KR20090126930A - Devicefor isolating stem cell - Google Patents

Abstract

PURPOSE: An apparatus for sorting stem cells is provided to minimize damage of adipose tissue and obtain fat-derived stem cell with high yield. CONSTITUTION: An apparatus for sorting stem cells comprises: a controller(100) which regulates rotation force and speed; a rotation part(200) having a device which receives rotation force from the controller and has a detachable insertion rack; and the insertion rack(300) having a container device for injection or container(400).

Description

 Stem cell separation device {Devicefor Isolating stem cell}

The present invention relates to a device for separating stem cells from adipose tissue, and more particularly, to a stem cell separation device that minimizes the transplant delay by blocking the air exposure opportunity.

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 that have 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. In comparison with the mesenchymal stem cells of bone marrow origin, adipose stem cells in functional aspect were found to have almost the same characteristics such as differentiation ability, cytological characteristics, immunological characteristics, and tissue regeneration ability. 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 there are no limitations on the supply and demand of tissues, and they are easy to culture in vitro. It will be superior to the cell.

In the conventional method of separating and culturing lipo-separated cells, the inhaled or excised fat is crushed again to disintegrate tissue into collagenase, and then centrifuged to precipitate and separate stromal vascular fraction (SVF). In general, methods for culturing cells have been proposed.

In addition, Korean Patent Invention No. 788632 discloses a fat-containing suspension suspended in physiological saline obtained by liposuction of human adipose tissue evenly and resuspended with an appropriate amount of physiological saline to put an appropriate amount into a cell culture flask or roller bottle. 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 order to solve the above problems, an object of the present invention is to provide a stem cell separation apparatus with improved yield while minimizing damage to adipose tissue.

Another object of the present invention is to provide a stem cell separation apparatus capable of obtaining an effective ingredient with high effectiveness in a short time.

Still another object of the present invention is to provide a stem cell separation apparatus having improved stability by blocking the possibility of contamination in the air.

Another object of the present invention to provide a stem cell separation device that is easy to sterilize through a modular configuration.

In order to achieve the above object, the present invention provides a stem cell separation apparatus, comprising: a control unit for controlling rotational force and rotational speed; A rotating unit which receives the rotational force from the control unit and has means for attaching and detaching the inserting rack; It includes an insertion rack formed with a receiving means for accommodating a syringe or a container containing a liposuction material, the syringe or container may be reversed in the direction of gravity in the upper and lower parts repeatedly, the gravity and rotational force is It provides a stem cell separation device that rotates obliquely so that all can work.

In another aspect, the present invention provides a stem cell separation device that rotates relative to the center of the syringe or container when the oblique rotation.

In another aspect, the present invention provides a stem cell separation device having the inclined angle of 30 to 60 degrees.

In another aspect, the present invention provides a stem cell separation device of the rotation time 30 to 90 minutes.

In another aspect, the present invention provides a stem cell separation device having the rotational speed of 5 to 15g.

In another aspect, the present invention provides a stem cell separation device having a coupling hole corresponding to the coupling hole formed in the rack on the opposite side is connected to the shaft formed in the control unit for receiving the rotational force from the control unit, the opposite side to detachable the rack. .

In another aspect, the present invention provides a stem cell separation device is inserted into a four-frame flame structure.

In another aspect, the present invention provides a stem cell separation apparatus having one or more first insertion grooves that can accommodate a syringe or a container on one surface of the receiving means of the insertion rack, the one or more second insertion grooves formed on a surface symmetrical thereto. .

In another aspect, the present invention provides a stem cell separation device that is a stem cell separation device that is an open chamber so that the receiving means of the inserting rack can accommodate a syringe or a container on one side.

In another aspect, the present invention provides a stem cell separation device wherein the insertion groove or the chamber is formed in the same shape or different shapes.

In another aspect, the present invention provides a stem cell separation device having a structure in which the shaft of the controller is orthogonal to the syringe or the container.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers.

Hereinafter, the method and apparatus of the present invention are described by way of fat, but the type of tissue is adipose tissue, blood, bone marrow, muscle, skin, liver, connective tissue, fascia and other soft liquid tissues or tissue components. It includes, but is not limited to these.

Liposuction material as used herein refers to all materials that occur during liposuction. Typically such liposuction material includes adipose tissue and suction by liposuction.

In the stem cell separation according to the present invention, first, a starting material should be obtained. The starting material can be obtained via liposuction as fat. The fat may be human fat, and may be autologous fat when it is cultured with adipose derived stem cells and used for molding purposes. On the other hand, liposuction can be made at the desired site, and fat can be collected through a syringe. The syringe which can be used at this time includes all types capable of collecting fat, and as a non-limiting example, Korean Patent Application Nos. 2003-5029, 2005-61134, 2005-34848, It may be a syringe disclosed in 2006-64946, Korean Utility Model Application No. 2003-21484, 2004-10685, 2006-26454. The syringe is characterized in that the liposuction material sucked at the same time as the liposuction is blocked from the atmosphere.

The aspirated liposuction material may be treated as an enzyme such as collagen after extracting only the active ingredient through centrifugation. The treatment process is generally maintained for about 2 to 4 hours, the treatment equipment is generally used an orbital shaker (Orbital shaker). The shaker generates rotational force in the horizontal direction to induce mixing. The shaker may not be an appropriate means in that the components of the liposuction material are significantly different in components or specific gravity such as free oil and body fluid. This is not appropriate in that time is spent for a long time. In addition, the horizontal rotational force may form a vortex due to the collision with the container may be a cause of cell damage.

It is therefore an object of the present invention to provide a new type of device to continuously use the syringe used for liposuction and to improve yield while minimizing mixing and separation time.

1 and 4 show the stem cell separation apparatus according to the present invention. The apparatus according to the present invention may be composed of a control unit 100, a rotating unit 200, the insertion rack 300. The controller 100 may include a driving motor and a control system (not shown) for driving the rotating unit 200. The drive motor and the control system may be formed in a conventional manner, and thus a detailed description thereof will be omitted.

The rotating unit 200 is connected to the shaft formed in the control unit 100 to receive the rotational force from the control unit 100, the opposite side is provided with a means for attaching and detaching the rack 300. The rotating part 200 includes a coupling hole 210 that matches the coupling hole 310 formed in the rack 300 to fix the rack 300. The coupling holes 210 and 310 are fastened by a bolt coupling method to securely couple the rotating part 200 and the rack 300. Desorption can be done in reverse. Meanwhile, the coupling method has been described as an example and is not limited thereto. The rack 300 is formed in a structure that can be separated from the rotating part 200, there is an advantage that can be individually sterilized by separating only the rack 300.

Insertion rack 300 may be formed in a four-frame frame structure, one side may be formed with a receiving means for accommodating a syringe or a container. In FIG. 4, as an example of the accommodation means, the first insertion groove 320 may have a second insertion groove 330 formed on a symmetrical surface thereof. The insertion grooves 320 and 330 serve to fix the upper and lower portions of the syringe to the container including the liposuction material. Meanwhile, the syringe to the container 400 is accommodated in the insertion grooves 320 and 330. The insertion grooves 320 and 330 may be formed in one or more, and may be formed in the same shape or in different shapes to accommodate various types of syringes or containers.

Meanwhile, the syringe or container 400 is accommodated in the insertion grooves 320 and 330 of the rack 300. Therefore, the syringe 400 rotates the -XY system from the XY system while being inclined at a predetermined angle with respect to the X axis. The constant angle θ is preferably 30 to 60 degrees, most preferably 45 degrees. Consideration should be given to the correlation between gravity and centrifugal forces that occur as the syringe 400 rotates. (See Figures 6 and 7)

As described above, when the syringe 400 itself is rotated at an inclined angle, a plurality of inhaling substances having a significantly different specific gravity may be well mixed with each other to minimize vortices that may occur inside the syringe. This causes a change in the relative position between the materials in the direction of gravity and the flow of material consists of Z, the long axis of rotation, while at the same time the internal material, which is symmetrical in axis Z, is repeatedly reversed up and down in the direction of gravity. Increasing the chance of even contact within the interior increases the chance of contact between the materials and eventually increases the degree of mixing between the materials.

Increasing the degree of mixing between these substances may shorten the enzyme treatment time, such as collagen may lead to improved cell survival rate during adipocyte separation. The mixing time using the apparatus according to the present invention is preferably 30 to 90 minutes, which is a significant opportunity to improve the cell survival rate in consideration of the processing time of 120 minutes to 240 minutes for the shaker.

On the other hand, the rotational speed of the rotating unit is preferably rotated to 5 to 15g in consideration of the mixing potential of the upper and lower in the container, that is, gravity and centrifugal force by the action of gravity. Where g is the acceleration of gravity.

1 to 4 is a rack 300 is rotated inclined as an embodiment of the present invention, the syringe 400 inserted orthogonal to the insertion groove of the rack 300 also shows an example of rotating inclined or the syringe or container If the configuration to rotate obliquely to the Z axis will be included in the technical idea of the present invention.

The liposuction material treated as an enzyme such as collagen by the apparatus according to the present invention can be centrifuged in a state contained in a syringe and extract the active ingredient without being exposed to the outside.

  Meanwhile, FIG. 5 shows another example of the accommodation means, and the rack 300 may be formed in the form of a chamber 350. In this case, one or more chambers may be formed, and may be formed in chambers of different sizes in the same shape or to accommodate containers of various sizes. In addition, the chamber may be formed in a shape having different inlets, such as symmetrically positioning the position of the syringe.

As described above, the stem cell separation apparatus according to the embodiment of the present invention has the advantage of improving the yield while minimizing damage to the adipose tissue because of the non-harsh mixing conditions and the mixing efficiency.

In addition, the device according to the present invention is characterized by the use of a syringe in which the suction procedure has been performed from the human body. That is, it is possible to obtain fat-derived stem cells with stability by blocking the opportunity of the liposuction material contained in the inside of the syringe in which the inhalation procedure is performed to make contact with the outside.

In addition, the device according to the present invention has the advantage that each component is modular, such as a syringe, a rack, a rotating part can be detachable, so that each component can be steam autoclaved separately.

In addition, the device according to the present invention has the effect that the operator can utilize the syringe as an enzyme treatment container without contacting the source during the pretreatment.

Example 1

After extracting 50cc of human adipose tissue, centrifugation of liposuction material to extract only the active ingredient, and then adding collagen to the syringe containing the active ingredient, the device according to the present invention, the syringe is rotated by 10g while the syringe is inclined at 45 degrees. Mix for 60 minutes while maintaining the speed. After that, only the active ingredient (stem cell) was extracted through centrifugation again. It carried out 6 times by the same method as the above.

Comparative Example 1

Same as Example 1, except that an orbital shaker was used instead of the apparatus according to the present invention.

As a result of the results for the above Example and Comparative Example treated cells, the final isolated cells and survival rate, mixing treatment time is shown in Table 1 below.

division Processing time (minutes) Total cell count (Unit 1x106 cells / ml) Viable Cell Count (Unit 1x10 ⁶ cells / ml) Survival rate (%)   Example 1 1 time 60 2.43 1.86 76.68 Episode 2 60 2.48 1.91 77.15 3rd time 60 2.68 2.11 78.86 4 times 60 2.64 2.07 78.54 5 times 60 2.37 1.80 76.09 6th 60 2.15 1.58 73.64 Average 60 - - 76.83   Comparative Example 1 1 time 120 1.27 0.76 59.84 Episode 2 120 1.36 0.85 62.50 3rd time 120 1.95 1.44 73.84 4 times 120 1.87 1.36 72.73 5 times 120 1.19 0.68 57.14 6th 120 1.08 0.57 52.78 Average 120 - - 63.14

As described above, when the apparatus according to the embodiment of the present invention was used, it was confirmed that the total cell number and survival rate were improved by more than 13 on average and the processing time was also improved by more than 2 times.

The present invention described above is not limited to the above-described embodiment and the accompanying drawings, and various substitutions, modifications, and changes are possible within the scope without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

1 is an exploded perspective view of a device according to an embodiment of the present invention.

Figure 2 is a perspective view of the device according to a preferred embodiment of the present invention.

3 is a perspective view showing a control unit and a rotating unit according to an embodiment of the present invention.

Figure 4 is a perspective view of the rack for insertion according to an embodiment of the present invention.

5 is a perspective view of the rack for insertion according to another embodiment of the present invention.

6 is a conceptual rotation of the syringe according to an embodiment of the present invention.

7 is a conceptual diagram rotated 180 degrees of FIG. 6.

<Description of Major Symbols in Drawing>

100: control unit 200: rotating unit

300: rack for insertion 400: syringe or container

Claims (11)

In the stem cell separation device, A control unit for adjusting the rotational force and the rotational speed. A rotating unit which receives the rotational force from the control unit and has means for attaching and detaching the inserting rack; Including an insertion rack formed with a receiving means for receiving a syringe or a container containing a liposuction material, The syringe or the container is a stem cell separation device that the upper and lower parts can be repeatedly inverted in the direction of gravity, the inclined rotation so that both gravity and rotational force can act. The method of claim 1, Rotating obliquely is a stem cell separation device that rotates based on the center of the syringe or container. The method of claim 1, The inclined angle of the stem cell separation device 30 to 60 degrees. The method according to claim 1 or 3, The rotation time is 30 to 90 minutes stem cell separation device. The method of claim 4, wherein The rotation speed is 5 to 15g stem cell separation device. The method of claim 1, The rotating unit is connected to the shaft formed in the control unit for receiving the rotational force from the control unit, the stem cell separation device having a coupling hole corresponding to the coupling hole formed in the rack on the opposite side to be removable. The method of claim 6, Insertion rack is a stem cell separation device formed of a four-frame flame structure. The method of claim 1, Receiving means of the insertion rack is a stem cell separation apparatus having at least one first insertion groove that can accommodate a syringe or a container on one surface, at least one second insertion groove is formed on a surface symmetrical to this. The method of claim 1, Receiving means of the inserting rack is a stem cell separation device is one side of the chamber open to accommodate the syringe or container. The method of claim 8 or 9, The insertion groove or the chamber is a stem cell separation device formed in the same shape or different shapes. The method of claim 1, The shaft of the control unit is a stem cell separation device having a structure orthogonal to the syringe or the container.

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