KR101762833B1

KR101762833B1 - Method for isolating adipose-derived stem cell and exosome

Info

Publication number: KR101762833B1
Application number: KR1020150087445A
Authority: KR
Inventors: 김수; 김형기; 윤상원; 이원종; 최은욱
Original assignee: (주)프로스테믹스
Priority date: 2014-06-20
Filing date: 2015-06-19
Publication date: 2017-07-28
Also published as: KR20150145720A

Abstract

The present invention relates to a method for separating fat-derived stem cells and exosomes from adipose tissue into high yields and various uses of isolated adipose stem cells and exosomes.

Description

[0001] METHOD FOR ISOLATING ADIPOSE-DERIVED STEM CELL AND EXOSOME [0002]

Adipose tissue can be enzymatically disrupted to produce two major populations of adult fat cells and adipose stem cells (SVF). SVF has been implicated in the pathogenesis of preadipocytes, adult endothelial cells (EC), endothelial progenitor cells (EPC), vascular smooth muscle cells (SMC), pericytes, wall cells, macrophages, Derived stem / stromal cells (ASC). ASC is a self-regenerating, multi-potent mesenchymal progenitor that can be easily distinguished into adipocytes, osteoblasts and chondrocytes. In addition, several investigators have also produced endothelium, myogenic, liver, and nervous system from ASC under specific induction conditions. In addition to its flexibility, ASC also produces bioactive molecules such as anti-immune and anti-apoptotic, anti-apoptotic, antiscarring, angio-genic, and mitotic factors . Thus, SVF and ASC from adipose tissue have significant efficacy in cell-based therapies.

In US Pat. No. 7,514,075 and US Application No. 20050084961, Hedrick et al. Describe an automated system and method for separating regenerative cells, such as stem and / or progenitor cells, from adipose tissue do. The systems and methods described herein describe rapid and reliable methods for separating and enriching regenerative cells suitable for re-fusion to a subject.

The device disclosed in the prior art uses centrifugal force for cell separation, which causes stress to the cells. Moreover, they are expensive and bulky.

In view of the above description, it is necessary to develop a system for treating mammalian tissues to isolate adipose stem cells (SVF), which are easy to operate and economical in clinical trials.

On the other hand, exosome is a nano-sized vesicle formed naturally in cells. It contains protein and genetic information, and it transmits various signals including genetic information to other cells to develop cells, proliferation, differentiation, , Angiogenesis, and progression of various diseases. The exosomes, which are bio-nanospores, can be avoided in the immune response, are excellent in human fitness, have a great interest in the drug delivery system due to advantages such as drug loading function, target delivery to specific cells, and stability in blood have.

One object of the present invention is to provide a method for separating adipose stem cells and adipose derived from adipose tissue into high yields.

Another object of the present invention is to provide a pharmaceutical composition for promoting cell proliferation, which comprises a fat-derived stem cell and an exosome isolated by the above method.

Another object of the present invention is to provide a pharmaceutical composition for promoting angiogenesis, which comprises the adipose derived stem cell and the exosome isolated by the above method.

It is still another object of the present invention to provide a pharmaceutical composition for anti-inflammation comprising the fat-derived stem cells and the exosome isolated by the above method.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

Various embodiments described in the present invention are described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" the embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

The inventors of the present invention have found that stem cells and exosomes can be separated into high yields when collagenase treatment and centrifugation are performed under specific conditions in adipose tissue, leading to the present invention.

Specifically, in accordance with an embodiment of the present invention,

Adding a phosphorylation buffer solution to the adipose tissue and centrifuging to obtain an adipose tissue layer;

Adding a collagenase to the adipose tissue layer and reacting; And

Adding phosphorylation buffer solution and centrifuging to obtain a first supernatant containing an exosome and a first pellet layer containing stem cells.

In the present invention, the adipose tissue may be a subcutaneous fat tissue discarded during liposuction, and the extracted fat may be used as the subcutaneous adipose tissue regardless of the extraction site, such as abdomen, hip, waist, thigh, Or the fat of the same person can be used together.

When the adipose tissue is prepared as described above, a phosphate buffered saline (PBS) is added thereto in the same amount as the fat tissue described above, and centrifuged at 1,000 to 1,500 rpm for 5 to 10 minutes, The oil layer, the fatty tissue layer, the PBS layer and the fourth pellet layer. In the present invention, the fatty tissue layer can be recovered.

Here, the above-mentioned fatty tissue layer contains a considerable amount of blood vessels as connective tissues. However, fat-derived stem cells and exosomes are distributed around the blood vessels, and therefore, fat cells derived from adipose can be effectively obtained by cutting off the connection sites between the adipocytes and blood vessels. The above-mentioned tissue of the connective region is mainly composed of collagen type II, which is not as rigid as fat, has fluidity, and is secreted in a large amount in chondrocytes which can be easily recombined or separated. Therefore, the collagen type II degrading enzyme collagenase type II is used to increase the yield of stem cells in order to break up the surrounding vascular tissue with a large amount of adipose-derived stem cells.

The amount of the collagenase to be used in the present invention is not particularly limited and can be adjusted according to the physical properties of the fat. For example, if the fat tissue is hard and can not be sucked into the pipette, Or 0.05% by weight if the fat tissue is in a light state so as to be close to a liquid phase. In the present invention, the concentration of the collagenase after the addition of the collagenase is 0.07 to 0.08% by weight. Preferably 0.075% by weight, although it is not limited thereto.

In addition, in the present invention, during the reaction after the addition of the above-described collagenase, a step of stirring the collagenase so as to sufficiently diffuse into the fatty tissue layer can be performed. For example, a Rokcker, an Orbital shaker, a magnetic stirrer, or the like can be used.

In the present invention, when the agitation speed is too high, the viability of the finally isolated stem cells can be lowered. If the agitation speed is too slow, the stem cells or exosomes can not be isolated sufficiently, so that a stirring speed of 50 to 100 rpm is preferable .

However, in the present invention, after the addition of the above-mentioned collagenase, the cells are reacted for 30 to 40 minutes at a temperature of 25 to 30 ° C, which is lower than the conventional treatment conditions, thereby effectively isolating the connective tissue, Can be greatly improved, and when the temperature or time range is out of the above range, the cell viability can be markedly lowered or the yield can be significantly reduced.

In the present invention, when the collagenase is treated and reacted under the above-mentioned temperature and time conditions, most of the fat cells are broken and the oil layer is ejected, and the fatty tissue is changed into the complete liquid phase.

More preferably, in order to more effectively isolate the peripheral blood vessel tissue, stem cells and exosome after the collagenase treatment as described above, the gauge is gradually increased from 18 gauge needle to 25 gauge needle, As shown in FIG.

In the present invention, after the collagenase reaction as described above, a phosphorylation buffer solution is added to an adipose tissue layer and centrifuged at 2,000 to 3,000 rpm for 3 to 10 minutes to form a first pellet layer containing stem cells and a second pellet layer containing exosome 1 < / RTI >

In the present invention, an additional step can be carried out using the obtained first pellet layer and the first supernatant, in order to increase the yield of fat-derived stem cells and exosomes.

Specifically, in the present invention, the first pellet layer containing the stem cells is passed through a 60 to 80 탆 mesh filter, and the filtered suspension is slowly added to a phycol solution, for example, a Histopaque Centrifuging at 1,500 to 2,500 rpm for 5 to 15 minutes to obtain a second supernatant containing stem cells and a second pellet layer of a gray mononuclear cell layer and exosomes .

On the other hand, the addition of phosphorylation buffer in the fat tissue in the present invention, and centrifuged to pellet the fourth layer is approximately 1 × 10 ⁵ ~ obtained And 4 x 10 ⁵ (per 10-20 ml of adipose tissue) fat-derived stem cells. Therefore, in the present invention, the fourth pellet layer may be added to the first pellet layer before passing the first pellet layer through the filter, thereby increasing the yield of finally obtained stem cells.

In addition, in the present invention, a phosphorylated buffer solution is added to the second pellet layer obtained as described above, followed by centrifugation at 1,000 to 2,000 rpm for 3 to 10 minutes to form a third pellet layer containing stem cells and a third pellet layer containing exosome 3 < / RTI > supernatant may be further performed.

However, in the present invention, the first to fourth pellet layers may be dissolved in a suitable solution, for example, a normal saline, a water for injection, a culture medium for stem cells, or a serum-free culture medium.

In the third pellet layer obtained in the present invention, the fat-derived stem cells are contained in a high content of 20 to 30% by weight, and the exosomes obtained through the first, second and third supernatants are nanoparticles , A protein and a miRNA, and is characterized in that the miRNA specifically includes Let7a.

According to another embodiment of the present invention, a first supernatant fluid containing a first pellet layer, a second pellet layer, a third pellet layer and an exosome containing adipose derived stem cells obtained by the separation method according to the present invention, A second supernatant, a third supernatant, and a third supernatant.

According to another embodiment of the present invention, a first supernatant fluid containing a first pellet layer, a second pellet layer, a third pellet layer and an exosome containing fat-derived stem cells obtained by the separation method according to the present invention, 2 < / RTI > supernatant, and a third supernatant.

Here, the pharmaceutical composition may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, suppository or sterilized injection solution, Can be used. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The route of administration of the pharmaceutical compositions according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated. And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and may be 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical compositions according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The separation method provided in the present invention enables separation of stem cells and exosomes from adipose tissue with high yield.

In addition, the fat-derived stem cells and exosome obtained by the above separation method of the present invention are excellent in cell proliferation promotion, angiogenesis promotion and anti-inflammatory effect.

FIG. 1 is a photograph of a fat-derived stem cell contained in a pellet layer obtained according to an embodiment of the present invention in Experimental Example 1. FIG.
FIG. 2 shows the results of confirming the distribution of stem cell markers existing in the stem cells contained in the pellet layer obtained in Experimental Example 1 according to an embodiment of the present invention.
FIG. 3 shows the results of analysis of the content distribution according to the particle size of the exosome in the supernatant obtained in Experimental Example 2 according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of confirming the number of nanoparticles contained in the supernatant obtained in Experimental Example 2 according to an embodiment of the present invention.
FIG. 5 is a graph showing the results of confirming the content of Let-7a, which is a microRNA marker of exosomes isolated from the supernatant obtained in Experimental Example 2 according to an embodiment of the present invention.
FIG. 6 shows the result of examining the markers of exosomes isolated from the supernatant obtained in Experimental Example 2 according to an embodiment of the present invention.
FIG. 7 shows the results of measurement of the content of stem cells in the pellet layer obtained in Example 3 of the present invention and the pellet layer obtained in Comparative Examples 1 and 2. FIG.
FIG. 8 shows the results of measuring the number of nanoparticles of the exosome in the supernatant obtained in Comparative Example 1 and the pellet layer obtained in Example 3 of the present invention in Experimental Example 3. FIG.
FIG. 9 is a graph showing the results of measuring the content of stem cells in the pellet layer obtained in Example 4 and the pellet layer obtained in Comparative Examples 3 and 4 according to an embodiment of the present invention.
10 is a graph showing the results of measurement of the number of nanoparticles of the exosome in the supernatant obtained in Comparative Examples 3 and 4 and the pellet layer obtained in Example 4 of the present invention.
FIG. 11 shows the results of confirming the effect on fibroblast proliferation by mixing the supernatant and the pellet layer obtained in Experimental Example 5 according to an embodiment of the present invention.
FIG. 12 shows the results of confirming the vascularity by mixing the supernatant and the pellet layer obtained in Experimental Example 6 according to an embodiment of the present invention.
FIG. 13 shows the results of confirming the ability of the pellet layer and the supernatant solution obtained in Experimental Example 7 to inhibit nitric oxide formation.

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

Example

Example 1. Preparation of fatty tissue

After obtaining the consent from the patient who had undergone subcutaneous fat removal, the abdominal fat tissue was removed from the patient by liposuction method, and the abdominal fat tissue was collected and stored at room temperature within 1 hour in a sterilized syringe.

Example 2. Obtaining the first pellet layer and the first supernatant

In a clean bench or BSC (Biosafety cabinet), 10-20 ml of adipose tissue was taken in a 50 ml cortical tube in a syringe containing adipose tissue. Phosphate buffered saline (PBS) was added to the transferred tubes in the same volume as the adipose tissue, mixed well using a pipette, and then centrifuged at 1,200 rpm for 5 minutes.

After centrifugation, the oil layer was removed with a pipette from the top, the fatty tissue layer, the PBS tissue layer, the PBS layer and the pellet layer, and the fatty tissue layer was carefully taken and transferred to a new tube. The PBS layer was carefully removed and the pellet layer containing a significant amount of red blood cells (RBCs) on the top of the tube was transferred to a new tube for later addition to the filtration process.

After that, the collagenase type II powder was dissolved in PBS to a final concentration of 1%, filtered through a 0.2-μm syringe filter, and added to a final amount of 0.075% relative to the volume of adipose tissue. The collagenase was then agitated to allow diffusion into the adipose tissue layer. After the addition of collagenase, the cells were allowed to react at 25 to 30 ° C for 40 minutes, and the adipose tissues and cells were physically isolated using a syringe. Specifically, starting from an 18 gauge needle, it was gradually increased to 25 gauge so that cells could be passed through a small aperture through physical external pressure to increase cell yield.

After that, about 25-35 ml of PBS was added to the tube containing the adipose tissue and mixed well, followed by centrifugation at 2,000 rpm for 5 minutes. After centrifugation, the supernatant and the bottom pellet layer were separated using a pipet, respectively, and then transferred to a 50 ml tube.

Example 3. Obtaining the second pellet layer and the second supernatant

To the pellet layer obtained in Example 2, PBS was added to give a final volume of 45 ml. The pellet was sufficiently washed with a pipette, centrifuged at 1,200 rpm for 5 minutes, and the PBS supernatant was discarded.

After washing, the obtained pellet layer was mixed with the pellet layer containing the RBC separately stored in Example 2, and then filtered through a 70 탆 mesh filter. After the filtration, the visible debris caught in the mesh was returned to the pipette, allowing the solution to come out well. The supernatant filtered was subjected to a ficoll gradient to separate RBCs, tissue particles, extracellular matrix and mononuclear cells containing stem cells according to specific gravity. Specifically, the suspension was slowly put on a Histopaque previously contained in a tube, followed by centrifugation at 2,000 rpm for 10 minutes at room temperature. After centrifugation, the supernatant of the solution obtained using the 5 ml pipette and the gray mononuclear cell group having the same color as the cell pellet in the middle were separated and transferred to a new 50 ml tube.

Example 4. Obtaining the third pellet layer and the third supernatant

PBS was added to the cell fraction group obtained in Example 3 until the final amount of 45 ml was obtained and centrifuged at 1,200 rpm for 5 minutes to obtain a supernatant and a pellet layer.

COMPARATIVE EXAMPLES 1 and 2 COMPARATIVE EFFECT OF COLLAGENASE PROCESSES ON TEMPERATURE AND TIME

The procedure of Examples 1 to 4 was repeated except that the collagenase treatment was carried out at a reaction temperature of 5 to 10 ° C or 35 to 40 ° C in Example 2. The pellet layer containing stem cells, A supernatant was obtained.

Comparative Examples 3 and 4. Comparison of Effect of Collagenase Treatment Time

The procedure of Examples 1 to 4 was repeated except that the reaction time after the collagenase treatment in Example 2 was changed to 10 minutes or overnight (overnight, approximately 10 hours), and the pellet layer containing stem cells , And the supernatant was obtained.

Experimental Example 1: Identification of adipose-derived stem cells

The pellet layer obtained in Example 4 was analyzed by an optical microscope to analyze the cells. The photographs are shown in Fig. 1. The distribution of stem cell markers existing in the stem cells contained in the pellet layer was confirmed, 2.

As shown in FIG. 1, when analyzed by a flow cytometer, it was found that the pellet layer obtained by the separation method according to the present invention contained a large amount of adipose stem cells, and as shown in FIG. 2, CD44, CD90, and CD34, which are representative markers of CD44, CD44 and CD44, were expressed in high content.

As a result, the pellet layer obtained by the separation method of the present invention contained a considerable amount of stem cells having typical biological indicators of adipose derived stem cells, and the quantitative content also had a high yield of 27% by weight.

Experimental Example 2: Examination of Exosomal Components

The results are shown in FIG. 3. The number, nanoparticles, and RNA content of the nanoparticles contained in the supernatant were determined according to the following Table 1 , And the number of the nanoparticles is also shown in FIG. 4 as a graph. In addition, the content of Let-7a, which is a microRNA marker derived from exosome of exosomes isolated from the supernatant, was confirmed. The results are shown in FIG. 5, and the exosomal marker was analyzed by Western blot The results are shown in Fig.

division Number of nanoparticles (NTA) protein RNA Example 4 8.5x10 ⁷ 117.8 g 1.33 g

As shown in FIG. 4, it was confirmed that the exosome was contained in the supernatant obtained by the separation method according to the present invention in an extremely high content. As shown in Table 1, FIG. 3 and FIG. 4, As shown in FIGS. 5 and 6, the exosome-derived miRNA marker Let-7a and the exosome marker HSP70, CD63 and CD9 were also detected.

Experimental Example 3: Comparison of stem cell contents according to collagenase treatment temperature

The content of stem cells in the pellet layer obtained in Example 4 and Comparative Examples 1 and 2 was measured. The results are shown in Fig. 7. The results are shown in Fig. 7. The exosomal nanoparticles in the supernatant obtained in Example 4 and Comparative Examples 1 and 2 The results are shown in Fig.

As shown in FIGS. 7 and 8, when the reaction temperature after the collagenase treatment was changed from 25 to 30 ° C (Example 4) according to the separation method of the present invention, stem cells and exosomes And it was confirmed that it was included in a very high content.

However, it was confirmed that stem cells and exosomes were contained in a very low content when the reaction temperature was in the low temperature range of 5 to 10 ° C (Comparative Example 1), which was lower than the above range, and the reaction temperature was 35 to 40 ° C (Comparative Example 2), the content of stem cells was decreased. Especially, the content of exosomes contained in the supernatant was significantly reduced compared with the case of the method of the present invention.

Experimental Example 4: Comparison of stem cell content with collagenase treatment time

The content of stem cells in the pellet layer obtained in Example 4 and Comparative Examples 3 and 4 was measured and the results are shown in Fig. 9. The results are shown in Fig. The results are shown in Fig.

As shown in FIGS. 9 and 10, when the reaction time after the collagenase treatment was changed to 40 minutes (Example 4) according to the separation method of the present invention, the obtained pellet layer and supernatant contained very high levels of stem cells and exosomes And it was confirmed that the content was included.

However, when the reaction time was less than the range of the present invention (Comparative Example 3), it was confirmed that both stem cells and exosomes were included in a very low content. When the reaction was carried out overnight (Comparative Example 4) Also, it was confirmed that the content of stem cells contained in the pellet layer was significantly reduced compared with the case of the method of the present invention.

Experimental Example 5: Confirmation of cell proliferation effect (1)

The pellet layer obtained in Example 4 and the supernatant were mixed and added to the culture solution of human fibroblasts. After culturing for 48 hours, the proliferation rate of the fibroblasts was confirmed, and the results are shown in FIG. As a control, DMEM (Dulbecco's Modified Eagle's Medium, Gibco, USA) containing 10% FBS, penicillin (50 U / ml) and streptomycin ) Were used.

As shown in FIG. 11, the mixture of the pellet layer and the supernatant obtained according to the separation method of the present invention effectively promoted the proliferation of fibroblasts.

Experimental Example 6: Confirmation of promoting effect of angiogenesis (2)

A mixture of the pellet layer and supernatant obtained in Example 4 was used to confirm the degree of angiogenesis in vascular endothelial cells, and the results are shown in Fig. As a positive control, VEGF growth factor, which is known to be excellent in angiogenic effect, was additionally used. Negative control group was EGM-2 Bullet Kit (Lonza, USA) containing 2% FBS as a basic culture medium for vascular endothelial cells .

As shown in FIG. 12, the mixture of the pellet layer and the supernatant obtained according to the separation method of the present invention exhibited an excellent angiogenic effect when compared with the VEGF-treated group having excellent angiogenic activity, although its activity was low there was.

Experimental Example 7: Confirmation of anti-inflammatory effect (3)

In order to confirm the anti-inflammatory effect, nitric oxide (NO) formation inhibition experiment was performed by GRIESS method using RAW264.7 cell line (ATCC number: CRL-2278).

Specifically, RAW264.7 cells, macrophages of mice, were subcultured several times, placed in 24-well plates at 3 × 10 ⁵ per well, and cultured for 24 hours. 1 μg of LPS (Lipopolysaccharide) was treated for 24 hours to induce inflammation. Then, the mixture of the pellet layer and supernatant obtained in Example 4 was diluted and replaced with a cell culture medium and cultured for 48 hours. 100 μl of the supernatant was transferred to a 96-well plate, 100 μl of each of the GRIESS solution was added thereto, and the reaction was allowed to proceed at room temperature for 10 minutes. The absorbance at 540 nm was measured to determine the NO inhibitory effect by the mixture of the pellet layer and the supernatant , And the results are shown in Fig.

As shown in FIG. 13, the mixture of the pellet layer and the supernatant obtained by the separation method of the present invention significantly reduced the amount of NO (NO 3) production compared to the comparison group in which the mixture of the pellet layer and the supernatant was not treated And it showed excellent anti-inflammatory effect.

As described above, the pellet layer and the supernatant obtained according to the separation method of the present invention activate fibroblast proliferation, promote angiogenesis, and have an excellent effect of inhibiting NO formation, thereby promoting cell proliferation, And the anti-inflammatory effect was very excellent.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

Preparing fatty tissue;
Adding a phosphorylation buffer solution to the adipose tissue and centrifuging at 1,000 to 1,500 rpm for 5 to 10 minutes to obtain a fatty tissue layer;
Adding collagenase to the adipose tissue layer and reacting at 25 to 30 ° C for 30 to 40 minutes;
Adding phosphorylation buffer solution and centrifuging at 2,000 to 3,000 rpm for 3 to 10 minutes to obtain a first pellet layer containing stem cells;
Passing the first pellet layer through a 60-80 micron mesh filter;
Adding a suspension through the filter to the Ficoll solution, and centrifuging at 1,500 to 2,500 rpm for 5 to 15 minutes to obtain a second pellet layer containing stem cells; And
Adding phosphorylation buffer solution to the second pellet layer and then centrifuging at 1,000 to 2,000 rpm for 3 to 10 minutes to obtain a third supernatant containing a third pellet layer containing stem cells and exosomes Derived stem cell and exosome.

delete

The method according to claim 1,
Wherein the collagenase is added so as to have a final concentration of 0.07 to 0.08% by weight.

The method according to claim 1,
Further comprising the step of adding a collagenase to the adipose tissue layer and reacting, and then isolating the adipose tissue and cells using a needle of 18 to 25 gauge.

The method according to claim 1,
Characterized in that prior to passing the first pellet layer through a mesh filter a phosphorylated buffer solution is added to the adipose tissue and centrifuged to mix the fourth pellet layer obtained with the adipose tissue layer with the first pellet layer, A method for separating fat-derived stem cells and exosomes.

delete

The method according to claim 1,
Wherein the third pellet layer comprises 20 to 30% by weight of adipose derived stem cells.

The method according to claim 1,
Wherein the exosome comprises at least one of nanoparticles, proteins, and miRNA.

12. The method of claim 11,
Wherein said miRNA is Let7a. &Lt; RTI ID = 0.0 > 11. < / RTI >

delete