KR101719743B1

KR101719743B1 - Method for obtaining stromal vascular fraction from adipose tissue

Info

Publication number: KR101719743B1
Application number: KR1020150048524A
Authority: KR
Inventors: 박준한
Original assignee: 박준한
Priority date: 2015-04-06
Filing date: 2015-04-06
Publication date: 2017-03-24
Also published as: KR20160119609A

Abstract

The present invention relates to a method for easily obtaining adipose stem cells from adipose tissue obtained from a human body and a method for producing a dietary composition for fat from adipose tissue obtained from a human body, The manufacturing method is simple and the manufacturing time can be shortened. Therefore, it can be usefully applied to a fat transplantation in a plastic surgery or the like. In addition, the lipid stem cell and lipid-digesting composition prepared is excellent in engraftment rate and does not aggregate so that it can be safely used for fat transplantation without side effects.

Description

METHOD FOR OBTAINING STROMAL VASCULAR FRACTION FROM ADIPOSE TISSUE [0002]

The present invention relates to a method for obtaining adipose stem cells from adipose tissue, and more particularly, to a method for obtaining adipose stem cells from adipose tissue obtained from a human body and a method for producing adipose stem cells from adipose tissue obtained from a human body .

Implantation using the subcutaneous fat of the subject himself or herself is usually performed in a plastic surgeon or the like for the purpose of facial cosmetic molding, breast molding, and removal of a trail due to a wound.

Adipose-derived stem cells (ASCs) are present in adipose tissue used for such transplantation. Adipose tissue is advantageous for harvesting stem cells because it is easy to harvest a large amount of tissue. ASC shows stable growth and proliferation during culture, and when differentiated, induces differentiation into various cells such as bone marrow stem cells. At present, ASC is obtained by separate culture of stromal vascular fraction (SVF, adipose stem cell) after removal of mature adipocytes and red blood cells of adipose tissue. However, this SVF contains various components depending on the stage of differentiation of fat cells such as lipid precursor cells and lipoblasts. For this purpose, a method of separating proteins using a specific antibody specific to a protein existing on the surface of the cell membrane has been developed, but it is limited in terms of high cost and difficult to use publicly. In addition, methods based on cell size and density are used as methods for separating cells having high survival rate and high purity in organs and tissues of humans and animals, but the manufacturing process is complicated and time consuming.

In case of performing transplantation by collecting subcutaneous fat as in the prior art, not only the fat cells of about 50% or more are damaged in the process of collecting subcutaneous fat, but also the cell clusters of various sizes remain so that the desired content is precisely transplanted (For example, a part under the eyes) in which the skin is thin and the subcutaneous fat is small. In addition, when only a subcutaneous fat lump is transplanted, adipocytes present in the fat tissue mass suffers from necrosis because it receives difficulties in receiving nutrients from the surrounding environment or discharging waste materials. In addition, the process of producing conventional fat-soluble edible compositions is very complicated and time consuming.

Adipocytes, adipocyte stem cells and adipose tissue can be obtained from liposuction and adipose tissue from adipose tissue, as described in known methods, for example, in International Patent Publication Nos. WO 2000/53795 and WO 2005/047730 Precipitation, enzymatic treatment such as collagenase, and removal of floating cells such as red blood cells by centrifugation. In addition, as disclosed in International Patent Publication No. WO 2006/084284, the human adipose tissue of the subcutaneous reservoir is subjected to lipid inhalation and sedimentation, sedimentation of blood, washing of fatty tissue with Krebs-Ringer buffer, After the treatment, the adipose tissue stromal cells are isolated by centrifugation to remove oil and adipocytes, suspended in a medium, and adhered to or cultured in a tissue culture dish or flask to remove floating cells.

Fat stem cells and adipocytes used for facial formation, scar removal, and the like are present in the remaining cell mass by separating the fat using the collagenase, and the improvement of the subcutaneous fat defect site It has been used in plastic surgeries for the purpose. Such adipose stem cells and adipocytes not only damage about 50% or more of adipocytes in the process of collecting adipose tissue, but also cause problems such as damage to adipose cells due to physical stress during the enzymatic treatment, There is a problem that the rate of living in a local area is lowered. These problems such as decreased cell activity and reduced engraftment rate can cause necrosis of isolated adult fat-derived stem cells.

Therefore, unlike the conventional method, there is a need to develop a method for producing adipocyte stem cells having excellent adherence rate and producing a fat-edible composition.

1. International Patent Publication No. WO 2000/53795 2. International Patent Publication WO2005 / 042730 3. International Patent Publication No. WO 2006/084284

It is an object of the present invention to provide a method for easily obtaining an adipocyte stem cell excellent in engraftment rate from adipose tissue derived from a human body.

Another object of the present invention is to provide a method for producing a composition for edible fat having excellent engraftment rate from a fat tissue derived from a human body.

In one embodiment, the present invention provides a method of making a composition comprising: (1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin; (2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber; And (3) separating the adipose stem cells (SVF) by adding collagenase to the separated fibers and culturing in an incubator equipped with a septal agitator at 36 to 38 ° C for 20 to 40 minutes with stirring, And a method for obtaining adipose stem cells from adipose tissue.

In another embodiment, the present invention provides a method of treating a subject comprising (1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin; (2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber; (3) adding collagenase to the separated fibers, and then culturing the cells in an incubator equipped with a septal agitator at 36 to 38 DEG C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF); (4) centrifuging the fat separated in step (2) and dehydrating it; (5) centrifuging the blood to obtain platelet-rich plasma (PRP); And (6) mixing the fat stem cells obtained in the step (3), the dehydrated fat obtained in the step (4), and the platelet-rich plasma obtained in the step (5) A method for preparing a composition for implantation is provided.

The method for preparing lipid stem cells and fat-soluble dietary composition according to the present invention can be advantageously applied to fat transplantation in plastic surgeries since the manufacturing process is simple and the manufacturing time can be shortened. In addition, the lipid stem cell and lipid-digesting composition prepared is excellent in engraftment rate and does not aggregate so that it can be safely used for fat transplantation without side effects.

The present invention provides a method for producing an adipocyte stem cell and a fat edible composition which can be used for cosmetic molding and the like.

One embodiment of the present invention is a method of treating a subject suffering from (1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin; (2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber; And (3) separating the adipose stem cells (SVF) by adding collagenase to the separated fibers and culturing in an incubator equipped with a septal agitator at 36 to 38 ° C for 20 to 40 minutes with stirring, And a method for obtaining adipose stem cells from adipose tissue.

Further, another embodiment of the present invention is a method for preparing a pharmaceutical composition comprising: (1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin; (2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber; (3) adding collagenase to the separated fibers, and then culturing the cells in an incubator equipped with a septal agitator at 36 to 38 DEG C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF); (4) centrifuging the fat separated in step (2) and dehydrating it; (5) centrifuging the blood to obtain platelet-rich plasma (PRP); And (6) mixing the fat stem cells obtained in the step (3), the dehydrated fat obtained in the step (4), and the platelet-rich plasma obtained in the step (5) A method for preparing a composition for implantation is provided.

First, a method of obtaining adipose stem cells from adipose tissue, according to one embodiment of the present invention, is described.

In the method for obtaining the adipose stem cells, the adipose tissue is mixed with physiological saline, gentamicin and cephalosporin in the step (1).

The adipose tissue may be obtained from the human body of the subject to be transplanted, for example, surplus fat obtained in front of the thigh, in the front abdomen, behind the thighs and the like. The adipose tissue may be obtained according to conventional liposuction methods, for example, an inflating solution made to allow liposuction by local anesthesia or sleep anesthesia, and an infusion solution for administering an anesthetic solution to the area of the fat to be inhaled By volume, followed by conventional liposuction. Specifically, Tumescent can be used as the swelling solution and the anesthetic solution. In general, lidocaine and prolocaine, which are used as local anesthetics, have a high absorption rate and can not be used in a large amount when used singly, while tumecente can be safely used even when its absorption rate is delayed to 5 times or more. For example, if you want to inhale 1000 cc of fat, 1000 cc of tume cent is injected to aspirate the fat.

The inhaled fat is placed in a suitable container, such as a 3000 cc sterile container, and then mixed with physiological saline, gentamicin and cephalosporin.

The physiological saline solution suspends adipose tissue and provides an environment in which antibiotics described later can act. The physiological saline can be used in an amount of 400 to 1000 cc per 500 to 1000 cc of fat tissue.

Gentamycin is an aminoglycoside antibiotic substance and inhibits the synthesis of proteins in the cytoplasm of gram-negative and positive bacteria and exhibits antibiotic action. The gentamicin may be used in an amount of 0.5 to 2 cc per 500 to 1000 cc of fat tissue.

The cephalosporin is an? -Lactam antibiotic and exhibits an antibiotic action against various bacteria. The cephalosporin may be used in an amount of 0.5 to 2 cc per 500 to 1000 cc of fat tissue.

The above-mentioned gentamycin and cephalosporin serve to remove pathogens that may be present in the inhaled adipose tissue.

At this stage, mention has been made of gentamicin and cephalosporin as representative antibiotics, but it will be appreciated by those skilled in the art that other antibiotics besides these antibiotics may be used.

In this step 2, the fat mixture is stirred with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber.

In adipose tissue inhaled from the human body, there are hematopoietic cells such as fibroids, red blood cells, and white blood cells in addition to adipocytes. These fibroids and hemocytes act as impurities in lipid transplantation, thereby lowering the transplantation engrafting rate. However, the conventional methods fail to provide a technique for removing such impurities, and since the inoculated fat tissue is washed with physiological saline to obtain adipocytes, when the adipocyte is used for transplantation, a high graft survival rate is not achieved have. On the contrary, the present invention is characterized in that fibrous and hematopoietic cells are easily removed from adipose tissue by a stirring and centrifugal separation method using a magnetic stirrer. Furthermore, the present invention can obtain the fat stem cells (SVF) from the removed fiber and use it in fat transplantation, thereby enabling the fiber to be recycled and the fat transplantation effect to be maximized.

Specifically, the fat mixture obtained in step 1 is placed in a container, for example, a 3000 cc pail, and then stirred at a low speed with a magnetic stirrer, that is, at 100 to 500 rpm for 5 to 20 minutes. In this process, the fibers are collected at the center of the container by centrifugation, and the hemocytes are destroyed and condensed in the stirring process and collected on the bottom of the container. When the shock is applied to the mature fat cells before the killing, free oil is collected in the upper layer. Thereafter, the glass oil collected in the upper layer is removed using gauze, and the fibers gathered at the center are removed by using a hand or a spatula, and then the remaining pure fat excluding the blood cells precipitated at the bottom can be separated have. The separated fibers may be used to obtain adipose stem cells at a later stage. In addition, the isolated fat may be used in the preparation of a fat-soluble composition according to another embodiment of the present invention. Furthermore, the separated glass oil can be used as a moisturizing / functional raw material for cosmetics.

While it is known to use conventional machines to separate fibers, this method requires time to separate or separate the fats again, which is time consuming and potentially contaminated, while the method is only as simple as recovering the fibers gathered naturally among Paul Therefore, it is not necessary to worry about the shortage of necessary fat, and the process can proceed naturally without stopping the machine for separation.

In step 3, collagenase is added to the separated fibrous material and cultured in an incubator equipped with a septal agitator at 36 to 38 ° C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF).

In this step, the adipose stem cells are separated from the fibrous material, which is an impurity.

Specifically, collagenase is added to the separated fibers in step 2. The collagenase is an enzyme that promotes the hydrolysis of collagen, and serves to decompose fibrous collagen to separate adipose stem cells. The collagenase may be a commercially available collagenase, for example, NB4 enzyme may be used.

Then, the collagenase-treated fiber is cultured in an incubator equipped with a septic agitator at 36 to 38 ° C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF). The incubator equipped with the seesaw agitator includes a stirrer in a conventional incubator and can perform agitation by a septic agitator and incubation in an incubator at the same time. In the case of adipose stem cells, in particular, adipose tissue is treated with collagenase, which is then centrifuged to obtain adipocyte stem cells. Since the fat is oxidized after 1 hour from exposure to air, It is difficult to obtain an adipocyte stem cell having excellent engraftment rate. However, the present invention can prevent oxidation of fat by shortening the time required for obtaining adipose stem cells by simultaneously performing stirring and culturing in step 3. Normally, it takes more than 1 hour to obtain adipose stem cells. In this step, it can be shortened by 20-30 minutes or more. The culturing can be performed at 36 to 38 ° C for 20 to 40 minutes, and the cultured stem cells can be separated by centrifugation.

The isolated adipose stem cells may be used alone for fat transplantation or as a composition for transplantation mixed with adipose tissue obtained from a human body to be described below. The method for obtaining adipose stem cells according to the present invention enables to obtain adipocyte stem cells having excellent engraftment rate by performing fibrous separation by a magnetic stirrer and stirring and culturing by a septal agitator simultaneously from the fibrous material.

Next, a method for preparing a fat edible composition from adipose tissue, according to another embodiment of the present invention, is described.

The above production method is produced by mixing the fat stem cells obtained by the above-described method with the fat obtained by the method described below.

The method for producing the edible composition comprises: (1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin; (2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber; (3) adding collagenase to the separated fibers, and then culturing the cells in an incubator equipped with a septal agitator at 36 to 38 DEG C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF); (4) centrifuging the fat separated in step (2) and dehydrating it; (5) centrifuging the blood to obtain platelet-rich plasma (PRP); And (6) mixing the adipose stem cells obtained in the step (3), the dehydrated fat obtained in the step (4), and the platelet-rich plasma obtained in the step (5).

Since the above steps (1) to (3) are as described above, steps (4) to (6) will be described in detail.

In this step 4, the fat removed in step 2 is centrifuged and dehydrated. The above process is a process for removing water contained in fat, and can be performed by a conventional centrifugal separation method.

In this step, the water-removed fat may be subjected to a filtering process using a filter, for example, a commercially available puresex.

In this step 5, the blood is centrifuged to obtain platelet-rich plasma (PRP).

Platelet-rich plasma is defined as the plasma component of an autologous blood having a large amount of platelets. It has been reported that the normal platelet concentration in our body is 200,000 cells / ul, and the clinical effect of platelet-rich plasma can be expected at a concentration at least 4 times higher than this. Platelet-rich plasma is obtained through centrifugation of autologous blood and contains a higher concentration of platelets than whole blood. Initially, platelets have been known to act only in the blood clotting process. However, as the understanding of wound healing becomes deeper, platelet rich growth factors and cytokines play a role in tissue regeneration and healing process It became clear that Platelet-rich plasma was first isolated in the 1970s, and Ferrari et al. First used it in open heart surgery in 1987 to avoid excessive blood transfusion. Since then, the area has gradually expanded to include orthopedics, plastic surgery, neurosurgery, dermatology, It has been widely used in various indications such as otolaryngology and various indications.

The platelet-rich plasma serves to provide growth factors and cytokines to the edible composition.

In this Step 6, the lipid stem cells obtained in the above step (3), the dehydrated fat obtained in the above step (4), and the platelet-rich plasma obtained in the above step (5) do.

The adipose stem cells obtained in the step (3) may be further separated by centrifugation, and the separated fat may be mixed with a PDRN (polydeoxyribonucleotide) injection and a chelation injection. The PDRN enhances the rate of engraftment of fat transplants, plays a role in fibroblast growth, cell regeneration, wound healing, and the like. In addition, the chelating injection solution serves to remove heavy metals, cholesterol, blood clots, etc. existing in cells.

The mixing ratio of the adipose stem cell, fat, and platelet-rich plasma may be in the range of 0.5 to 1: 0.5 to 1: 0.5 to 1, but is not limited thereto.

The composition for edible fat produced by the method of the present invention is excellent in engraftment after implantation because it contains adipose stem cells isolated from the fibrous body, fat separated from adipose tissue and platelet-rich plasma separated from blood.

In addition, harvesting apparatuses such as Habes Jet, Hurisell, and Tea Station are currently used in the market, but Harvest Jet is not a device that obtains stem cells only by filtering out debris from adipocytes, Is self-washing and concentrating, and it extracts fat stem cells at one time. But it is expensive at 7-8 million won, and it has weak point of filtering to filter out adipocytes, and it takes about 2 hours. The equipment costs from at least 4-5 million to 200 million, but the filtration is weak and the viability of the resulting composition drops.

On the other hand, the fat-soluble dietary composition prepared according to the present invention exhibits a transplantation rate of 70% or more at the time of fat transplantation, has a higher engraftment rate than the fat transplantation operation performed with other machines and can shorten the operation time by 1 hour or more have. Furthermore, since the fat is not calcified after the postprandial calcification or the fat accumulates and the lump is not formed after the transplantation of the chest or the hip, it is very safe and efficient in the transplantation of the fat.

Hereinafter, the present invention will be described in more detail with reference to the following embodiments. However, the following description of the embodiments is intended to illustrate specific embodiments of the present invention and is not intended to limit or limit the scope of the present invention.

Example 1: Isolation of adipose stem cells (SVF) from adipose tissue

600cc of the inhaled fat was put into a 3000cc bottle, 500cc of physiological saline, 1 ampoule of gentamicin and 1 vial of cephalosporin 1 were mixed. The fat mixture was stirred for 10 minutes at 200 rpm using a magnetic stirrer. After stirring, the oil layer formed on the upper layer was removed using gauze, and the produced fibrous material was recovered in the center portion. Then, the fat except for the blood cells precipitated on the bottom was carefully separated.

The fibrous material was divided into 50 cc tubes by 25 cc, and NB4 enzyme was added thereto. The fibrous material to which the enzyme had been added was placed in an incubator in which a sieving stirrer was placed, and cultured at 36 DEG C for 30 minutes with stirring. The culture was centrifuged to separate SVF as supernatant.

Example 2: Preparation of fat-soluble edible composition from adipose tissue

The fat removed in Example 1 was centrifuged to remove water. The dehydrated and concentrated fats were around 6-7 in size with 60 cc syringes.

Meanwhile, the SVF isolated in Example 1 was centrifuged to remove fat, and centrifuged again to obtain 2 SVF tubes of about 5 cc below the tube. The 10 cc solution was collected separately and mixed with 1 ampoule of PDRN injection and 1 ampoule of chelation solution.

On the other hand, 60 cc of the previously collected blood was centrifuged to obtain 20 cc of a platelet-rich plasma solution.

20 cc of the obtained platelet-rich plasma solution was mixed with 20 cc of the obtained SVF and 20 cc of fat to obtain a fat-soluble composition.

Test Example 1: Effect of transplantation of fatty food composition

The fat-edible composition of Example 2 was transplanted to 20 subjects to the chest or hips, and whether or not there was an immune rejection reaction and whether the adipose tissue was maintained was observed for 3, 6, and 9 months.

Generally, after fat transplantation, calcification or fat accumulation occurs, resulting in a cluster after breast or hip transplantation. However, even if the fat is uniformly transplanted, it may accumulate in the process of engraftment after migration and angiogenesis.

However, as a result of implanting the edible composition of the present invention, it was confirmed that 90% of the initial amount of adipose tissue was maintained, and there was no clumping phenomenon at all.

Claims

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(1) mixing adipose tissue with physiological saline, gentamicin and cephalosporin;
(2) stirring the fat mixture with a magnetic stirrer at 100 to 500 rpm for 5 to 20 minutes to separate fat and fiber;
(3) adding collagenase to the separated fibers, and then culturing the cells in an incubator equipped with a septal agitator at 36 to 38 DEG C for 20 to 40 minutes with stirring to separate the adipose stem cells (SVF);
(4) centrifuging the fat separated in step (2) and dehydrating it;
(5) centrifuging the blood to obtain platelet-rich plasma (PRP); And
(6) mixing the adipose stem cells obtained in the step (3), the dehydrated fat obtained in the step (4), and the platelet-rich plasma obtained in the step (5);
/ RTI >
The physiological saline, gentamicin and cephalosporin are used in an amount of 400 to 1000 cc, 0.5 to 2 cc and 0.5 to 2 cc, respectively, per 500 to 1000 cc of fat tissue,
In step (2), the fibers are formed in the center of the container by stirring and then separated,
In step (3), the isolated adipose stem cells are mixed with a PDRN (polydeoxyribonucleotide) injection solution and a chelation injection solution,
Wherein the mixing ratio of adipose stem cells, fat and platelet-rich plasma is in the range of 0.5: 1: 0.5 to 1: 0.5: 1 in step (6).

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