. That is, it was confirmed that the ratio of the S phase, which means division, in the cells cultured using the immersion solution increased compared to the non-containing group.

Cell migration and vascular structure formation test using tissue-derived immersion solution

5-1: Confirmation of cell migration

Transwell migration analysis was performed to confirm whether the tissue-derived immersion solution of the present invention has the ability to move cells to a specific disease site or target into which the culture solution was injected, like the conventional cell-derived condition culture solution. .

Rabbit fat-derived mesenchymal stem cells, pig-derived chondrocytes, and human umbilical vein vascular endothelial cells were directed towards DMEM-F12 medium containing 50% immersion solution or 30% immersion solution containing no FBS or external growth factors. It was observed for 5 hours whether it could be cultured through a PET membrane (SPL) of µm. After removing the unmoved cells and fixing the cells moving to the lower well for 30 minutes using 4% PFA, staining for 30 minutes with 0.5% crystal violet (SIGMA), and photographing under a 100-fold microscope, Image J. J) The ratio of cells per field of view was analyzed after 3 repeated experiments using the program.

In the case of mesenchymal stem cells and chondrocytes, an experiment was performed using a medium containing 10% FBS as a positive control and a serum-free medium as a negative control. Human umbilical vein endothelial cells were tested using a medium containing Low Serum Growth Supplemental Factor (Cascade Biologics ^TM ) as a positive control in M200 medium, which is a dedicated medium, and M200 medium without any addition as a negative control. The basic medium to which was added was also used as M200 medium.

Check the degree of cell migration (the ratio of cells per field of view) Mesenchymal stem cells Chondrocytes Human umbilical vein endothelial cells Positive control 10.573% 16.100% 10.573% Negative control 0.477% 6.393% 0.477% 50% concentration immersion liquid 13.674% 19.037% 13.674% 30% concentration immersion liquid 7.783% 16.000% 7.783%

As a result of the analysis, the ratio per field of view of adipose-derived mesenchymal stem cells (Fig. 7a), chondrocytes (Fig. 7b), and human umbilical vein vascular endothelial cells (Fig. 7c) passing through the membrane toward the immersion solution for 5 hours is normal cell culture conditions. Phosphorus was measured higher than the positive control, and it was confirmed that the number of migrated cells significantly increased compared to the control.

That is, the tissue-derived immersion solution of the present invention contained various factors (see Fig. 2) that were proven to affect the movement of cells, and even through an actual cell experiment, the cartilage tissue-derived immersion solution was Movement induction).

5-2: Confirmation of angiogenic ability

In addition, it was confirmed through an angiogenesis (Tube formation) analysis whether the VEGF factor contained in the immersion fluid can affect the formation of a blood vessel structure targeting vascular endothelial cells.

First, gelation was performed by adding 100 μl of growth factor reduced Matrigel ^TM Basement Membrane Matrix (BD Biosciences) to a 24-well culture plate, and then 2 μM of calcein-AM fluorescent substance-stained human umbilical vein endothelium. Cells were cultured by dispensing so as to be 50000 cells per well using media containing exclusive media (including Low Serum Growth supplementary factor; positive control), M200 (not supplemented; negative control), 50% and 30% immersion solution. The vascular structure and the mesh range formed during the culture for 8 hours were observed at a rate of 100 times through a fluorescence microscope. The photographed photographs were compared and analyzed for the total range of cells cultured in a definite mesh form through an Angiogenesis Analyzer (Gilles Carpentier Research Web Site) plug-in of the Image J program.

The mesh ranges per field of view for the positive control group, the negative control group, the medium containing 50% immersion solution and the medium containing 30% immersion solution were identified as 103274, 3568.67, 74279 and 61328.7, respectively, and as shown in FIG. 7d, cartilage It was confirmed that the formation of vascular structures in human umbilical vein vascular endothelial cells was promoted by the tissue-derived immersion solution.

According to the above results, it was confirmed that the immersion solution containing cytokines or growth factors promotes proliferation of several cell groups, improves culture yield, and induces cell migration.

According to reports, stem cells cultured or pretreated with growth factors and cytokines have been reported to increase the viability of cells and improve the differentiation ability into a specific lineage, which means that the immersion solution is used as a cell therapy agent as well as a simple mass culture. This means that there is potential for development as an effective cell therapy agent and a medium that can obtain stem cells or cell populations with improved therapeutic efficiency.

Induction of cartilage differentiation using tissue-derived immersion solution

In the present invention, it was confirmed whether the immersion solution can induce cartilage differentiation of synovial-derived mesenchymal stem cells.

First, the synovial membrane-derived mesenchymal stem cells were cultured for 1 week in a medium containing a general culture growth medium (DMEM-F12) and a cartilage-derived immersion solution, respectively, and then total RNA using an RNA extraction kit (Qiagen). Was extracted. After 1 μg of RNA was synthesized using a cDNA synthesis kit (maxime RT Pre mix kit; Qiagen), a real-time polymerase chain reaction was performed using a 7500 real-time PCR equipment (Applied Biosystems®).

As shown in FIG. 8, it was confirmed that the expression of collagen II and aggrecan, which are cartilage-related indicators, was relatively increased in the cell group cultured through the immersion solution.

Comparison of continuous passage culture and proliferation capacity of adipose-derived stem cells using immersion solution

In the present invention, the ability of continuous passage and proliferation of adipose-derived mesenchymal stem cells was confirmed using a cartilage-derived immersion solution.

Specifically, a culture solution containing a general 10% FBS (DMEM-F12, GIBCO) and a cartilage tissue immersion solution at a concentration of 30% (10% FBS + 30% ACCM) was prepared and the cells were cultured. The obtained three rabbit adipose-derived mesenchymal stem cells (MSCs) were obtained (Passage 0), and the MSCs of the same individual were divided into two culture groups to perform culture (n=3).

Cultured through a culture dish with a size of 90 mm at a ratio of 5 × 10 ³ cells per cm ² area, and if more than 90% confluency is achieved, only adherent cells are removed through trypsin and trypan blue And the number of cells was measured using a hemocytometer. The fat-derived MSC in the Passage 0 state was confirmed to be 90% or more confluency for 72 hours under both culture conditions, and then the passage culture time was fixed at 72 hours and continued culture.

Then, the subculture was performed with the same seeding number and set to repeat up to a total of 7 times. In order to confirm the differentiation ability of MSC, some cells were separated and cultured for each passage.

As shown in Figure 9, when using the cartilage tissue-derived immersion liquid, it was confirmed that the confluency state was reached at a higher rate within the same time period, and in all subculture steps from step 1 to step 7 It was confirmed that more than 90% confluence was shown.

In addition, it was confirmed that the adipose-derived MSC was able to perform 7 passages normally in both experimental groups, and could be confluency of 90% or more within 72 hours in all subculture steps. Thus, for comparison of proliferation ability, the number of cells per experimental group was counted during subculture, and the doubling time was calculated.

Cell count and doubling time for each subculture 10% FBS 10% FBS + 30% ACCM Cell number Doubling time Cell number Doubling time Passage 1 1,382,000 32 2,333,000 23.97 Passage 2 951,000 41.97 1,883,000 26.57 Passage 3 839,333 46.78 1,640,000 28.73 Passage 4 795,333 49.39 1,399,333 31.59 Passage 5 748,667 52.51 1,299,667 33.11 Passage 6 699,667 56.87 1,351,000 32.54 Passage 7 697,000 57.33 1,217,667 34.67

As shown in FIG. 9D, as a result of counting the number of proliferated and cultured cells for each subculture, it was confirmed that the number of amplified MSCs in all subculture steps was greater when cartilage tissue immersion was used, and a shorter doubling time ( doubling time) was confirmed.

In addition, as shown in Figure 9e, as a result of calculating the cumulative cell number through 7 passages, when culturing 287.5 × 10 ³ MSCs (Passsage 0) using a cartilage immersion solution, the general 10% FBS supplementation conditions and In comparison, it was confirmed that about 1.78 times as many cells were obtained (10% FBS: 6.4 × 10 ⁶ / 10% FBS + 30% ACCM: 1.14 × 10 ⁷ ).

Confirmation of differentiation ability during amplification culture of adipose-derived stem cells using immersion liquid

In the present invention, when the adipose-derived mesenchymal stem cells were subcultured using a cartilage tissue-derived immersion solution, it was confirmed for each subculture to maintain the differentiation ability into osteoblasts and adipocytes.

As mentioned in Example 7 above, some cells were separated and cultured for each passage.

The induction period of differentiation was set to 14 days, and the medium was replaced at a 3-day cycle. Osteoblast differentiation medium was purchased from Irvine Scientific and used, and adipocyte differentiation medium was 44.5 mL α-MEM, 5 mL FBS, 0.5 mM IBMX, 1 uM dexamethasone, 200 μM delivery. Metacin (Indomethacin) and 1 mM insulin (Insuline) were added and used. The presence or absence of differentiation was observed by fixing the cells with 4% PFA, followed by staining with Alizarine red S and ORO (Oil red O).

FIG. 10A is data confirmed by staining the degree of differentiation into osteoblasts with Alizarine red S, and FIG. 10B is data confirmed by staining the degree of differentiation into adipocytes with Oil red O.

As a result of observation, in the case of adipose-derived MSC, the differentiation ability was maintained even during a total of 7 passages, and it was confirmed that the differentiation ability was maintained normally even when amplified culture was supplemented with cartilage tissue immersion fluid.

Therefore, it was confirmed that the application of the cartilage tissue immersion liquid did not simply amplify the number of cells, but also functionally maintain the differentiation ability of mesenchymal stem cells and obtain cells with high yield.

Claims (14)

It is prepared by immersing the frozen section of animal cartilage tissue-derived tissue, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG (CXCL9), IP-10 (CXCL 10) and osteooff. Rotegerin (Osteoprotegerin) cartilage tissue-derived immersion solution, characterized in that it contains.
delete delete The method of claim 1, wherein the cartilage tissue-derived immersion solution
(a) preparing animal cartilage tissue, washing it with a washing solution containing antibiotics, and then preparing a 50-200 μm-thick flake using a frozen section;
(b) adding the flake to the cell culture medium and immersing it for 1 to 7 days; cartilage tissue-derived immersion solution, characterized in that it is prepared by a method comprising.
delete The method of claim 4, wherein the medium in step (b) is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), Improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (Advanced RPMI1640), F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) cartilage tissue-derived immersion solution, characterized in that at least one selected from the group consisting of.
delete delete The cartilage tissue-derived immersion liquid according to claim 4, wherein in step (b), 1 to 5 ml of cell culture medium per 100 mg of flakes is added.
It is prepared by immersing the frozen section of animal cartilage tissue in a cell culture medium, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG (CXCL9), IP-10 (CXCL 10). ) And osteoprotegerin (Osteoprotegerin), a composition for cell culture comprising an immersion solution derived from cartilage tissue as an active ingredient,
The cells are vascular endothelial cells, adipose-derived stem cells, synovial membrane-derived mesenchymal stem cells, or chondrocytes, characterized in that the cell culture medium composition.
delete delete It is prepared by immersing the frozen section of animal cartilage tissue-derived tissue, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG (CXCL9), IP-10 (CXCL 10) and osteooff. A composition for promoting vascular structure formation of vascular endothelial cells, comprising as an active ingredient a cartilage tissue-derived immersion solution containing rotegerin (Osteoprotegerin).
It is prepared by immersing the frozen section of animal cartilage tissue-derived tissue, TIMP-2, Decorin, MCP-1 (CCL2), VEGF-A, MIG (CXCL9), IP-10 (CXCL 10) and osteooff. A composition for cartilage differentiation of synovial mesenchymal stem cells derived from synovial membranes comprising a cartilage tissue-derived immersion solution containing rotegerin (Osteoprotegerin) as an active ingredient.

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