KR20120069330A - Isolating and culturing system for fat stem cells - Google Patents

Abstract

PURPOSE: An adipose-derived stem cell isolating/culturing system is provided to minimize time and space for moving a sample. CONSTITUTION: An adipose-derived stem cell isolating/culturing system comprises: a fat bag for containing adipose by inhalation; an adapter for the fat bag; an adapter insertion part for containing the adapter; a centrifugal separator(40) for centrifuging the sample in the fat bag; a heating block having the adapter; a culture machine(60) for culturing stem cells in the fat bag; a cell counter(70) for counting the number of stem cells; and a drawer(80) which is integrally mounted with the culture machine.

Description

Isolating and culturing system for fat stem cells}

The present invention relates to a system for extracting and culturing fat stem cells from organisms such as bone marrow, umbilical cord blood, peripheral blood, embryos, and more particularly, a centrifuge for separating and extracting fat stem cells from organisms, and extracted fat stem cells. Integrate the incubator to incubate while maintaining the temperature required to minimize the moving space and travel time of the worker and the sample to perform the centrifugation, temperature control and culture of the sample in a batch to perform the extraction process of fat stem cells It relates to an adipose stem cell isolation culture system that can be efficiently performed.

Stem cells have the ability to self-replicate and have the ability to differentiate into two or more cells. Recently, research has been conducted to obtain stem cells from adipose tissue in addition to bone marrow. Adipose-derived stem cells isolated from adipose tissue are multipotent stem cells with the ability to differentiate into various types of cells such as fat cells, muscle cells, chondrocytes, and bone cells.

Compared with the mesenchymal stem cells of bone marrow origin, the adipose stem cells have excellent self-renewal ability and easy in vitro culture, and are rich in adipose tissue capable of separating adipose stem cells, and are simple and safe to collect. The functional stem cells were found to have almost the same characteristics, such as differentiation ability, cytological characteristics, immunological characteristics, and tissue regeneration ability, even when compared with the mesenchymal stem cells of bone marrow origin. In other words, adipose stem cells are very useful for stem cells having a high possibility of replacing mesenchymal stem cells of bone marrow origin.

Also, compared with mesenchymal stem cells, adipose stem cells are easier and safer to acquire tissues, and have merit without restriction on tissue supply and facilitation of in vitro culture. It is said to be above the cell.

The conventional method of separating and culturing lipo-isolated cells is to break down the inhaled or excised fat and break down the tissue with collagenase. Methods of culturing cells have been generally proposed.

In addition, Korean Patent No. 788632 discloses a fat-containing suspension suspended in physiological saline obtained by liposucti on of human adipose tissue evenly and resuspended in an appropriate amount of saline to put an appropriate amount into a flask or roller bottle for cell culture. Next, stationary culture or rotary culture was performed. In the case of stationary culture, the cells are allowed to settle for at least 6 to 12 hours and then the cell layer (fat-derived MSC, fibroblast) attached to the flask surface is recovered by treatment with trypsin. At this time, to directly recover the suspended in a small amount of physiological saline, or if you want to reduce the volume of the cell layer, using only the pellet layer to be centrifuged for 10 minutes by centrifuging the cell layer recovered with physiological saline at 1000rpm. The separated cell layer contains adult stem cells and fibroblasts. A method of preparing a composition for cosmetic or cosmetic skin containing adult stem cells, fibroblasts and fat or fat cells by mixing the separated cell layers with fat has been proposed.

Meanwhile, in WO2005 / 042730, A) obtaining an aspirate by liposuction; B) transferring to a centrifuge to obtain a cell fraction of liposuction; C) obtaining a cell fraction by centrifugation according to specific gravity; D) A method for producing a collagen-free stem cell, which consists of collecting a cell layer having a lower specific gravity than the erythrocyte layer, has been proposed.

In order to produce adipose derived stem cells as in the above method is to be centrifuged to obtain an active ingredient from inhaled or excised fat. In addition, the mixture should be formed for secondary use development, such as for molding purposes.

Part of the problem that can be a big problem in this process is that it is not easy to separate the effective active ingredient in the first centrifugation process, and clinically, it is known that 500,000 cells can be separated from about 100cc of inhaled adipose tissue. It is an inefficient method of obtaining only one millionth of cells. That is, not only takes a long time to separate in the centrifugation process, but also damages the effective cells due to severe conditions at the time of separation.

In addition, there is a problem that mixing between the components to be mixed in the process of fat extraction is not easy.

Another problem can be fatal. In other words, there is a risk of microbial contamination while the cells are exposed to the outside during the acquisition, separation, extraction of active ingredients, culture, and re-injection of fat, which can cause side effects due to secondary infection or contamination. Can be.

In consideration of the above situation, as a conventional apparatus for separating stem cells with improved yield while minimizing damage to adipose tissue, a "stem cell separation apparatus" of Korean Patent Publication No. 10-2009-126930 has been published in a public patent publication.

"Stem cell separation apparatus" of the Patent Publication No. 10-2009-126930, in the stem cell separation apparatus as shown in Figure 1, the control unit 100 for adjusting the rotational force and rotational speed; A rotating part 200 which receives the rotational force from the control part 100 and has means for attaching and detaching the inserting rack 300; Consists of the insertion rack 300 is formed with a receiving means for accommodating a syringe or container 400 containing a liposuction material, the insertion rack 300 is one that can accommodate a syringe or container on one surface One or more second insertion grooves 330 are formed in a plane in which the first insertion grooves 320 are symmetrical thereto, and the shaft 110 of the controller 100 has a structure orthogonal to a syringe or a container, and the syringe Alternatively, the container 400 may be rotated inclined so that the upper and lower portions may be repeatedly reversed in the direction of gravity, and the gravity and the rotational force may act by centrifuging while being accommodated inside the syringe in which the suction procedure is performed from the human body. Stability is secured by blocking the opportunity for the liposuction material to come into contact with the outside, and the mixing conditions and the mixing efficiency are possible, so that the yield is improved while minimizing damage to the fat tissue. will be.

However, since the "stem cell separation device" of Korean Patent Publication No. 10-2009-126930 has to separate stem cells and transfer the separated stem cells to a separate incubator, the stem cells are exposed to the outside during the transfer process. Safety issues for humans, such as the risk of contamination, are present, as well as the time and labor required to transfer stem cells from the centrifuge to the incubator because the centrifuge and incubator are not located within the same working radius. There was a problem that the extraction process of fat stem cells is made inefficient due to unnecessary waste.

"Centrifuge Separator and Centrifugal Separation Method", thus developed, is disclosed in Korean Patent Laid-Open Publication No. 10-2010-005952.

Patent Publication No. 10-2010-005952 "Centrifuge and centrifugation method for centrifugation" is centrifugal separation for separating the target cells from the tissue extracted from the organism using centrifugation as shown in Figure 2 In the apparatus, the upper separation space 111 is formed by using a flexible material, and the tissue injection port 112 for injecting the extracted tissue into the upper separation space 111, the processing separately from the tissue injection port 112 An upper separation bag (110) including a treatment solution inlet (113, 114) for injecting a solution, and a solution outlet (115) for discharging residual material in the upper separation space (111) after centrifugation; It is formed integrally with the upper separation bag 110 by using a flexible material, and forms a lower separation space 121 connected to the upper separation space 111, and the target cells from the lower separation space 121. Lower separator bag 120 is formed to discharge the discharge port 122; And a partition member 130 for selectively opening and closing a passage connecting the upper separation space 111 and the lower separation space 121, wherein the upper separation bag 110 includes the tissue inlet 112 and the A treatment solution inlet (113, 114), and a valve member (101) for selectively opening and closing the solution outlet (115), the lower separation bag 120 is a valve for selectively opening and closing the target outlet (122) It comprises a member 101, consisting of the upper separation space 111 and the lower separation space 121 formed in the film separating bag formed in a shaker incubator to inject the enzyme and react, and in the rotary axis of centrifugation The film separation bag is placed in the order of the upper separation bag (110) and the lower separation bag (120) in order to recover the fat stem cells by centrifugation.

However, this "centrifugal separator and centrifugal separation method" is a centrifugal separator 100 consisting of the upper separation bag 110 and the lower separation bag 120 described in the specification is a container having a special structure Because of this, not only the structure of the shake incubator for positioning it, but also the structure of the centrifugal rotation shaft is unknown, so that a special container composed of the upper separating bag 110 and the lower separating bag 120 is provided in the shaking incubator. It is understood that it is located and cultured, mounted in a centrifuge and centrifuged, but this also means that the time taken to transfer stem cells from the centrifuge to the incubator is not located within the same working radius. There is a problem that extraction processing of fat stem cells is inefficient due to unnecessary waste of manpower. As well as the incubator and the centrifuge having a structure that can easily and easily employ a special container consisting of the upper separation bag 110 and the lower separation bag 120, as well as the same centrifuge and incubator It was necessary to increase the extraction efficiency of fat stem cells by excluding the unnecessary waste of time and manpower required to move stem cells from the centrifuge device to the incubator by placing them within the working radius of.

The present invention has been invented to solve various defects and problems caused by the conventional stem cell separation apparatus and culture apparatus in view of the above situation, and the object thereof is to inhale the adipose tissue and to keep the adipose tissue embedded in the fat bag. Stem cells are extracted and cultured in the required temperature atmosphere, but the centrifuge is integrated into the incubator so that the separate extraction and cultivation of fat stem cells are performed in the same workspace. It minimizes the chance of fat stem cells coming into contact with the outside during the process of movement while minimizing the incubation of the sample by minimizing the process of cultivating the sample. It can improve the yield while minimizing damage of fat stem cells. The purpose is to provide a separate culture system of fat stem cells.

Another object of the present invention is to integrate the incubator and the centrifuge and to mount the heating block while the adipose tissue is embedded in the plastic bag and to cultivate stem cells in the incubator to facilitate the control of the culture temperature while promoting convenience in use. In addition, the present invention provides a culture system for separating adipose stem cells capable of culturing a large amount in a short time by increasing the culture efficiency of the incubator.

Fat stem cell isolation culture system of the present invention for achieving the above object is a fat bag in which adipose tissue is sucked as a sample (10; fat bag); An adapter 20 for the bag, in which the bag 10 is mounted; An adapter insertion stand (30) in which the padback adapter (20) mounted therein is mounted; A centrifuge (40) mounted with the adapter insert (30) to centrifuge the sample in the plastic bag (10); A heating block (50) on which a pet adapter (20) is mounted, in which the pet bag (10) in which the stem filaments separated from the sample are embedded is mounted; An incubator (60) for culturing stem cells in the plastic bag (10) in the heating block (50) which is installed to be integrated with the centrifuge on the upper right side of the centrifuge (40); A well-known cell counter 70 installed integrally with the incubator 60 at the top of the incubator 60 and counting stem cells in the plastic bag 10; The lower part of the incubator 60 is characterized in that it consists of a drawer 80 for storing the article integrally installed with the incubator 60.

In the present invention, the adipose tissue is sucked and the adipose tissue is separated and extracted from the adipose stem cells while embedded in the plastic bag, and cultured in the required temperature atmosphere, but the extraction and culture work of the adipose stem cells in one same workspace By integrating the centrifuge and incubator to minimize the movement space and travel time of the worker and the sample, it is possible to streamline the processing such as cultivation of the sample and to provide the opportunity for fat stem cells to come into contact with the outside during the movement of the work. It is possible to improve the yield while minimizing the damage of fat stem cells due to the stability because there is no possibility of unnecessary germs being introduced into the sample during the movement, and the adipose tissue is embedded in the plastic bag by integrating the incubator and the centrifuge. Incubation of stem cells in the incubator While being able to easily adjust, it aims at the convenience of use, and has a special advantage of increasing the incubation efficiency of the incubator to enable a large amount of culture in a short time.

1 is an exploded perspective view of a conventional stem cell separation apparatus,
2 is a view showing a conventional centrifugal separation apparatus used for separation of adipose stem cells,
Figure 3 is a perspective view of the adipose stem cell isolation culture system of the present invention,
4 is an exploded perspective view of a centrifuge according to the present invention;
5 is a plan view of the incubator according to the present invention,
Figure 6 is a cross-sectional view of the adipose stem cell isolation culture system of the present invention.

Hereinafter, with reference to the accompanying drawings will be described in detail a preferred embodiment of the adipose stem cell isolation culture system of the present invention.

Figure 3 is a perspective view of the adipose stem cell isolation culture system of the present invention, Figure 4 is an exploded perspective view of the centrifuge according to the present invention, Figure 5 is a plan view of the incubator according to the present invention, Figure 6 of the present invention is a fat stem cell isolation culture system As a cross-sectional view, the fat stem cell isolation culture system of the present invention comprises a fat bag (10; fat bag) into which adipose tissue is sucked and embedded as a sample; An adapter 20 for the bag, in which the bag 10 is mounted; An adapter insertion stand (30) in which the padback adapter (20) mounted therein is mounted; A centrifuge (40) mounted with the adapter insert (30) to centrifuge the sample in the plastic bag (10); A heating block (50) on which the adapter (20) for mounting the padback (10) mounted therein is embedded with a stem filament centrifuged from the sample; An incubator (60) for culturing stem cells in the plastic bag (10) in the heating block (50) which is installed to be integrated with the centrifuge on the upper right side of the centrifuge (40); A well-known cell counter 70 installed integrally with the incubator 60 at the top of the incubator 60 and counting stem cells in the plastic bag 10; The lower part of the incubator 60 is configured with a drawer 80 for storing the article integrally installed with the incubator 60.

The fat bag 10 is made of polyvinyl chloride, and includes a tissue inlet 12 for injecting the tissue extracted into the inner hollow part 11; A treatment solution inlet 13 for injecting treatment solution separately from the tissue inlet 12; Stem cell outlet 14 for discharging stem cells cultured separately; A first valve 15 for selectively opening and closing the tissue inlet 12; A second valve (16) for selectively opening and closing the treatment solution inlet (13); It consists of a third valve 17 for selectively opening and closing the stem cell outlet (14).

The plastic bag adapter 20 is used to mount the fat bag 10 to the centrifuge 30, and is separated into the front case 21 and the rear case 22 and the front case 21. The plastic bag 10 is mounted between the rear case 22 and the front and rear cases 21 and 22 are fixed to each other by a locking means.

In addition, the adapter insert 30 is for mounting the patch bag adapter 20, the built-in plastic bag 10 to the centrifuge 30, made of a rectangular tubular shape of the insertion grooves on both sides of the upper side ( 31) is formed.

The centrifugal separator 40 includes a disc portion 41; A centrifugal separation means (43) having a plurality of bucket frames (42) having a 'Y' shape radially from the center; and a motor installed under the centrifugal separation means (43) to rotate the centrifugal separation means (43) And a control panel (45) having a means (44) and a microprocessor for controlling the motor means (44).

Here, the bucket frame 42 is formed with a projection (42a) for hanging the adapter insert 30.

In addition, the heating block 50 is to maintain the stem cells in the plastic bag 10 at a specific temperature, and has a space for accommodating the adapter 20 for a plastic bag in which the plastic bag 10 is embedded, The space is heated by a plate heater or cooled by a plate cooler to maintain the required temperature.

And, the incubator 60 is installed on the bottom surface (60a) of the inside of the chamber base plate 61 to move forward, backward, left, right and rotation by the eccentric interlocking means; A plurality of discs 62 installed on the base plate 61 at regular intervals in a horizontal direction; A receiving groove 63 formed to accommodate the heating block 50 in the disc 62; A door 64 for opening and closing the chamber; It consists of a control panel 65 provided with a microprocessor.

Next will be described in detail the action of the adipose stem cell isolation culture system of the present invention configured as described above.

First, the first valve 15 of the plastic bag 10 is opened and the adipose tissue is injected into the hollow part 11 of the plastic bag 10 through the tissue inlet 12, and then the first valve 15 is closed. 2 valve 16 is opened to inject the treatment solution reacting with the tissue through the treatment solution inlet 13 and then the second valve 16 is closed.

Next, the front case 21 and the rear case 22 of the plastic adapter 20 are separated, and the front and rear surfaces of the front and rear case 21 and the rear case 22 are inserted into the bag 10 by the locking means. Engage and secure the cases 21 and 22.

Subsequently, after inserting the adapter 20 for the bag, in which the bag 10 is mounted, to the adapter insert 30, the lid 41 of the centrifuge 40 is opened to open the bucket frame 42 of the centrifuge 40. The protrusion 42a in the fitting inserts the insertion groove 31 of the adapter insert 30 into which the adapter 20 for the bag is inserted and mounts the adapter insert 30 in the centrifuge 40, and the lid portion 41. To close it.

Subsequently, when the centrifuge 40 is operated by operating the control panel 45, the motor means 44 is operated so that the bucket frame 42 of the centrifuge 40 is rotated so that it is inserted into the adapter insert 30. The fat bag 10 mounted on the fat bag adapter 20 is rotated together with the fat bag adapter 20 so that the fat tissue in the fat bag 10 is centrifugal to centrifuge the fat stem cells from the fat tissue.

Here, the rotational speed of the bucket frame 42 by the motor means 44 is 300? According to the set value set in the microprocessor in the control panel 45. It can be arbitrarily adjusted in the range of 11,000 rpm, and the sample can be centrifuged while controlling the rotational speed of the motor by dividing it into a low speed step and a high speed step as necessary.

In this state, the third valve 16 of the plastic bag 10 is opened to discharge substances other than the stem cells centrifuged to the stem cell outlet 14, so that only the fat stem cells are provided in the hollow part 11 in the plastic bag 10. After this is left, put the plastic bag adapter (20) in the heating block 50 with the plastic bag (10) mounted to open the door 64 of the incubator 60, the heating block equipped with a plastic bag adapter (20) 50 is placed in the receiving groove 63 of the incubator 60, and the control panel 65 is operated to set the culture atmosphere (temperature humidity, etc.) in the incubator 60, and then the door 64 of the incubator 60. Close and start the incubator 60.

Accordingly, the eccentric interlocking means of the lower part of the incubator 60 is operated so that the base plate 61 moves forward and backward, left and right, and rotates. In addition, the front and rear, left and right movement and rotational movement with the plastic bag 10, in this state, the atmosphere in the chamber is adjusted to a setting atmosphere suitable for the culture of the sample to incubate the sample.

That is, when the temperature in the chamber becomes higher than the set temperature, the plate-shaped cooler installed in the heating block 50 operates to lower the temperature to the set temperature, and when the temperature becomes lower than the set temperature, the heating block 50 is installed. The plate heater is operated to raise the temperature to the set temperature.

In this manner, the adipocyte stem cells cultured in the incubator 60 can check the culture state of the adipocyte stem cells by counting the stem cells in the plastic bag 10 with a known cell counter 70 as necessary.

While the present invention has been described as a preferred embodiment, the present invention is not limited thereto, and various modifications can be made without departing from the spirit of the invention.

10 fat bag 11 hollow part
12: tissue injection port 13: treatment solution injection port
14 stem cell outlet 15 the first valve
16: second valve 17: third valve
20: pad adapter 21: front case
22: rear case 30: adapter insert
31: insertion groove 40: centrifuge
41: lid 42: bucket frame
42a: protrusion 43: centrifugation means
44: motor means 45: control panel
50: heating block 60: incubator
60a: bottom 61: base plate
62: disc 63: receiving groove
64: door 65: control panel
70: cell counter 80: drawer for storing goods

Claims (7)

A fat bag (10; fat bag) into which adipose tissue is sucked and embedded as a sample; An adapter 20 for the bag, in which the bag 10 is mounted; An adapter insertion stand (30) in which the padback adapter (20) mounted therein is mounted; A centrifuge (40) mounted with the adapter insert (30) to centrifuge the sample in the plastic bag (10); A heating block (50) on which the adapter (20) for mounting the padback (10) mounted therein is embedded with a stem filament centrifuged from the sample; An incubator (60) for culturing stem cells in the plastic bag (10) in the heating block (50) which is installed to be integrated with the centrifuge on the upper right side of the centrifuge (40); A well-known cell counter 70 installed integrally with the incubator 60 at the top of the incubator 60 and counting stem cells in the plastic bag 10; Fat stem cell separation culture system consisting of a drawer (80) for storing the goods installed integrally with the incubator (60) in the lower portion of the incubator (60). According to claim 1, wherein the fat bag (10; fat bag) is made of polyvinyl chloride (polyvinyl chloride), the tissue inlet 12 for injecting the tissue extracted into the inner hollow portion (11); A treatment solution inlet 13 for injecting treatment solution separately from the tissue inlet 12; Stem cell outlet 14 for discharging stem cells cultured separately; A first valve 15 for selectively opening and closing the tissue inlet 12; A second valve (16) for selectively opening and closing the treatment solution inlet (13); Fat stem cell separation culture system, characterized in that consisting of the third valve 17 for selectively opening and closing the stem cell outlet (14). The method of claim 1, wherein the adapter for the bag 20 is used to mount the fat bag (10; fat bag) to the centrifuge 30, separated into a front case 21 and a rear case 22 Fat and fat stem cell separation culture system characterized in that the front and back cases 21 and 22 are coupled to the front and back case 21 and the rear case 22 is fitted with a pad back (10) as a locking means. The method according to claim 1, wherein the adapter insert 30 is for mounting the packet adapter (20) in which the bag 10 is built in the centrifuge 30, formed in a rectangular tubular shape on the upper both sides of the outer side Adipose stem cell separation culture system, characterized in that the insertion groove 31 is formed in. According to claim 1, wherein the centrifugal separator (40) and the disc-shaped lid portion 41; A centrifugal separation means (43) having a plurality of bucket frames (42) having a 'Y' shape radially from a center thereof; A fat stem cell separation culture system comprising a means (44) and a control panel (45) containing a microprocessor for controlling the motor means (44). The method according to claim 5, wherein the bucket frame 42 is a fat stem cell separation culture system, characterized in that the projection (42a) is formed to hang the adapter insert (30). According to claim 1, The incubator 60 is installed on the bottom surface (60a) of the inside of the chamber base plate 61 for forward, backward, left, right and rotational movement by the eccentric interlocking means; A disc 62 disposed on the base plate 61 at regular intervals in the horizontal and horizontal directions; A receiving groove 63 formed to accommodate the heating block 50 in the disc 62; A door 64 for opening and closing the chamber; Adipose stem cell isolation culture system, characterized in that consisting of a control panel (65) having a microprocessor.

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