KR102566931B1 - Multi chamber type cleaning material container, apparatus and method for seperating stem cell using the same - Google Patents

Abstract

The present invention relates to a multi-stage detergent container for separating stem cells, and to a multi-stage stem cell separation apparatus and method including the same, and more particularly, to effectively wash collagenase or impurities mixed in the process of extracting stem cells from fat. It relates to a multi-stage washing container for separating stem cells, and a multi-stage stem cell separation device and method including the same.

Description

Multi-stage washing container for stem cell separation, multi-stage stem cell separation device and method including the same

The present invention relates to a multi-stage detergent container for separating stem cells, and to a multi-stage stem cell separation apparatus and method including the same, and more particularly, to effectively wash collagenase or impurities mixed in the process of extracting stem cells from fat. It relates to a multi-stage washing container for separating stem cells, and a multi-stage stem cell separation device and method including the same.

In general, stem cells refer to cells that have the ability to differentiate into all types of cells constituting the body, such as nerves, blood, cartilage, and the like, if necessary, while being maintained without differentiation into specific cells.

There are two major methods for obtaining these stem cells. The first is a method of obtaining stem cells (embryonic stem cells) from an embryo, and the second is a method of obtaining from stem cells (adult stem cells) kept in each part of the adult human body. Although these methods are different in terms of functionality, both embryonic stem cells and adult stem cells have the characteristics of being able to differentiate into various types of cells.

Embryonic stem cells have the advantage of excellent differentiation ability and long telomeres, but they have ethical problems, making it difficult to obtain a large amount of cells. In comparison, adult stem cells can obtain a large number of cells, but there is a risk of infection when transplanted to others and their differentiation ability is relatively low.

However, despite the above disadvantages, adult stem cells are characterized by being very safe for medical applications. In addition, since it has site-specific differentiation ability to differentiate according to the characteristics of the surrounding tissues and does not cause cancer even when injected in an undifferentiated state, necessary cells can be produced immediately after transplantation. In addition, it has the self-renewal ability to regenerate and store undifferentiated stem cells that are needed later.

On the other hand, adipose tissue plays an important role in normal growth and physiology in vivo, but its importance has been overlooked so far. The most common type of fat is located under the skin (subcutaneous fat), and there is also white adipose tissue located in the abdominal cavity (visceral fat) or in the reproductive organs (gonadal fat).

A somewhat less common form in adults is brown adipose tissue, which plays an important role in generating heat in the neonatal period (Gimble, New Biol. 2(4):304-12, (1990)). However, fertility and maturation processes are closely related to an individual's adipose tissue storage. Puberty in females and males 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, many studies have been conducted on the use of adipose tissue, and recently, as it was confirmed that adult stem cells exist in adipose tissue (Zuk PA, et al., Molecular Biology of Cell, Vol 13: 4279-4295 ( 2002): RodriguezAM, et al., Biochimie, 87: 125-128 (2005)) Various studies on the utilization of adipose-derived stem cells have begun to be conducted.

In addition, based on the development of biochemistry and molecular biology, a small amount of signal substances (growth factors) that exist in our body have been discovered, and based on this, the biological aging theory is being reestablished (Stanley Cohen, Nobel Lecture 1986, Dec. 8).

Signal substances (growth factors) decrease in vivo as age increases, and it has been revealed that the decrease in growth factors 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).

In this way, research on the structure of signal substances (growth factors) has been conducted, but since most growth factors have a protein structure, the structure is three-dimensionally complex, resulting in various problems in chemical synthesis. The reality is that the cost involved is also very high.

Accordingly, in the process of research on how to obtain an active human growth factor at a low price while maintaining its activity in the human body, it was known that adipose-derived adult stem cells secrete growth factors (Rehman, J. et al. ., Circulation, 109:1292-1298 (2004)).

Therefore, research on methods of obtaining stem cells from adipose tissue is being conducted, and representative methods of extracting adipose stem cells through processes such as fat extraction, enzyme treatment, adipose stem cell washing and separation, and adipose stem cell filtration this is being used

Specifically, an extraction step of extracting fat from adipose tissue, an enzyme treatment step using collagenase, etc., adipose stem cell separation and washing step using centrifugation, and a filtration step to remove clumped (lump) adipose stem cells, etc. includes

Among them, the adipose stem cell separation and washing step using centrifugation is a process that is performed multiple times, and a centrifugal separator is used to extract adipose stem cells from fat extracted with a luer lock syringe or to wash the extracted adipose stem cells with a washing solution. .

However, in the method of extracting adipose stem cells (SVF) according to the prior art, in the process of separating SVF from fat, a separation vessel and a filtration vessel had to be separately used to separate and filter the adipose stem cells (SVF).

Therefore, the separation and filtration process is cumbersome, requiring a lot of extraction time, and the use of many containers not only wastes extraction cost, but also reduces the amount of stem cell extraction.

In addition, since the detergent and the stem cell stock solution are not uniformly mixed during the stem cell washing process, there is a problem in that sufficient washing is not possible and the number of washings that can be performed in one washing container is not sufficient.

Republic of Korea Patent No. 10-1354959 Republic of Korea Patent No. 10-1598053 Republic of Korea Patent No. 10-1376169 Republic of Korea Patent No. 10-1557190 Republic of Korea Patent No. 10-1838220

The present invention is to solve the above problems, a multi-stage detergent container for separating stem cells that can effectively remove collagenase or impurities mixed in the process of extracting stem cells from fat, and multi-stage stem cell separation including the same It is intended to provide an apparatus and method.

In addition, the present invention uses a multi-tiered detergent container to wash the stem cells multiple times, put a strainer in the extraction container to remove impurities, and put a leak prevention film on the detergent container to prevent the leakage of the detergent during use. It is intended to provide a multi-stage detergent container for separation, and a multi-stage stem cell separation device and method including the same.

To this end, the multi-stage detergent container for separating stem cells according to the present invention includes a detergent container body having a receiving part in which the detergent is accommodated; a partition wall dividing the accommodating part into a first accommodating part and a second accommodating part in the interior of the cleaning agent container body; a first detergent injection unit connected to the first accommodating part at one side of the detergent container body and coupled to a stem cell extracting container to supply detergent; and a second detergent injection unit connected to the second accommodating part at the other side of the detergent container body and coupled to the stem cell extracting container to supply detergent.

At this time, a first cleaning agent container cap assembled to open and close the first cleaning agent injection unit; and a second cleaning agent container cap assembled to open and close the second cleaning agent injection unit.

In addition, a plurality of mesh holes installed inside the first accommodating portion to block the discharge portion of the first cleaning agent injection unit and having a size that prevents the free fall of the cleaning agent inside the first accommodating portion due to gravity are formed. 1 anti-leak film; and a second portion installed inside the second accommodating portion to block the discharge portion of the second cleaning agent injection unit and having a plurality of mesh holes having a size to prevent the free fall of the cleaning agent inside the second accommodating portion due to gravity. It is preferable to include; a leakage prevention film.

On the other hand, the multi-stage stem cell separation device according to the present invention includes a multi-stage detergent container as described above; A degrading enzyme container in which fat is accommodated together with the degrading enzyme; and an extraction container coupled to the degrading enzyme container to extract stem cells degraded from the fat and washing the stem cells by receiving a detergent from the multi-stage detergent container.

At this time, the decomposition enzyme container accommodates the decomposition enzyme and the fat therein, and one end of the cylinder having a tip for input and output; a gasket provided inside the cylinder; It is preferable to include; and a plunger (Plunger) for advancing and retracting the gasket.

In addition, the extraction container and the extraction container body in which the stem cells are accommodated; A fastener provided on one side of the extraction container body and coupled to the tip of the decomposition enzyme container; It is preferable to include; and a strainer installed inside the extraction vessel body to block the discharge portion formed through the fastener.

In addition, it is preferable to further include an extraction vessel cap assembled to open and close the fastener of the extraction vessel.

Furthermore, the multi-stage stem cell separation method according to the present invention includes the steps of: 1) injecting fat into a degrading enzyme container in which the degrading enzyme is accommodated; 2) coupling and connecting an extraction vessel to the input/output tip of the decomposition enzyme vessel; 3) extracting the stem cells in the extraction vessel by installing the extraction vessel in a centrifugal separator with the extraction vessel facing down; 4) separating the extraction vessel from the degrading enzyme vessel; 5) connecting the extraction vessel to a first cleaning agent injection unit provided on one side of a cleaning agent container having an interior divided into two parts; 6) firstly washing the stem cells by placing the extraction vessel in a centrifuge with the vessel facing up; 7) secondarily washing the stem cells by placing the extraction vessel in a centrifugal separator in a downward state; 8) separating the extraction vessel from the cleaning agent container and sealing the first cleaning agent injection part; 9) connecting the extraction container to a second cleaning agent injection unit provided on the other side of the cleaning agent container; 10) tertiary washing of the stem cells by placing the extraction vessel in a centrifuge with the vessel facing up; 11) fourth washing of the stem cells by placing the extraction vessel in a centrifuge with the vessel facing down; 12) separating the extraction vessel from the cleaning agent vessel; and 13) extracting and storing purified stem cells in the extraction container.

The present invention as described above has the effect of washing the extracted stem cell stock solution several times using a multi-stage detergent container. Therefore, it is possible to effectively remove collagenase or impurities mixed in when stem cells are extracted from fat.

In particular, during the washing process, the high-density stem cell stock solution and the low-density detergent are alternately placed in the centrifuge, so that the stem cell stock solution and the detergent are effectively mixed according to the difference in density while washing is performed.

In addition, a sieve is provided inside the extraction container to remove impurities from the stem cell stock solution, and a leak prevention film is placed on the detergent container to prevent leakage of the detergent during use.

1 is a configuration diagram showing a multi-stage stem cell separation device according to the present invention.
2 is a cross-sectional view showing another stem cell separation device according to the present invention.
3 is a diagram showing a multi-stage washing agent container for separating stem cells of the present invention.
Figure 4 is a diagram showing the degradation enzyme container of the present invention.
5 is a view showing the extraction vessel of the present invention.
Figure 6 is an embodiment showing the coupling state of the decomposition enzyme vessel and the extraction vessel in the present invention.
Figure 7 is an embodiment showing the combined state of the cleaning agent container and the extraction container in the present invention.

Hereinafter, with reference to the accompanying drawings, a multi-stage detergent container for separating stem cells according to a preferred embodiment of the present invention, and a multi-stage stem cell separation device and method including the same will be described in detail.

First, referring to FIGS. 1 and 2, the multi-stage stem cell separation device according to the present invention will be described. (30).

Therefore, after the adipose stem cells are introduced from the degrading enzyme container 20 into the extraction container 30, the stem cells are washed multiple times by combining the extraction container 30 with the multi-stage cleaner container 10 containing the detergent. to lose

To this end, the multi-stage detergent container (10, hereinafter referred to as 'detergent container') is partitioned into a first accommodating part 11a and a second accommodating part 11b, each containing a detergent, and the decomposition enzyme container 20 ) contains fat with decomposing enzymes inside.

The extraction container 30 is coupled to the degrading enzyme container 20 to extract the decomposed stem cells from the fat, and then receives a detergent such as saline from the detergent container 10 to wash the stem cells.

As shown in (a) to (c) of FIG. 3, the detergent container 10 supplies a detergent used for washing stem cells, and is a detergent for washing stem cells extracted (derived) from fat, that is, adipose stem cells. is accepted.

As a cleaning agent, saline may be used as an example. At this time, the stem cells extracted from the fat may exist in the state of a stem cell stock solution containing collagenase, which is a degrading enzyme, and the detergent washes the collagenase as described above.

To this end, the cleaning agent container 10 includes a cleaning agent container body 11, a partition wall 12, a first cleaning agent injection unit 13 and a second cleaning agent injection unit 14, more preferably a first cleaning agent container cap. (15), a second cleaning agent container cap 16, a first leakage prevention film 17 and a second leakage prevention film 18.

Here, the cleaning agent container body 11 has a receiving portion, which is a space in which the cleaning agent is accommodated, and is made of a cylindrical shape in an embodiment, and may be made of a transparent material so that the inside can be confirmed.

The partition wall 12 is installed inside the cleaning agent container body 11, and divides the receiving portion inside the cleaning agent container body 11 into a first accommodating portion 11a and a second accommodating portion 11b, so that they are Let them be partitioned from each other without being connected (leakage/mixing).

The detergent contained in the first accommodating part 11a and the second accommodating part 11b may be the same as saline solution, but may be different from each other if necessary. If different, different types or concentrations of the first cleaning agent and the second cleaning agent may be applied.

Hereinafter, the cleaning agent prepared in the first accommodating part 11a and the second accommodating part 11b will be described as an example. In addition, an example of washing a total of 4 times with detergents stored in 2 locations is described in detail below.

The first detergent injection unit 13 is connected to the first accommodating part 11a at one side of the detergent container body 11 and is coupled to the extraction container 30 for extracting stem cells to supply the detergent. For example, the first cleaning agent injection part 13 protrudes from the top of the first accommodating part 11a for a certain length, has a flow path formed therein, and has means for coupling such as a screw thread.

The second detergent injection unit 14 is connected to the second accommodating part 11b on the other side of the detergent container body 11, and is coupled to a container for extracting stem cells to supply the detergent. For example, the second cleaning agent injection part 14 protrudes from the lower end of the first accommodating part 11a, has a flow path formed therein, and is provided with means for coupling such as a screw thread.

The first cleaning agent container cap 15 is assembled to open and close the first cleaning agent injection unit 13, and a structure such as a screw coupling method as shown in the drawing and an interference fitting method can be applied if leakage of the cleaning agent can be prevented. can

The second cleaning agent container cap 16 is assembled to open and close the second cleaning agent injection unit 14, and various other structures such as a screw coupling method as shown and an interference fit method may also be applied.

The first leakage prevention film 17 separates the first detergent container cap 15 and removes the detergent contained in the first accommodating part 11a of the detergent container 10 during use (during the stem cell washing process). 1 Prevents leakage through the detergent injection part 13.

For example, combining or separating the cleaning agent container 10 from the extraction container 30 so that the cleaning agent container 10 is alternately washed multiple times using the first cleaning agent injection unit 13 and the second cleaning agent injection unit 14 of the cleaning agent container 10 Prevent spillage of detergent during the process.

To this end, the first leakage prevention film 17 is installed inside the first accommodating part 11a to block the discharge part (flow path) formed in the first cleaning agent injection part 13 . At the same time, a plurality of mesh holes are formed in the first accommodating part 11a to prevent the free fall of the cleaning agent due to gravity.

The mesh hole may be set to a size of approximately 50 um to 100 um, and due to the ultra-fine mesh hole, the cleaning agent flows only by removing the first cleaning agent container cap 15 unless it is rotated at high speed in a centrifuge or sucked with a syringe or the like. don't get off

Similarly, the second accommodating portion 11b is provided with a second leakage preventing film 18, and the second leakage preventing film 18 discharges the second cleaning agent injection part 14 from the inside of the second accommodating portion 11b. It is installed to prevent wealth. In addition, a plurality of mesh holes are formed in the second accommodating part 11b to prevent free fall of the detergent due to gravity.

On the other hand, the multi-stage stem cell separation device according to the present invention uses the above-described multi-stage detergent container 10 as one component to extract stem cells from fat together with the degrading enzyme container 20 and the extraction container 30 and wash them Construct a stem cell extraction kit that

As shown in (a) and (b) of FIG. 4 , the decomposition enzyme container 20 is to contain fat together with the decomposition enzyme therein, and a luer lock syringe may be used as an example.

Accordingly, the decomposition enzyme container 20 includes a cylinder 21 having a tip 21a for input and output and a gasket 22 provided inside the cylinder 21 to accommodate the decomposition enzyme and the fat therein. and a plunger 23 advancing and retracting the gasket 22.

The plunger 23 has a male thread formed at the center of the front portion thereof, so that it can be coupled to or separated from the female thread formed at the center of the rear portion of the gasket 22 in a screw fastening method. It can be mounted in a centrifuge in a separated state.

As shown in (a) to (c) of FIG. 5, the extraction container 30 has a configuration in which the stem cells (or stock solution) are accommodated, and is coupled to the degrading enzyme container 20 to extract the stem cells decomposed from the fat, Furthermore, the cleaning agent is supplied from the multi-stage cleaning agent container 10 so that the stem cells are washed.

To this end, the extraction vessel 30 includes an extraction vessel body 31, fasteners 32 and 33, and strainers 36 and 37 as an example, and preferably further includes an extraction vessel cap 34 and 35. can do.

Here, the extraction vessel body 31 has a cylindrical shape to accommodate stem cells or a stock solution containing the stem cells therein, for example, to be inserted into a bucket and then mounted in a centrifuge to facilitate use.

The fasteners 32 and 33 are provided on one side of the extraction container body 31 and are coupled to the tip 21a of the degrading enzyme container 20, and the inside of the fasteners 32 and 33 made of a connector shape A passage through which entry and exit is possible is formed through which the stem cells and cleaning agent flow.

These fasteners 32 and 33 are detachably coupled to the input/output tip 21a of the aforementioned degrading enzyme container 20, or the first cleaning agent injection unit 13 and the second cleaning agent injection unit of the cleaning agent container 10 A screw thread may be formed to be detachably coupled to (14). Of course, the illustration is omitted, but the structure of the interference fit method that is interlocked with each other may also be applied.

In addition, the fasteners 32 and 33 may be provided only in the extraction vessel body 31, but may be composed of a first fastener 32 and a second fastener 33 respectively formed at the top and bottom.

At this time, the first fastener 32 and the second fastener 33 may be used regardless of order or direction, but the first fastener 32 is used as an inlet and the second fastener 33 is used as an outlet. can be used, and vice versa.

The strainers 36 and 37 are installed to block the passage formed through the fasteners 32 and 33, that is, the discharge part, inside the extraction vessel body 31 as above, and the strainers 36 and 37 pass only the stem cells. and block other foreign substances from entering. To this end, the strainers 36 and 37 may be designed to have holes of about 100um.

Therefore, when the stem cell stock solution accommodated in the extraction vessel 30 is moved to a storage syringe (not shown), impurities are removed in the process of passing through the sieve 36 and 37 to obtain high purity stem cells.

However, when the first fasteners 32 and the second fasteners 33 are provided at the top and bottom of the extraction container body 31, the strainers 36 and 37 also have an accommodation space inside the extraction container body 31. A first strainer 36 and a second strainer 37 may be provided at the upper and lower portions of the middle, respectively.

This is also true of the extraction vessel caps 34 and 35 that open and close the fasteners 32 and 33 of the extraction vessel 30. That is, the extraction vessel caps 34 and 35 include the first extraction vessel cap 34 and the second extraction vessel cap 35 to be coupled to the first fastener 32 and the second fastener 33, respectively. can

The extraction container caps 34 and 35 are not used during centrifugation or block the fasteners 32 and 33 of the extraction container 30 during storage or movement to prevent external contamination and to prevent leakage of internally contained substances. It is closely coupled to the fasteners 32 and 33.

Hereinafter, a multistage stem cell separation method for separating stem cells from fat using the aforementioned stem cell separation device will be described.

First, the present invention fills the degrading enzyme in the degrading enzyme container (20). As an example, the decomposition enzyme may be composed of saline and collagenase, and fills 20cc.

As described above, the fat absorbed from the human body is injected (injected) into the decomposition enzyme container 20 in which the decomposition enzyme is accommodated. As an example, 60 cc of fat may be injected and subdivided into 3 kits of 20 cc each, and fat may be injected before decomposition enzyme.

When fat and decomposing enzyme are injected into the decomposing enzyme container 20, the gasket 22 is moved to the top, and in this state, the plunger 23 screwed to the gasket 22 is fastened so that it can be mounted on the centrifuge. can be released and removed.

After that, the fasteners 32 and 33 of the extraction vessel 30 are coupled to the input and output tip 21a of the decomposition enzyme container 20 and connected to each other, and if there are several fasteners 32 and 33, any one of them combine into one The coupling may be performed by inserting the male thread of the extraction container 30 into the female thread formed on the tip 21a of the decomposition enzyme container 20 and screwing it (see FIG. 6).

In this way, in a state in which the degraded enzyme container 20 and the extraction container 30 are combined, the extraction container 30 is mounted on a centrifuge with the extraction container 30 positioned at the bottom and centrifuged. Through this, the stem cells are extracted in the extraction container 30 .

That is, the relatively low-density saline solution goes up, and the high-density stem cells go down, and as a result, the stem cells are extracted while being accommodated in the extraction container 30 disposed on the lower side.

Next, after the stem cells are extracted, the extraction container 30 is separated from the degrading enzyme container 20, and the fastener 32 of the extraction container 30 is inserted into the first detergent injection part formed on one side of the detergent container 10. (13) is entered into.

The stomach cleaning agent container 10 is spatially partitioned into a first accommodating portion 11a and a second accommodating portion 11b by a partition wall 12 provided therein, and the first cleaning agent and the second cleaning agent are accommodated therein, respectively. In an embodiment, both the first cleaning agent and the second cleaning agent may be accommodated as saline solution by 20 cc.

In this assembled state, the extraction container 30 is mounted in a centrifuge with the upward facing state to perform primary washing of the stem cells. is connected to the extraction vessel 30 and proceeds (see (a) of FIG. 7).

When the centrifugation for the first washing proceeds, since the extraction container 30 is placed on top, the high-density stem cells sink back down and are sufficiently mixed with the first washing agent to perform washing.

After the first washing is completed, after the separation in the centrifugal separator, the stem cell stock solution is washed again with the first washing agent by placing the extraction container 30 in the centrifuge with the downward direction opposite to the first washing (Fig. 7). see (b)).

That is, two washings are performed with the first cleaning agent prepared in the first accommodating part 11a. In the first washing, the extraction container 30 is mounted facing upward, and in the second washing, the extraction container 30 is downward. It is to be installed facing up and cleaned.

In this way, during the first and second cleaning with the first cleaning agent, the second cleaning agent injection unit 14 is kept sealed by the second cleaning agent container cap 16 .

After completing the first and second washing with the first cleaning agent, the extraction vessel 30 is separated from the first cleaning agent inlet 13, and the first cleaning agent inlet 13 is connected to the first cleaning agent container cap 15 sealed with

However, since any one of the first cleaning agent injection unit 13 and the second cleaning agent injection unit 14 can be selected and used first, the order or selection can be freely changed, but as an embodiment, the first cleaning agent injection unit ( 13) is used, and then the second cleaning agent injection unit 14 is used.

Next, the second cleaning agent container cap 16 sealing the second cleaning agent injection unit 14 is separated, and the fastener 33 of the extraction container 30 is fastened. At this time, there is a first leakage prevention film 17 and a second leakage prevention film 18 inside the cleaning agent container 10 to prevent the detergent from flowing out only by gravity.

Thereafter, the extraction vessel 30 is mounted in a centrifuge with the upward facing state, and the stem cell stock solution is washed three times with the second cleaning agent prepared in the second accommodating part 11b. Even during the third washing, since stem cells with high density are mounted on top, washing is performed while the stem cells sink to the bottom and are sufficiently mixed with the second washing agent.

After the third washing, they are separated from the centrifuge, and opposite to the third washing, the stem cell stock solution is washed a fourth time with a second detergent by placing the stem cells in a centrifuge with the extraction container 30 facing down.

That is, it is washed twice with the second cleaning agent. In the third washing, the extraction container 30 is mounted facing upward, and in the fourth washing, the extraction container 30 is mounted facing downward.

Therefore, the stem cells can be washed at least four times using one multi-stage detergent container 10 equipped with the first accommodating part 11a and the second accommodating part 11b and each prepared with detergent.

In addition, during the first and third washing, the stem cell stock solution is mounted so that it is positioned above the cleaning agent, so that the stem cell stock solution sinks to the bottom and is sufficiently mixed with the cleaning agent for effective cleaning.

In this way, after washing the stem cell stock solution for the fourth time, the extraction container 30 is separated from the second detergent injection part 14, and the second detergent injection part 14 is sealed with the second detergent container cap 16. do.

Finally, the purified stem cells are extracted through the extraction port formed on the other side of the extraction vessel 30 and stored in a separate storage syringe (not shown) to complete the present invention. At this time, the present invention is provided inside the extraction vessel 30 Since the impurities contained in the stem cell stock solution are filtered through the strainers 36 and 37, the purity of the purified stem cells is high.

In the above, specific embodiments of the present invention have been described in detail. However, the spirit and scope of the present invention is not limited to these specific embodiments, but those skilled in the art can make various modifications and variations without changing the gist of the present invention. You will understand when you grow up.

Therefore, since the embodiments described above are provided to completely inform those skilled in the art of the scope of the invention to which the present invention pertains, it should be understood that it is illustrative and not limiting in all respects, The invention is only defined by the scope of the claims.

10: multi-stage cleaning agent container
11: cleaning agent container body
12: bulkhead
13: first cleaning agent injection unit
14: second cleaning agent injection unit
15: first cleaning agent container cap
16: second cleaning agent container cap
17: first leakage prevention film
18: second leakage prevention film
20: degrading enzyme container
21: cylinder
21a: tip for entry and exit
22: gasket
23: Plunger
30: extraction vessel
31: brewing vessel body
32, 33: fasteners (first fastener, second fastener)
36, 37: strainer (first strainer, second strainer)
34, 35: extraction vessel cap (first extraction vessel cap, second extraction vessel cap)

Claims (8)

a cleaning agent container body 11 having a receiving portion in which the cleaning agent is accommodated;
a partition wall 12 dividing the accommodating part into a first accommodating part 11a and a second accommodating part 11b inside the cleaning agent container body 11;
a first detergent injection unit 13 connected to the first accommodating part 11a at one side of the detergent container body 11 and coupled to an extraction container for extracting stem cells to supply a first detergent; and
A second detergent injection unit 14 connected to the second accommodating part 11b at the other side of the detergent container body 11 and coupled to an extraction container for extracting the stem cells to supply a second detergent; includes and
The extraction vessel 30 is connected to the first detergent injection unit 13 to wash the stem cells with the first detergent accommodated in the first accommodating unit 11a,
Multi-stage washing agent for separating stem cells, characterized in that the extraction container 30 is connected to the second washing agent injection part 14 and the stem cells are washed using the second washing agent accommodated in the second receiving part 11b. courage. According to claim 1,
a first cleaning agent container cap 15 assembled to open and close the first cleaning agent injection unit 13; and
The multi-stage detergent container for separating stem cells, characterized in that it further comprises; a second detergent container cap 16 assembled to open and close the second detergent injection part 14. According to claim 2,
It is installed inside the first accommodating part 11a to block the discharge part of the first cleaning agent injection part 13 and has a size to prevent the free fall of the cleaning agent inside the first accommodating part 11a due to gravity. a first leakage prevention film 17 in which a plurality of mesh holes are formed; and
It is installed inside the second accommodating part 11b to block the discharge part of the second cleaning agent injection part 14, and has a size that prevents the free fall of the cleaning agent inside the second accommodating part 11b due to gravity. A multi-stage detergent container for separating stem cells, characterized in that it includes; a second leakage prevention film (18) in which a plurality of mesh holes are formed. A multi-stage cleaning agent container 10 according to any one of claims 1 to 3;
A degrading enzyme container 20 in which fat is accommodated with degrading enzymes therein; and
An extraction container (30) coupled to the degrading enzyme container (20) to extract stem cells decomposed from the fat and washing the stem cells by receiving a detergent from the multi-stage detergent container (10). A multi-stage stem cell separation device. According to claim 4,
The degrading enzyme container 20,
a cylinder (21) having a tip (21a) formed at one end for accommodating the decomposing enzyme and the fat therein;
A gasket 22 provided inside the cylinder 21; and
Multi-stage stem cell separation device comprising a; plunger (Plunger, 23) for advancing and retracting the gasket (22). According to claim 4,
The extraction vessel 30,
an extraction container body 31 in which the stem cells are accommodated;
Fasteners 32 and 33 provided on one side of the extraction vessel body 31 and coupled to the tip 21a of the decomposition enzyme vessel 20; and
The multi-stage stem cell separation device comprising: a strainer (36, 37) installed inside the extraction container body (31) to block the discharge portion penetrating the fasteners (32, 33). According to claim 6,
The multi-stage stem cell separation device further comprising extraction vessel caps 34 and 35 assembled to open and close the fasteners 32 and 33 of the extraction vessel 30. 1) injecting fat into the decomposition enzyme container 20 in which the decomposition enzyme is accommodated;
2) coupling and connecting the extraction vessel 30 to the input/output tip 21a of the decomposition enzyme vessel 20;
3) extracting the stem cells in the extraction vessel 30 by placing the extraction vessel 30 in a centrifuge with the downward facing state;
4) separating the extraction vessel 30 from the degrading enzyme vessel 20;
5) Connecting the extraction vessel 30 to the first cleaning agent injection unit 13 provided on one side of the cleaning agent vessel 10 whose interior is divided into two parts, the first housing unit 11a and the second housing unit 11b doing;
6) firstly washing the stem cells using a first detergent contained in the first accommodating part 11a by mounting the extraction container 30 in a centrifugal separator in an upward state;
7) mounting the extraction vessel 30 in a centrifugal separator in a downward state, and secondarily washing the stem cells using a first cleaning agent accommodated in the first accommodating part 11a;
8) separating the extraction container 30 from the cleaning agent container 10 and sealing the first cleaning agent injection part 13;
9) connecting the extraction vessel 30 to the second cleaning agent injection unit 14 provided on the other side of the cleaning agent container 10;
10) thirdly washing the stem cells using a second detergent contained in the second accommodating part 11b by mounting the extraction container 30 in a centrifugal separator in an upward state;
11) fourthly washing the stem cells using a second cleaning agent contained in the second accommodating part 11b by installing the extraction container 30 in a centrifugal separator in a downward state;
12) separating the extraction vessel 30 from the cleaning agent vessel 10; and
13) Step of extracting and storing the purified stem cells in the extraction container 30; multi-stage stem cell separation method characterized in that it comprises.

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