KR20180127884A - Container and method for extracting adipose stem cells - Google Patents

Abstract

The present invention relates to a container for extracting adipose stem cells and a method for extracting adipose stem cells using the same. Specifically, the present invention provides a container for extraction which comprises: a main body; a lower stopper hermetically closing a lower portion of the main body and having a filter net fused on the upper portion to cover a receiving groove formed in a central portion of the upper portion. In the centrifugation process, the adipose stem cells (SVF) are moved to the receiving groove of the lower stopper by the centrifugal force and are separated and filtered naturally, thereby simplifying the process of extracting the adipose stem cells (SVF) from fat compared to the conventional process. Accordingly, the filtration rate and extraction amount of adipose stem cells (SVF) can be enhanced while reducing extraction time and cost.

Description

TECHNICAL FIELD The present invention relates to a container for extracting adipose stem cells and a method for extracting adipose stem cells using the same.

The present invention relates to a container for extracting adipose stem cells and a method for extracting adipose stem cells using the same. More particularly, the present invention relates to a method for easily isolating adipose stem cells (Stromal Vascular Fraction, SVF) The present invention relates to a container for extracting adipose stem cells and a method for extracting adipose stem cells using the same.

In general, a stem cell refers to a cell capable of differentiating into all kinds of cells constituting the body, such as nerve, blood, and cartilage, if necessary, without being differentiated into specific cells. There are two major ways to obtain such stem cells. First, there is a method of obtaining stem cells (embryonic stem cells) from embryos generated from embryos. Second, there is a method obtained from stem cells (adult stem cells) which are retained in each part of the adult human body. Although there are functional differences, embryonic stem cells and adult stem cells all have differentiation characteristics into different types of cells.

Embryonic stem cells have excellent ability to differentiate, and although Telomer has long advantages, it has an ethical problem and it is difficult to acquire a large amount of cells. On the other hand, adult stem cells can obtain a large number of cells, but they have a disadvantage in that the risk of infection or the ability of differentiation is relatively low when they are transplanted into other persons. Despite these shortcomings, adult stem cells are characterized by their high safety for medical applications. In addition, there is a site-specific differentiation that differentiates itself according to the characteristics of the surrounding tissues. In addition, since it does not cause cancer even if it is injected under undifferentiated state, There is a self-renewal ability to recreate and store the necessary undifferentiated stem cells.

Although adipose tissue plays an important role in normal growth and physiology in vivo, its importance has been overlooked so far. The most common form of fat is the white adipose tissue located in the subperitoneal (visceral fat) or in the reproductive organs (gonadal fat), located under the skin (subcutaneous fat). A somewhat less common form in adults is brown adipose tissue and plays an important role in generating heat in the neonatal period (Gimble, New Bio1.2 (4): 304-12, (1990)). However, reproductive and maturation processes are closely linked to the storage of adipose tissue. Adolescent puberty in women and men is closely related to the production and secretion of adipose tissue-derived hormones, and also plays an important role in glucose metabolism and energy balance.

Accordingly, much research has been conducted on the use of adipose tissue. (2002): Rodriguez AM, et al., Biochimie, 87: 125-9, 2002). [0004] Recently, adult stem cells have been found to be present in adipose tissue (Zuk PA, et al., Molecular Biology of Cell, Vol 13: 4279-4295 128 (2005)) Several studies have begun on the use of adipose-derived stem cells. In addition, based on the advances in biochemistry and molecular biology, we have found a small amount of signaling substances (growth factors) that exist in the living body, and the theory of biodegradation based on them is being redefined (Stanley Cohen, Nobel Lecture 1986, Dec, 8). In addition, this signaling substance (growth factor) decreases in vivo with increasing age, and it has been shown that this decrease in growth factor is closely related to our aging (Sporn M and Roberts A, Handbook of Experimental Pharmacology , Vol. 1, Vol. 95/1, 1990, Springer-Verlag, DE, Berlin., Pp. 667-698).

As described above, studies on the structure of the signal substance (growth factor) have been carried out. However, since most of the growth factors have a protein structure, their structures are three-dimensionally complex, In addition, the cost for such synthesis is also very expensive. Thus, it is known that adipose-derived adult stem cells secrete growth factors in the process of obtaining active growth factors at low cost while maintaining activity in the human body (Rehman, J. et al , Circulation, 109: 1292-1298 (2004)). Accordingly, studies on obtaining stem cells from adipose tissue have been carried out. Representative methods include, for example, lipid extraction from fat tissue, enzyme treatment, lipid stem cell washing and isolation, and fat stem cell filtration Methods for extracting stem cells are widely used.

As described above, the method for extracting the adipose stem cells comprises the steps of extracting fat from adipose tissue, enzyme treatment using collagenase and the like, separation and washing of adipose stem cells using centrifugation, and culturing of adulthood adipose stem cells And the like. In addition, centrifugation is used to separate and clean the adipose stem cells, which is a multiple-step procedure. In order to extract the adipose stem cells from the fat extracted with the Luracle syringe or to wash the extracted adipose stem cells with the washing solution, centrifugation is used .

However, in the method of extracting adipose stem cells according to the prior art, in the process of separating adipose stem cells (SVF) from fat extracted from adipose tissue, the adipose stem cells (SVF) are separated Therefore, the extraction cost is wasted due to the use of many containers, and the extraction amount of adipose stem cells is also decreased.

KR 10-0660117 B1, Dec. 14, 2006. KR 10-1019085 B1, 2011. 02. 24.

Accordingly, the present invention has been proposed in order to solve the problems of the prior art, and it is an object of the present invention to simplify the process of extracting SVF from fat to reduce the extraction time and cost while reducing the filtration rate of SVF The present invention provides a container for extracting adipose stem cells and a method for extracting adipose stem cells using the same.

According to an aspect of the present invention, there is provided a container for extracting adipose stem cells comprising: a main body having a receiving space therein; A lower stopper inserted into the lower portion of the main body to seal the lower portion of the main body; And an upper stopper for sealing an upper portion of the injection port provided in the main body, wherein the lower stopper is provided with a receiving groove at an upper center portion thereof and an outer side surface thereof inserted into the lower portion of the main body, And a filter net welded to the upper end of the packing to cover the receiving groove.

In addition, the packing may include an engaging part made of an elastic material and tightly brought into close contact with the inside of the body. And a support portion formed of a material having a strength higher than that of the coupling portion to improve the strength of the coupling portion and coupled to an upper portion of the coupling portion and fused to the filter network, And the upper end of the support portion may be formed with a downwardly recessed seating jaw to seat the filtering net.

In addition, the main body has an injection port protruding upward, and the accommodation space can be partitioned into an upper liquid chamber and a lower liquid chamber by the injection port.

According to another aspect of the present invention, there is provided a method for extracting adipose stem cells comprising the steps of: (a) extracting fat from adipose tissue; (b) sonicating the extracted fat using an ultrasonic device; (c) injecting the ultrasonically decomposed fat into a first extraction container and performing primary centrifugation; (d) extracting only the adipose stem cells (SVF), the supernatant and the pure adipose stem cells (SVF) separated by the first centrifugation from the first extraction container; And (e) injecting the stem cell (SVF) extracted from the first extraction container and the supernatant into a second extraction container and performing secondary centrifugation; And (f) extracting only adipose stem cells (SVF) from the second extraction container among the adipose stem cells (SVF) separated by the second centrifugation and the supernatant.

The method may further include, after the step (f), activating the fat stem cells (SVF) extracted from the second extraction container by repeatedly relaxing using an activator.

In addition, the first and second extraction containers each include a main body having a receiving space therein; A lower stopper inserted into the lower portion of the main body to seal the lower portion of the main body; And an upper stopper for sealing an upper portion of the injection port provided in the main body, wherein the lower stopper is provided with a receiving groove at an upper center portion thereof and an outer side surface thereof inserted into the lower portion of the main body, And a filter net welded to the upper end of the packing to cover the receiving groove.

In the step (f), only the adipose stem cells (SVF) filled in the receiving groove of the second extraction container may be extracted using a syringe.

As described above, according to the present invention, there is provided an extraction container including a main body and a lower stopper which hermetically seals the lower portion of the main body and has a filter net welded thereon so as to cover a receiving groove formed in the upper central portion, (SVF) is moved to the receiving groove of the lower stopper by the centrifugal force and is naturally separated and filtered, thereby simplifying the extraction process of the SVF from the fat, thereby reducing the extraction time and cost On the other hand, the filtration rate and extraction amount of the SVF can be improved.

1 is a perspective view illustrating a container for extracting adipose stem cells according to an embodiment of the present invention;
FIG. 2 is a sectional view showing a container for extracting adipose stem cells shown in FIG. 1. FIG.
FIG. 3 is a perspective view illustrating the lower stopper shown in FIG. 2; FIG.
Fig. 4 is a cross-sectional view of the lower stopper shown in Fig. 3 as seen from the front when cut in the vertical direction. Fig.
FIG. 5 is a perspective view illustrating a container for extracting adipose stem cells according to another embodiment of the present invention. FIG.
FIG. 6 is a cross-sectional view of the vessel for extracting adipose stem cells shown in FIG. 5; FIG.
FIG. 7 is a flowchart illustrating a method for extracting adipose stem cells according to an embodiment of the present invention. FIG.
FIGS. 8 to 18 are diagrams for explaining each process shown in FIG. 7. FIG.

Hereinafter, the technical features of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a perspective view illustrating a container for extracting adipose stem cells according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view illustrating a container for extracting adipose stem cells shown in FIG.

1 and 2, a container 10 for extracting adipose stem cells according to an embodiment of the present invention includes a centrifuge for centrifugation through a centrifuge in a state in which fat extracted from adipose tissue is decomposed through ultrasonic decomposition As a separating container, for example, a capacity of 60 cc is produced.

As shown in FIG. 2, the container 10 for extracting adipose stem cells includes a main body 11 provided with a receiving space therein, a lower cap inserted into the lower inside of the main body 11 to seal the lower portion of the main body 11 And an upper stopper 13 for sealing the upper portion of the injection port 111 provided in the main body 11. [ Further, it may further include upper and lower lids (14, 15) covering upper and lower portions of the main body (11).

The body 11 has a cylindrical structure as shown in Fig. 2, and has a structure in which the upper part and the lower part are respectively opened. The main body 11 may be made of a transparent material or a translucent material through which the inner space is viewed. For example, it may be made of PE (polyethylene) or the like.

The main body 11 is provided with an injection port 111 for injecting the ultrasonically decomposed fat into the main body 11 at an upper portion thereof. At this time, the injection port 111 may have a structure that becomes narrower toward the upper part, and is sealed by an upper stopper 13 made of a silicone material or a rubber material.

A lower stopper 12 made of silicone or rubber is fitted and sealed inside the lower end of the main body 11.

FIG. 3 is a perspective view illustrating the lower stopper shown in FIG. 2, and FIG. 4 is a cross-sectional view of the lower stopper shown in FIG.

2 to 4, the lower stopper 12 is inserted into the lower portion of the main body 11 to seal the opening provided in the lower portion of the main body 11. [ The lower stopper 12 includes a packing 121 having an accommodating groove 121a formed at its upper center and an outer side inserted into the lower end portion of the main body 11 to be closely contacted with each other, (Not shown).

The packing 121 is inserted into the lower portion of the main body 11 and then tightly adhered to the inner surface of the main body 11 by elasticity and stably supports the filter net 122, The structure may be formed by stacking layers of different materials having different strengths.

2, the packing 121 may be formed of a resilient material and may include a fitting portion a which is tightly brought into close contact with the inside of the main body 11, And a support part (b) made of a strong material and coupled to the upper part of the coupling part (a) and fusing the filtering wire (122). The support portion b may be made of a synthetic resin material such as PE (PolyEthylene), PP (PolyproPylen), or the like, which is stronger than the joint portion. The support portion b may be made of, for example, And the like.

As shown in FIG. 4, at the center of the packing 121, a receiving groove 121a for receiving the adipose stem cells (SVF) separated from the fat is provided below the filtering net 122. At this time, the volume of the receiving groove 121a is determined according to the amount of SVF that can be extracted from fat extracted from adipose tissue. For example, it is formed to have a size of about 5cc.

4, on the outer surface of the outer surface of the packing 121, that is, the outer surface of the joint portion of the packing 121 closely contacting the inner surface of the body 121, a plurality of tight contact protrusions 121c are formed outwardly, It is possible to easily insert into the inside of the main body 11 and improve the bonding force between the inside surfaces of the main body 11. [ A seating recess 121b recessed downward is formed at the upper end of the packing 121, that is, at the upper end of the supporting portion of the packing 121, so that the filtering net 122 is seated.

As shown in Figs. 2 and 4, the filter net 122 is fused to the mounting tab 121b of the packing 121. Fig. The filter network 122 is made of a synthetic resin material such as PE (PolyEthylene), PP (PolyproPylen), etc. After the centrifugation, the stem cells (SVF) (200 neck) or 150 mesh (150 neck).

As shown in FIG. 2, the main body 11 may further include an auxiliary injection port 112 formed at one side of the upper end. Further, it may further include an auxiliary stopper 113 for sealing the auxiliary injection port 112.

The upper lid 14 may be coupled to cover the upper portion of the main body 11 in a tight fitting manner and the lower lid 15 may be coupled to the lower portion of the main body 11 in a screw- .

FIG. 5 is a perspective view illustrating a container for extracting adipose stem cells according to another embodiment of the present invention, and FIG. 6 is a sectional view illustrating a container for extracting adipose stem cells shown in FIG.

5 and 6, a vessel 20 for extracting adipose stem cells according to another embodiment of the present invention includes a vessel 10 for extracting adipose stem cells shown in FIGS. 1 and 2, Like the vessel 10 for extracting adipose stem cells, the container for separating adipose stem cells (SVF) from the supernatant by secondary centrifugation of adipose stem cells (SVF) is composed of a main body 21, a lower stopper 22, (23), and further includes a top lid (24) and a bottom lid (25).

The container 20 for extracting adipose stem cells according to another embodiment is formed such that an injection port 211 of the main body 21 is exposed through the upper lid 24 to the outside. Then, the exposed injection port 211 is sealed by the upper stopper 23. The main body 21 is partitioned into an upper liquid chamber 21a and a lower liquid chamber 21b and the volume of the lower liquid chamber 21b in which the supernatant liquid and the adipose stem cells (SVF) For example, 30 cc, as compared with the capacity of the lower liquid chamber of the vessel 10 for extracting adipose stem cells.

In addition, the lower stopper 22 has the same structure as the lower stopper 12 of the vessel 10 for extracting adipose stem cells, and the upper lid 24 and the lower lid 25 may have a similar structure. The upper liquid chamber 21a of the main body 21 is sealed by the auxiliary cap 26. [

FIG. 7 is a flowchart illustrating a method for extracting adipose stem cells according to an embodiment of the present invention, and FIGS. 8 to 18 are diagrams for explaining each step shown in FIG.

First, as shown in FIG. 8, a kit for extracting adipose stem cells (SVF) from fat extracted from adipose tissue is prepared. At this time, the kit is shown in Table 1 below.

subject Extraction container
(60cc) Extraction container
(30 cc) Vessel
(200 cc) Activator
syringe Quantity (EA) 2 One One One 5

In the above Table 1, the extraction container 10 shown in Figs. 1 and 2 is used for the 60 cc extraction container, and the extraction container 20 shown in Figs. 5 and 6 is used for the 30 cc extraction container. use. The activator uses the patent No. 10-1429253 (registered on August 4, 2014) registered by the present applicant. Syringes use commonly used medical syringes.

Thereafter, as shown in Figs. 7 and 9, 100 cc of fat (1) is extracted from adipose tissue (ST1).

Thereafter, 100 cc of the extracted fat (1) is placed in a container (30) having a capacity of 200 cc, and then 20 cc of saline is introduced. Then, the fat 1 is ultrasonically decomposed using the ultrasonic device 50 (ST2).

 7 and 10, the ultrasonic decomposed fat 2 is sucked using the syringe 60, and 50 cc of each of the 60 cc extraction containers 10 is uniformly injected into the extraction containers 10 (ST3) .

Thereafter, as shown in Figs. 7 and 11, the fat 2 injected into the extraction container 10 is firstly centrifuged (ST4) by using the centrifuge 70. Then, as shown in Fig. At this time, the primary centrifugation is carried out at 3,200 rpm for 5 minutes.

As shown in FIG. 11, the primary centrifuged fat 2 is separately layered from the lower part of the extraction container 10 into the adipose stem cells (SVF) 2c / the supernatant liquid 2b / the pure fat 2a . At this time, the adipose stem cells (SVF) 2c located at the lowermost part of the extraction container 10 are introduced into the receiving groove 12a of the lower stopper 12 through the filter net 122 of the lower stopper 12 during the primary centrifugation, (SVF) 2c is firstly filtered by the filter network 122. In this process,

Thereafter, as shown in Figs. 7 and 12, among the pure fat 2a, the supernatant 2b and the adipose stem cell (SVF) 2c separated from the fat 2 through the primary centrifugation, (SVF) 2c and a supernatant 2b (a liquid containing a part of the adipose stem cells (SVF)) are extracted from the extraction container 10 using the syringe 80 and then transferred to the 30 cc extraction container 20 (ST5).

FIG. 13 is a view illustrating a process of extracting adipose stem cells and supernatant from an extraction container using the syringe shown in FIG. 12, and FIG. 14 is a diagram illustrating a process of extracting 30 cubic centimeters of adipose stem cells extracted from a syringe and a supernatant, And FIG.

(SVF) 2c filled in the receiving groove 121a of the lower stopper 12 of each of the two 60 cc extraction containers 10 by using the syringe 80 as shown in Fig. 13, Only the supernatant 2b is extracted. At this time, it is preferable that the syringe 80 is used by inserting the needle into the lower stopper 12 so as not to penetrate the filter net 122, and it is preferable to use two 60 cc extraction containers 10, And 15cc of the supernatant (2b) stacked thereon is extracted.

14, the syringe 80 is drawn into the injection port of the 30 cc extraction container 20, and the extracted adipose stem cells (SVF) 2c and the supernatant 2b are injected into the 30 cc extraction container 20 do.

On the other hand, when the adipose stem cells (SVF) 2c and the supernatant 2b are injected from the syringe 80 into the 30 cc extraction container 20, the centrifugal separator 70 is used to inject 30 cc The stem cells (SVF) 2c injected into the extraction container 20 and the supernatant 2b are subjected to secondary centrifugation (ST6). For reference, the left container in FIG. 15 is a container containing clear saline and is shown for comparison of color between the stem cell (SVF) 2c and the extraction container 20 filled with the supernatant liquid 2b.

After the secondary centrifugation, the adipose stem cells (SVF) contained in the supernatant liquid 2b are separated from the supernatant liquid 2b and stored in the receptacle 20 through the filter network of the lower cap 22 of the extraction container 20 And the lipid stem cells (SVF) 2c are secondarily filtered by the filter net 122 in this process.

Subsequently, as shown in Figs. 7 and 16, only the adipose stem cells (SVF) 2c separated into the receiving grooves of the lower stopper 22 are extracted and separated by using the syringe 90 in the same manner as in Fig. 13 ST7).

17 is a view showing a process of extracting adipose stem cells from an extraction container using the syringe shown in Fig. 16. As shown in Fig. 17, a needle of a syringe 90 is inserted into a lower stopper 22, 5cc of the adipose stem cell (SVF) 2c filled in the receiving groove of the cap 22 is extracted.

Thereafter, as shown in FIGS. 7 and 18, the adipose stem cells (SVF) 2c extracted by the injector 90 are relaxed 20 times by using the activator 40 to activate the adipose stem cells (SVF) 2c (ST8) (see Japanese Patent Registration No. 10-1429253 for the structure and action of the activator 40).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

10, 20: container for extracting adipose stem cells 11, 21:
12, 22: Lower stopper 13, 23: Upper stopper
14, 24: upper lid 15, 25: lower lid
21a: upper liquid chamber 21b: lower liquid chamber
111: inlet 112: auxiliary inlet
113: auxiliary stopper 121: packing
121a: receiving groove 121b:
121c: contact protrusion 122:

Claims (3)

A main body having a receiving space therein;
A lower stopper inserted into the lower portion of the main body to seal the lower portion of the main body; And
An upper cap for sealing an upper portion of the injection port provided in the main body; Lt; / RTI >
The lower stopper
A packing having an accommodating groove formed at an upper center portion thereof and having an outer surface inserted into the lower portion of the main body to be in tight contact therewith; And
A filtering net welded to an upper end of the packing to cover the receiving groove;
Wherein the stem cell extract is a bacterium.
The method according to claim 1,
The packing
An engaging part made of an elastic material and tightly brought into close contact with the inside of the body; And
A support portion formed of a material having a strength higher than that of the coupling portion and coupled to an upper portion of the coupling portion to fuse the filter net to improve the strength of the coupling portion; Lt; / RTI >
Wherein the fastening protrusions are formed on the outer surface of the coupling portion so as to closely contact the inner surface of the main body, and a seating jaw recessed downward is formed on an upper end of the support portion so that the filter net is seated. Container for cell extraction.
3. The method of claim 2,
Wherein the main body has an injection port protruded upward, and the accommodation space is divided into an upper liquid chamber and a lower liquid chamber by the injection port.

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