KR102290830B1 - Method and apparatus of isolating stromal cells from biological tissue without using phosphatase - Google Patents

Abstract

In one embodiment of the present invention, in order to separate stromal cells from biological tissues without using an enzyme, microscopically cut biological tissues are attached to a plurality of attachment members to which the biological tissues can be attached in a culture solution, thereby forming a substrate. It includes moving the stromal cells to the outside of the living tissue by inducing the spontaneous migration of the cells, and separating the stromal cells that have moved out of the living tissue from a plurality of attachment members, and the living tissue has a smaller average specific gravity than the culture solution. When the plurality of attachment members have a smaller average specific gravity than the culture solution, when the biological tissue has a larger average specific gravity than the culture solution, the plurality of attachment members have a larger average specific gravity than the culture solution, and the living tissue has the same average specific gravity as the culture solution Provided is a method and apparatus, characterized in that when having, the plurality of attachment members have the same average specific gravity as the culture solution. This solves the problems of toxicity, cost, processing time, and concerns about heterologous viruses, and the problem of instability due to the use of enzymes with components extracted from gastric juices of heterogeneous animals, and at the same time, it removes stromal cells from living tissues. Separation efficiency can be improved by separating stromal cells in a relatively intact natural state without using

Description

Method and apparatus for isolating stromal cells from living tissue without using enzymes

The present invention relates to a method and an apparatus for isolating stromal cells from a living tissue, and more particularly, to a method and an apparatus for separating stromal cells from a living tissue without using an enzyme.

There are two methods for separating stromal cells existing in the living tissue of an animal from the living tissue, one using an enzyme and one without using an enzyme.

A method of isolating stromal cells from animal tissues using an enzyme includes a method using an enzyme in an initial separation step or a method using an enzyme in a harvest or subculture step.

In the initial stage of isolating stromal cells from living tissue, the stromal cells are melted from the living tissue by using an enzyme such as collagenase for the strongly attached collagen tissues surrounding the stromal cells in the living tissue, and this After washing the enzyme from the dissolved stromal cells, stromal cells are obtained. However, in this case, problems such as toxicity and cost of the enzyme, processing time, and concerns about heterologous viruses occur.

When the stromal cells isolated in the above initial stage of separating stromal cells from living tissues are proliferated in an incubator, when the cell density is high and the passage is required, the passage is performed by using an enzyme such as trypsin. . However, since the enzyme used at this time is a component extracted from the gastric juice of a heterogeneous animal, a problem of poor stability occurs.

In the method of isolating stromal cells from animal tissues without using enzymes, in the initial separation stage, collagen is destroyed by micro-cutting biological tissues using ultrasound, laser, or strong negative pressure, and stromal cells are centrifuged through centrifugation. will separate However, in this case, the probability that the stromal cells are separated from the collagen is extremely low, and the damage to the stromal cells is increased, so that the yield is less than 5% compared to the method using an enzyme, and the process is complicated.

In the method of isolating stromal cells from animal tissues without using enzymes, in the harvesting or passage stage, the cells are proliferated on the surface of microbeads with different specific gravity in order to culture and proliferate the separated stromal cells, and in this way, the cells are proliferated. By repeating mixing the microbeads together with the liquid, the cells that come out are collected by colliding the microbeads with each other, or after culturing and propagating the stromal cells on a flat surface, the stromal cells are scraped off with a scraper. However, in this case, since the microbeads are spherical, the effect of dislodging the cells proliferated on the surface of the microbeads during the mutual collision process cannot be maximized. There is a problem of concern.

On the other hand, culturing living tissue, such as adipose tissue, floats in the culture solution by putting the adipose tissue in a container filled with the culture solution using the characteristic of floating in the culture medium so that the cultured adipose tissue is attached to the upper inner surface of the culture vessel. induce However, in this case, since the surface to which the cultured adipose tissue is attached is flat, there is a problem that the surface to which the cultured adipose tissue is attached cannot be maximized and the culture efficiency cannot be maximized.

Korean Patent Laid-Open No. 10-2015-0114947

One problem to be solved by the present invention is to further increase the possibility that the microscopically cut living tissue adheres to the attachment member in the culture medium, so that the spontaneous growth of stromal cells surrounded by the collagen of the living tissue on the attachment member without using an enzyme By increasing the possibility of spontaneous migration and further promoting the movement of stromal cells to the outside of the living tissue, problems such as enzyme toxicity, cost, processing time, and concerns about heterologous viruses and To provide a method and apparatus capable of improving separation efficiency by solving the problem of instability due to the use of enzymes and at the same time separating stromal cells from living tissues into relatively intact stromal cells without the use of enzymes and without damage. will be.

Another problem to be solved by the present invention is by effectively scraping the stromal cells that have moved out of the living tissue by spontaneous migration on the attachment member in the culture medium from the living tissue, so that the toxicity, cost, and treatment of the enzyme A method and apparatus capable of improving the separation efficiency of stromal cells from living tissues while solving problems such as time, concerns about heterologous viruses, and instability caused by using an enzyme having components extracted from gastric juice of a heterogeneous animal is to provide

One embodiment of the present invention for solving the above problem is a method of separating stromal cells from a living tissue without using an enzyme, comprising: (1) finely cutting the living tissue; (2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution; (3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and (4) separating the stromal cells moved to the outside of the living tissue from the plurality of attachment members, wherein when the living tissue has an average specific gravity smaller than that of the culture solution, the plurality of attachment members has a smaller average specific gravity than the culture solution. It provides a method, characterized in that it comprises.

Another embodiment of the present invention for solving the above problem is a method of isolating stromal cells from a living tissue without using an enzyme, comprising: (1) finely cutting the living tissue; (2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution; (3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and (4) separating the stromal cells that have moved to the outside of the living tissue from the plurality of attachment members, wherein when the living tissue has an average specific gravity greater than that of the culture solution, the plurality of attachment members has an average specific gravity greater than that of the culture solution. It provides a method, characterized in that it comprises.

Another embodiment of the present invention for solving the above problem is a method of isolating stromal cells from a living tissue without using an enzyme, comprising: (1) finely cutting the living tissue; (2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution; (3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and (4) separating the stromal cells moved to the outside of the living tissue from the plurality of attachment members, wherein when the living tissue has the same average specific gravity as the culture solution, the plurality of attachment members has the same average specific gravity as the culture solution. It provides a method characterized in that.

In the present embodiments, the living tissue of (1) may be finely cut so that at least a portion of the stromal cells are exposed to the outside between the collagen surrounding the stromal cells in the living tissue.

In the present embodiments, the separation of the stromal cells in (4) is performed by applying a physical force to the stromal cells by colliding with each other by the turbulent movement of the culture medium, a plurality of attachment members in which the stromal cells are moved to the outside of the living tissue are collided with each other. can be done

In the present embodiments, (5) may further include collecting the stromal cells isolated from the living tissue.

In the present embodiments, (2) to (4) may be repeated in order.

In the present embodiments, after replacing at least one selected from the group consisting of a living tissue, a culture medium, and a plurality of attachment members, steps (2) to (4) may be repeated in order.

In the present embodiments, the biological tissue may include at least one selected from the group consisting of skin, fat, cartilage, mucous membrane, blood vessel, ligament, heart, brain, placenta, umbilical cord, amniotic membrane, muscle, and peripheral nerve.

In the present embodiments, the culture medium may include at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium) and fetal bovine serum.

Another embodiment of the present invention for solving the above problem is a device for separating stromal cells from living tissue without using an enzyme, by inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in a culture solution. A plurality of attachment members for moving stromal cells to the outside of the living tissue are provided, and when the living tissue has an average specific gravity smaller than that of the culture solution, the plurality of attachment members has an average specific gravity smaller than that of the culture solution. provide the device.

In this embodiment, the material of the plurality of attachment members is polypropylene, polyethylene, polyurethane, ECM (Extracellular Matrix), collagen, polydioxanone, Polycaprolactone, PLLA (poly(L-lactide)), PLGA (poly(lactic-co-glycolic acid)), PLA (poly(lactic acid)), PGA (pterolyglutamic acid), hyaluronic acid acid) and may include at least one selected from the group consisting of silicon.

Another embodiment of the present invention for solving the above problem is a device for separating stromal cells from living tissue without using an enzyme, by inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in a culture solution. A plurality of attachment members for moving stromal cells to the outside of the living tissue are provided, and when the living tissue has an average specific gravity greater than that of the culture solution, the plurality of attachment members include those having a larger average specific gravity than the culture solution. provide the device.

In this embodiment, the material of the plurality of attachment members is Teflon, polycarbonate, polyethylene, phthalate, polystyrene, polyurethane, ECM (Extracellular Matrix) ), collagen, polydioxanone, polycaprolactone, PLLA (poly (L-lactide)), PLGA (poly (lactic-co-glycolic acid)), PLA (poly ( lactic acid)), pterolyglutamic acid (PGA), hyaluronic acid, and silicone may be included.

Another embodiment of the present invention for solving the above problem is a device for separating stromal cells from living tissue without using an enzyme, by inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in a culture solution. A device comprising a plurality of attachment members for moving stromal cells to the outside of the living tissue, wherein when the living tissue has the same average specific gravity as the culture solution, the plurality of attachment members have the same average specific gravity as the culture solution to provide.

In the present embodiments, the plurality of attachment members may be to attach the microscopically cut living tissue so that at least a portion of the stromal cells are exposed to the outside between the collagen surrounding the stromal cells in the living tissue.

In the present embodiments, the plurality of attachment members not only move the stromal cells to the outside of the living tissue by inducing the spontaneous migration of the stromal cells of the living tissue by attaching the living tissue in the culture medium, but also the moved substrate It may be to isolate cells from living tissue.

In the present embodiments, the plurality of attachment members include a main body constituting a region in which stromal cells moved to the outside of a living tissue by spontaneous migration are arranged, and a body extending outwardly from the main body and having a thickness smaller than the thickness of the main body. It may be provided with a scraping unit having a shape capable of scraping the stromal cells arranged on the attachment member.

In the present embodiments, the angle formed by the corner portion of the scraping portion may form an acute angle.

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In the present embodiments, it may be to further include a container for accommodating the culture solution, the living tissue, and a plurality of attachment members therein.

In the present embodiments, the container forms a space in which the culture solution, the living tissue, and a plurality of attachment members are accommodated, and may have an inclined portion formed to enable centrifugation by rotation.

In the present embodiments, the vessel may be one capable of inducing turbulence of the culture solution by forward rotation and reverse rotation.

In the present embodiments, the container is arranged at a position immersed in the culture solution in a state in which the biological tissue is not permeated and the culture solution is permeated and the container is stopped to further include a blocking film that blocks the biological tissue floating in the culture solution from floating in the culture solution can

In the present embodiments, the container may be formed at a position where the centrifugal force is maximum and have a converging part for convergence of the stromal cells separated from the living tissue by the centrifugal force.

In the present embodiments, the container may be positioned on the path from the receiving space to the convergence part to allow movement of stromal cells by centrifugal force and further include a filter that blocks the movement of living tissues and a plurality of attachment members. there is.

In the present embodiments, the container may be further provided with a stromal cell discharge unit formed in the convergence part to discharge the stromal cells converged in the convergence part to the outside.

In the present embodiments, the container may be one that is extended from the outside to the receiving space to further include a culture medium through tube for injecting or discharging the culture solution.

In the present embodiments, the container may further include a gas injection unit for injecting a gas for internal disinfection.

One effect of the present invention is to further increase the possibility that the microscopically cut living tissue adheres to the attachment member in the culture medium, so that spontaneous migration of stromal cells surrounded by collagen of the living tissue on the attachment member without using an enzyme (spontaneous) By increasing the possibility of migration) and further facilitating the movement of stromal cells to the outside of the living tissue, problems such as toxicity, cost, processing time, and concerns about heterogeneous virus of the enzyme and the use of enzymes with components extracted from gastric juice of heterogeneous animals It is possible to provide a method and apparatus capable of improving the separation efficiency by solving the problem of instability caused by .

Another effect of the present invention is that by effectively scraping the stromal cells that have migrated to the outer surface of the living tissue by spontaneous migration on the attachment member in the culture medium from the living tissue, the toxicity and cost of the enzyme, the processing time, To provide a method and apparatus capable of improving the separation efficiency of stromal cells from living tissues while solving problems such as concerns about heterologous viruses and instability caused by using an enzyme having components extracted from gastric juice of a heterogeneous animal it can be done

1 is a photograph showing an image of stromal cells protruding from a living tissue.
2 is a photograph showing an image of adipocytes attached to a chromic suture.
3 is a diagram schematically showing that stromal cells of a living tissue move out of a living tissue by spontaneous migration on an attachment member.
FIG. 4 is a diagram schematically showing that stromal cells of a living tissue move out of a living tissue by spontaneous migration on another type of attachment member.
FIG. 5 is a diagram schematically showing that stromal cells that have migrated to the outside of a living tissue by spontaneous migration on an attachment member are scraped by another attachment member.
6 is a view showing a state in which a culture solution, a living tissue, and an attachment member are arranged in each layer in a container accommodated therein.
7 is a view showing a state in which the culture solution in the container, the living tissue, and the attachment member are centrifuged.
8 is a view showing a state in which the stromal cells separated from the living tissue in the container are converged in the converging part together with the culture solution.

Detailed contents for carrying out the invention will be described. These descriptions are provided by way of example so that those of ordinary skill in the art to which the invention pertains can understand the specific content for carrying out the invention and may be modified in various other forms, so that the scope of the present invention is not limited to the following. It is not limited by the description of

1. A method for isolating stromal cells from living tissues without using enzymes

This method is a method of isolating stromal cells from living tissue without using an enzyme, and is characterized by using the inducing spontaneous migration of stromal cells of living tissue to move stromal cells to the outside of living tissue. .

The living tissue may include at least one selected from the group consisting of skin, fat, cartilage, mucous membrane, blood vessel, ligament, heart, brain, placenta, umbilical cord, amniotic membrane, muscle, and peripheral nerve.

Spontaneous migration of stromal cells in living tissue means that stromal cells spontaneously move to the outside by breaking through collagen surrounding stromal cells in living tissue. An image in which stromal cells continue to extend from a living tissue by spontaneous migration of such stromal cells is exemplarily shown in FIG. 1 .

The movement of the stromal cells to the outside of the living tissue by the spontaneous migration of the stromal cells can be a very important characteristic for isolating the stromal cells from the living tissue without using an enzyme. The present invention provides a method for isolating stromal cells from a living tissue without using an enzyme by inducing the spontaneous migration of the stromal cells of the living tissue to move the stromal cells to the outside of the living tissue.

Spontaneous migration of stromal cells can be made more effectively in the absence of a material capable of attaching living tissue to the living tissue. For example, as exemplarily shown in FIG. 2 , when adipose tissue is attached to a chromic catgut, adipocytes may migrate and adhere to the outside of the adipose tissue due to spontaneous migration of adipocytes. As described above, the spontaneous migration of stromal cells can be induced more effectively in the absence of a material capable of attaching living tissue to the living tissue, so that the spontaneous migration of stromal cells can be more effectively induced. can be separated. Here, the member of the material to which the living tissue can be attached may be various members. For example, it may be a member of the same material as the chromic suture.

Induction of spontaneous migration of stromal cells is preferably performed in a culture medium in which stromal cells can survive. Thereby, by separating the stromal cells that have moved out of the living tissue together with the culture solution, the stromal cells can be collected and cultured without being damaged.

The culture medium may include at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium) and fetal bovine serum.

The culture medium uses the same average specific gravity as the living tissue, so that the living tissue is distributed throughout the culture, so that the stromal cells that have moved out of the living tissue by spontaneous migration can be efficiently separated together with the culture solution.

If the spontaneous migration of stromal cells is made in a state in which the living tissue is attached to the absence of a material capable of attaching the living tissue in the culture medium, the culture medium may use an attachment member and one having the same average specific gravity as the living tissue, or use the attachment member and the biological tissue. It is preferable to use a tissue having an average specific gravity larger than that of a living tissue. Because, in this case, the attachment member and the living tissue are distributed as a whole in the culture medium, or the attachment member and the living tissue are distributed near the water surface of the culture solution, so that the possibility that the living tissue comes into contact with the attachment member is increased, so that the living tissue is more attached to the attachment member. This is because they are effectively attached and spontaneous migration of stromal cells can be induced more effectively.

It is more preferable that the living tissue be attached to the attachment member in a state of being finely cut so that at least a portion of the stromal cells are exposed to the outside through the collagen surrounding the stromal cells. This is because the stromal cells can be more efficiently separated from the living tissue by better inducing the spontaneous migration of the stromal cells in the living tissue. In this case, the cutting of the living tissue may be performed using a laser or the like.

After the stromal cells are moved to the outside of the living tissue by spontaneous migration of the stromal cells, the stromal cells can be separated from the living tissue by applying a physical force to the stromal cells. For example, after the stromal cells are moved to the outside of the living tissue by spontaneous migration in the culture medium, the stromal cells can be separated from the living tissue by applying a physical force to the stromal cells by turbulent movement with the culture solution. If the stromal cells are moved to the outside of the living tissue by spontaneous migration on the attachment member in the culture medium, a physical force is applied to the stromal cells on the attachment member by turbulent motion of the attachment member together with the culture solution, or a plurality of attachment members are applied. By colliding with each other by turbulent motion together with the culture solution, a stronger physical force is applied to the stromal cells on the attachment member to efficiently separate the stromal cells from the living tissue.

After the stromal cells are separated from the living tissue, they are collected outside. The stromal cells collected in this way can be proliferated through culture or subculture in a culture medium. If the stromal cells are separated from the living tissue in the culture solution, the stromal cells can be collected together with the culture solution, so that it is possible to more efficiently separate and culture or subculture.

2. A device for separating stromal cells from living tissues without using enzymes

This device is a device that separates stromal cells from living tissue without using an enzyme, and an attachment member for inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in a culture medium to move stromal cells to the outside of living tissue It is characterized in that it is provided.

The material of the attachment member may be made in various ways so as to induce the spontaneous migration of stromal cells of the living tissue by attaching the living tissue in the culture medium to move the stromal cells to the outside of the living tissue. For example, the attachment member may be made of the same or similar material to the living tissue in order to stably or efficiently attach the living tissue. If the attachment member is arranged in the culture solution while the biological tissue is attached to at least a part of the surface of the attachment member, the biological tissue distributed in the culture solution can be more easily attached to the surface of the attachment member to which the same tissue is attached. .

The shape of the attachment member may be variously formed so as to induce the spontaneous migration of stromal cells of the living tissue by attaching the living tissue in the culture medium to move the stromal cells to the outside of the living tissue. 3, the attachment member 110 is attached to a plurality of microscopically cut biological tissues 120, and the stromal cells 130 of the attached plurality of biological tissues 120 are spontaneously migrated. The body 111 may be formed of a body 111 having a surface forming a region to be moved and arranged outside the living tissue 120 . To this end, the attachment member 110 may be formed in the shape of a flat cube as a whole. Illustratively, the attachment member 110 shown in FIG. 3 has a shape of a square cube, but is not limited thereto, and the attachment member may have various shapes, such as a circular cube, a triangular cube, and a pentagonal cube.

The thickness t of the attachment member 110 is thick so that a plurality of microscopically cut biological tissues 120 are attached and has rigidity to withstand the turbulent movement of the culture medium, and at the same time it is thin so that it can move smoothly by the turbulent movement of the culture medium. It is desirable to do

If the concave-convex shape is formed on at least a part of the surface of the attachment member, the area in which the living tissue comes into contact with the surface of the attachment member increases, so that it can be attached more easily.

The specific gravity of the attachment member may be varied so as to induce the spontaneous migration of the stromal cells of the living tissue by attaching the living tissue in the culture medium to move the stromal cells to the outside of the living tissue.

If the biological tissue has an average specific gravity smaller than that of the culture solution and is mostly distributed near the water surface of the culture solution, it is preferable that the attachment member has an average specific gravity smaller than that of the culture solution. Because, in this case, since the attachment member is mostly distributed near the water surface of the culture medium like the living tissue, the possibility that the attachment member comes into contact with the living tissue is increased, so that the possibility that the living tissue is attached on the attachment member is increased. because it becomes Therefore, in this case, there is a greater possibility that the stromal cells of the living tissue migrate to the outside of the living tissue by spontaneous migration on the attachment member.

When the average specific gravity of the attachment member is smaller than that of the culture medium, the attachment member is polypropylene, polyethylene, polyurethane, ECM (Extracellular Matrix), collagen, polydioxanone ( polydioxanone), polycaprolactone, PLLA(poly(L-lactide)), PLGA(poly(lactic-co-glycolic acid)), PLA(poly(lactic acid)), PGA(pterolyglutamic acid), hyaluronic acid It may include at least one selected from the group consisting of hyaluronic acid and silicone.

If the biological tissue has a larger average specific gravity than the culture solution and is mostly distributed near the bottom of the culture solution, it is preferable that the attachment member has an average specific gravity larger than that of the culture solution. Because, in this case, the attachment member is mostly distributed near the bottom of the culture solution like the living tissue, so the possibility that the attachment member comes into contact with the living tissue is increased, so that the possibility that the living tissue is attached on the attachment member is increased. because it becomes Therefore, in this case, there is a greater possibility that the stromal cells of the living tissue migrate to the outside of the living tissue by spontaneous migration on the attachment member.

When the average specific gravity of the attachment member is greater than that of the culture medium, the attachment member is Teflon, polycarbonate, polyethylene, phthalate, polystyrene, polyurethane, ECM. (Extracellular Matrix), collagen, polydioxanone, polycaprolactone, PLLA (poly(L-lactide)), PLGA (poly(lactic-co-glycolic acid)), PLA (poly (lactic acid)), PGA (pterolyglutamic acid), hyaluronic acid (hyaluronic acid) and may include at least one selected from the group consisting of silicone.

If the biological tissue has the same average specific gravity as the culture solution and is thus dispersed throughout the culture solution, it is preferable that the attachment member has the same average specific gravity as the culture solution. Because, in this case, since the attachment member is also dispersed and distributed throughout the culture solution like the living tissue, the possibility that the attachment member comes into contact with the living tissue increases, and the possibility that the living tissue is attached to the attachment member increases. am. Therefore, in this case, there is a greater possibility that the stromal cells of the living tissue migrate to the outside of the living tissue by spontaneous migration on the attachment member.

The living tissue to be attached to the attachment member is preferably a living tissue finely cut so that at least a portion of the stromal cells are exposed to the outside between the collagen surrounding the stromal cells in the living tissue. This is because the spontaneous migration of stromal cells is better induced and the stromal cells can be better separated from living tissues.

The attachment member attaches the living tissue in the culture medium to induce spontaneous migration of the stromal cells of the living tissue to move the stromal cells to the outside of the living tissue, as well as the stromal cells moved to the outside of the living tissue by spontaneous migration may be separated from living tissue. Accordingly, the attachment member can promote not only the movement of stromal cells to the outside of the living tissue by spontaneous migration, but also the separation of the stromal cells moved to the outside of the living tissue from the living tissue.

Separation of the stromal cells, which have been moved to the outside of the living tissue by the attachment member spontaneously, from the living tissue, can be accomplished in various ways.

For example, the attachment member may serve to scrape the stromal cells that have moved out of the living tissue on the attachment member. Thereby, the stromal cells are separated from the living tissue by receiving a physical force and move into the culture medium. For this purpose, as illustrated in FIG. 4 , the attachment member 210 attaches to a region to which the living tissue 220 is attached and the stromal cells 230 moved to the outside of the living tissue 220 by spontaneous migration are arranged. In addition to the main body 211 forming, it extends outward from the main body 211 and has a thickness thinner than the thickness t of the main body 211 and has a shape capable of scraping the stromal cells arranged on other attachment members. It may further include a scraping portion 212 having. The scraping portion 212 may have an acute angle that is less than 90 degrees at an angle formed by a corner portion of its cross-section. The stromal cells 230 separated from the living tissue 220 by spontaneous migration by the scraping unit 212 and arranged on the outer surface of the biological tissue 220 or on the attachment member 210 are transferred to the living tissue ( 220) or can be easily separated from the attachment member 210 and moved into the culture medium. As illustrated in FIG. 5 , the stromal cells 230 separated from the living tissue 220 on the first attachment member 210 and arranged on the outer surface of the biological tissue 220 or on the attachment member 210 . ) has a thickness thinner than the thickness t' of the second body 211' and extends outward in the process of colliding with the adjacent second attachment member 210', which performs turbulent motion along with the turbulent motion of the culture medium. By being easily scraped by the second scraping unit 212 ′, it is easily separated from the living tissue 220 or the attachment member 210 to move into the culture medium. The first scraping unit 212 also serves to scrape the stromal cells arranged on the other attachment member. The first scraping part 212 and the second scraping part 212' have a thickness smaller than the thickness t of the first body 211 and the thickness t' of the second body 211', respectively. The angles (A) (A') of the corners of the cross-section form an acute angle smaller than 90 degrees. However, the shape of the scraping part is not limited to the shape shown in FIGS. 4 and 5 by way of example, and may have various other shapes capable of separating the stromal cells arranged on the attachment member.

The device may further include a container for accommodating the culture solution, the living tissue, and the attachment member therein.

The container has a space in which the culture solution, the living tissue, and the attachment member are accommodated. As illustratively shown in FIG. 6 , the container 350 may include a accommodating space 351 in which the culture solution 340 , the living tissue 320 , and the attachment members 310 and 311 can be accommodated. However, the accommodating space 351 is not limited thereto and may have various shapes in which the culture solution 340, the living tissue 320, and the attachment members 310 and 311 can be accommodated.

The accommodation space 351 has an inclined portion 352 formed to enable centrifugation by rotation, and has a circular cross-section whose radius increases upward. The upper portion of the accommodation space 351 is covered with a cover 353 . The cover 353 functions to prevent the culture solution 340 and the living tissue 320 and the attachment members 310 and 311 accommodated in the accommodation space 351 from leaking to the outside. Furthermore, the cover 353 functions to block the leakage of the culture solution 340 to the outside due to the turbulent movement of the culture solution 340 due to the forward and reverse rotation of the container 350 . The cover 353 may have an inclined portion having an inclination opposite to the inclination portion 352 of the receiving space 351 and having a circular cross-section whose radius becomes smaller toward the top may be advantageous for centrifugation by rotation.

The vessel may induce turbulence of the culture solution inside the receiving space by repeating forward rotation in one direction and reverse rotation in the opposite direction. Due to the turbulence of the culture solution, the living tissue and the attachment member also undergo turbulent flow. Accordingly, the stromal cells arranged on the attachment member are separated from the living tissue or the attachment member and separated into the culture medium by spontaneous migration from the biological tissue attached to the attachment member to the outside of the biological tissue. In addition, the vessel rotates in one direction to apply a centrifugal force to the stromal cells distributed in the culture solution, thereby separating the stromal cells from the living tissue and the attachment member to be collected together with the culture solution.

The container may further include a barrier film arranged at a position where it is submerged in the culture solution inside the accommodation space in a stationary state. The blocking film has an average specific gravity smaller than that of the culture solution and blocks the biological tissue floating in the culture solution from floating on the surface of the culture solution, so that the living tissue can contact the attachment member and attach to the attachment member. To this end, the blocking membrane has a through hole through which the living tissue is not permeated and the culture solution is permeated. The attachment member does not pass through the through hole of the blocking film.

6, the blocking film 360 is submerged in the culture solution 340, such as near the water surface below the water surface of the culture liquid 340 inside the accommodation space 351 in a state where the container 350 is stopped. arranged in position. A plurality of through-holes are formed in the blocking film 360 , and these through-holes have a size adjusted so that the culture solution 340 is permeated but the living tissue 320 and the attachment members 310 and 311 are not permeated. The biological tissue 320 injected into the culture solution 340 under the blocking membrane 360 is blocked by the blocking membrane 360 when the average specific gravity is smaller than that of the culture solution 340 , and the blocking membrane 360 does not float above the water surface of the culture solution 340 . ) are gathered in the vicinity of the lower side. Among the attachment members 310 and 311 injected into the culture solution 340 under the blocking membrane 360, the attachment member 311 having an average specific gravity smaller than that of the culture solution 340 is blocked by the blocking membrane 360 and the culture solution 340. It does not float above the water surface and gathers near the lower side of the blocking film 360 . If the biological tissue 320 has a smaller average specific gravity than the attachment member 311 , the attachment member 311 is collected under the layer in which the biological tissue 320 is collected. Accordingly, the biological tissue 320 is adjacent to or overlaps with the attachment member 311 to form a layer, so that the possibility of contact with the attachment member 311 is greatly increased, and the possibility of being attached to the attachment member 311 is greatly increased. As a result, the possibility that the stromal cells move out of the living tissue 320 by spontaneous migration in the living tissue 320 attached to the attachment member 311 is greatly increased.

In addition, the blocking film blocks the detachment of the attachment member to the upper part of the culture medium when the attachment member is in turbulent movement due to the turbulence of the culture medium, thereby blocking the stromal cells that have moved to the outside of the living tissue on the attachment member from leaving the culture solution. also do

The container may have a converging part that is formed at a position where the centrifugal force is maximum and converges the stromal cells separated from the living tissue by the centrifugal force. Thereby, the stromal cells separated from the living tissue are converged and can be easily discharged to the outside.

Exemplarily as shown in FIG. 6 , the converging portions 354a and 354b include a first converging portion 354a arranged at a position where the centrifugal force is maximum among the upper edges of the accommodation space 351 , and the accommodation space 351 . A second converging portion 354b is provided at a position where the centrifugal force is maximum while being symmetrical with the first converging portion 354a among the upper edges of the . The first converging unit 354a and the second converging unit 354b each have a space capable of accommodating the converged stromal cells together with the culture solution 340 . Since the first and second converging parts 354a and 354b are arranged at a position where the centrifugal force is maximum, they are separated from the living tissue 320 when the centrifugation is performed by the rotation of the vessel 350 and the culture solution 340 . The stromal cells distributed in the are induced to converge together with the culture medium (340). The convergence part is not limited to that shown in FIG. 6 by way of example and may include one or three or more convergence parts.

The container may further include a filter positioned on the path from the receiving space to the converging part to allow the movement of the stromal cells by centrifugal force and block the movement of the living tissue and the attachment member. Thereby, the culture medium in which the stromal cells are distributed in the converging part is converged, and the living tissue and the attachment member can be controlled so that they do not converge.

As illustratively shown in FIG. 6 , the filters 370a and 370b include the first filter 370a positioned on the path from the accommodation space 351 to the first converging part 354a, and the accommodation space 351 . ) and a first filter 370b positioned on the path from the second converging part 354b. The first filter 370a is positioned at the inlet of the first converging part 354a, and the second filter 370b is positioned at the inlet of the second converging part 354b. A plurality of through-holes are formed in the first and second filters 370a and 370b, and these through-holes allow the culture medium 340 in which the stromal cells are distributed to pass through, and the living tissue 320 and the attachment member 310 ( 311) has a size that is adjusted not to be penetrated. Accordingly, the first and second filters 370a and 370b allow the movement of the culture medium 340 in which the stromal cells are distributed and block the movement of the living tissue 320 and the attachment members 310 and 320, thereby allowing the first And the second convergence portion (354a) (354b), except for the biological tissue 320 and the attachment members 310 and 311, the culture medium 340 in which the stromal cells are distributed to converge. The filter is not limited to that shown in FIG. 6 by way of example and may be arranged at various positions on the path from the accommodation space to the convergence part.

The container may further include a stromal cell discharge unit formed in the convergence part to discharge the stromal cells converged in the convergence part to the outside. Thereby, it is possible to easily discharge the stromal cells converged in the converging part to the outside.

As illustratively shown in FIG. 6 , the stromal cell discharge units 355a and 355b are formed in the first converging unit 354a to discharge the stromal cells converged in the first converging unit 354a to the outside. It includes a stromal cell discharge unit (355a) and a second stromal cell discharge unit (355b) formed in the second converging unit (354b) to discharge the stromal cells converged in the second converging unit (354b) to the outside. The first stromal cell discharging unit 355a discharges the culture medium 340 in which the stromal cells converged in the first converging unit 354a are distributed to the outside, and the second stromal cell discharging unit 355b is connected to the second converging unit ( 354b), the culture medium 340 in which the converged stromal cells are distributed is discharged to the outside. The first and second stromal cell outlets 355a and 355b may be formed of tubes connected to outlets formed in the first and second converging parts 354a and 354b. The stromal cell discharge unit is not limited to that shown in FIG. 6 by way of example, and may be formed in a converging unit and may have various configurations capable of discharging the stromal cells converged in the converging unit to the outside.

The container may further include a culture medium through tube extending from the outside to the receiving space to inject or discharge the culture solution. This makes it possible to easily inject or discharge the culture solution into the receiving space.

As illustratively shown in FIG. 6 , the culture medium through tube 380 includes a tube extending into the receiving space 351 through the cover 353 from the outside. The culture medium through tube 380 may extend through the central portion of the cover 353 and the blocking film 360 to near the central bottom of the receiving space 351 in turn. A sealing member 390 made of rubber is arranged in the central portion of the cover 353 through which the culture medium through tube 380 passes, thereby firmly sealing the central portion of the receiving space 351 through which the culture medium through tube 380 passes. can By easily injecting or discharging the culture solution from the outside into the receiving space 351 without opening the cover 353 by the culture solution through tube 380, it is possible to easily inject or replace the culture solution whenever necessary. The culture medium through tube is not limited to that shown in FIG. 6 by way of example and may be formed of various configurations that extend from the outside to the receiving space to inject or discharge the culture medium.

The container may further include a gas injection unit for injecting a gas for internal disinfection. Thereby, it is possible to easily perform the disinfection of the inside of the container.

3. Example

An embodiment of the present invention provides a method for separating stromal cells from a living tissue without using an enzyme, comprising the steps of: (1) finely cutting the living tissue; (2) attaching the microscopically cut biological tissue to an attachment member made of a material to which the biological tissue can be attached in a culture solution; (3) inducing spontaneous migration of the stromal cells on the attachment member to move the stromal cells to the outside of the living tissue; (4) separating the stromal cells that have moved to the outside of the living tissue from the living tissue; and (5) collecting stromal cells isolated from living tissue.

(1) The step of finely cutting living tissue refers to cutting finely so that at least a portion of the stromal cells are exposed to the outside through the collagen surrounding the stromal cells in the living tissue. The cutting of the living tissue may be performed using a laser or the like. As described above, it is possible to effectively induce the spontaneous migration of the stromal cells in the living tissue by finely cutting the living tissue so that at least a part of the stromal cells are exposed to the outside between the collagen surrounding the stromal cells.

(2) The step of attaching the microscopically cut biological tissue on the attachment member of the material to which the biological tissue can be attached in the culture solution is exemplarily shown in FIG. made within the container.

First, a container 350 having a receiving space 351 for accommodating the living tissue 320 and the culture solution 340 and the attachment members 310 and 311 is prepared. After separating the cover 353 and the blocking film 360 of the container 350 from the container 350, the previously prepared microscopically cut biological tissue 320 and an attachment member made of a material to which the biological tissue 320 can be attached ( 310 and 311 are positioned in the accommodation space 351 . Thereafter, the cover 353 and the blocking film 360 are combined with the container 350 , and then through the culture medium through tube 380 extending to the receiving space 351 through the central portion of the cover 353 and the blocking film 360 in turn. A culture medium 340 in which stromal cells can survive is injected. At this time, the culture solution 340 is injected so that the water surface of the culture solution 340 is higher than the blocking film 360 .

When the culture solution 340 is injected while the biological tissue 320 and the attachment members 310 and 311 are positioned in the receiving space 351 of the container 350 as described above, the average specific gravity is smaller than that of the culture solution 340 . The living tissue 320 and the attachment member 311 rise toward the water surface of the culture solution 340 and are arranged in layers on the lower side of the blocking film 360 and its vicinity. At this time, the biological tissue 320 having a smaller average specific gravity than the attachment member 311 is positioned on the upper layer of the attachment member 311 , and the attachment member 311 is positioned on the lower layer of the biological tissue 320 . In this case, the layer in which the biological tissue 320 is arranged partially overlaps or is adjacent to the layer in which the attachment member 311 is arranged, so that the area in which the biological tissue 320 comes into contact with the attachment member 311 is greatly enlarged. The attachment member 310 having an average specific gravity greater than that of the culture solution 340 is arranged on and near the bottom of the accommodation space 351 under the culture solution 340 . A portion of the attachment member 310 having an average specific gravity greater than that of the culture solution 340 may be arranged on the upper surface of the blocking film 360 . As time elapses in the state in which the culture solution 340, the living tissue 320, and the attachment member 311 are arranged, the biological tissue 320 is attached to the attachment member 311.

(3) The step of inducing spontaneous migration of stromal cells on the attachment member to move the stromal cells to the outside of the living tissue is exemplarily performed on the attachment member 311 to which the living tissue 320 is attached, as shown in FIG. 6 . This means that the stromal cells in the living tissue 320 are moved to the outside of the living tissue 320 by spontaneous migration.

The stromal cells in the living tissue 320 move into the outer surface of the living tissue 320 or the surface of the attachment member 311 or the inside of the culture medium 340 by spontaneous migration. The movement of the stromal cells is actively caused by the stromal cells exposed to the outside through the collagen of the living tissue 320 attached to the attachment member 311 .

(4) The step of separating the stromal cells that have moved to the outside of the living tissue from the living tissue is exemplarily performed within the living tissue 320 on the attachment member 311 to which the living tissue 320 is attached, as shown in FIG. This means that the stromal cells moved to the outside of the living tissue 320 by spontaneous migration of the stromal cells are separated from the living tissue 320 and moved into the culture solution 340 .

Separating the stromal cells moved to the outside of the living tissue 320 from the living tissue 320 and moving them into the culture solution 340 exemplarily performs the forward and reverse rotation of the vessel 350 as shown in FIG. 6 . This is done by applying a physical force to separate the stromal cells from the living tissue 320 to the outside of the living tissue 320 by alternately and repeatedly. When the forward and reverse rotations of the vessel 350 are alternately repeated, turbulence is generated in the culture solution 340 and the attachment member 311 submerged in the culture solution 340 performs a turbulent motion, so on the attachment member 311 The stromal cells moved to the outside of the living tissue 320 are subjected to a physical force to be separated from the living tissue 320 .

In addition, as the attachment members 311 collide with each other due to the turbulent motion of the plurality of attachment members 311, the living tissue on the other attachment member 311 by the scraping part (see FIG. 5) formed in the attachment member 311 By scraping the stromal cells moved to the outside of the 320 , the separation of the stromal cells from the living tissue 320 into the culture medium 340 is further promoted.

Furthermore, the attachment members 311 are attached to the inner surface of the inclined portion 352 of the container 350 by the turbulent motion of the plurality of attachment members 311 and the average specific gravity is greater than that of the culture solution 340 while performing turbulent motion. The stromal cells moved to the outside of the living tissue 320 on the attachment member 311 as they come into contact with or collide with the member 310, the outer surface of the culture medium penetration tube 380 or the lower surface of the blocking film 360 are transferred to the living tissue ( Separation of the culture medium 340 from the 320 is promoted.

(5) The step of collecting the stromal cells separated from the living tissue is to collect the stromal cells separated from the living tissue into the culture solution by separating them from the living tissue and the attachment member. Similarly, (a) the step of converging the stromal cells 330 separated into the culture medium 340 from the living tissue 320 into the converging parts 354a and 354b by centrifugation, (b) the converging part 354a ) consists of a step of discharging the converged stromal cells (330) to the outside (354b).

(a) The step of converging the stromal cells 330 separated into the culture medium 340 from the living tissue 320 into the converging parts 354a and 354b by centrifugation is illustratively as shown in FIG. Similarly, by applying centrifugal force to the stromal cells 330 by rotating the vessel 350 containing the stromal cells 330 separated from the biological tissue 320 in one direction of forward rotation or reverse rotation, the substrate The cells 330 are arranged at positions symmetrical to each other to converge to the first converging portion 354a and the second converging portion 354b where the centrifugal force acts to the maximum, respectively.

As the vessel 350 rotates in one direction, the culture solution 340, as well as the living tissue 320 and the attachment members 310 and 311, have the first converging portion 354a and the second converging portion 354b by centrifugal force. ) will be moved to At this time, the culture solution 340 is a first filter 370a arranged at the inlet of the first converging unit 354a together with the stromal cells 320 and a second filter 370b arranged at the inlet of the second converging unit 354b. respectively, and converge to the first converging portion 354a and the second converging portion 354b, respectively. However, the biological tissue 320 and the attachment members 310 and 311 are both the first filter 370a arranged at the inlet of the first converging part 354a and the second arranged at the inlet of the second converging part 354b. None of the first converging part 354a and the second converging part 354b does not pass through the filter 370b. Because, the first and second filters 370a and 370b both penetrate the culture medium 340 and the stromal cells 320, but have a through-hole to prevent the living tissue 320 and the attachment members 310 and 311 from penetrating them. Because the size is adjustable.

As described above, only the stromal cells 320 together with the culture medium 340 converge in the first and second converging parts 354a and 354b, respectively.

(b) discharging the stromal cells 330 converged to the convergence parts 354a and 354b to the outside is exemplarily performed in the first and second convergence parts 354a and 354b as shown in FIG. 8 . This means discharging the converged culture medium 340 and the stromal cells 320 to the outside, respectively.

When only the culture medium 340 and the stromal cells 320 converge in the first and second converging parts 354a and 354b by one-way rotation of the vessel 350 , the rotation of the vessel 350 is stopped. Then, the culture medium 340 and the stromal cells 330 converged in the first converging unit 354a are discharged to the outside through the first stromal cell discharge unit 355a formed in the first converging unit 354a, 2 The culture medium 340 and the stromal cells 330 converged in the second converging unit 354b are discharged to the outside through the second stromal cell discharge unit 355b formed in the second converging unit 354b.

Through the above process, the stromal cells 330 separated from the living tissue 320 are finally collected.

By successively executing the steps (1) to (5) above, it is possible to continuously separate stromal cells from living tissues, thereby improving the separation efficiency.

Steps (2) to (5) above may be repeatedly executed in sequence. Thereby, the stromal cells 330 remaining in the same attachment members 310 and 311 or living tissue 320 or culture solution 340 existing in the receiving space 351 of the container 350 can be repeatedly collected. It is possible to improve the yield of stromal cells.

After replacing at least one of the living tissue 320, the culture medium 340, and the attachment members 310 and 311 in the steps of (2) to (4) above, the steps of (2) to (5) are sequential You can also run it over and over again. Thereby, the stromal cells 330 are repeatedly collected while replacing at least any one of the living tissue 320 , the culture medium 340 , and the attachment members 310 and 311 existing in the receiving space 351 of the container 350 . This can greatly improve the yield.

The present invention can be applied to a method and apparatus for isolating stromal cells from a living tissue without using an enzyme.

110, 210, 210', 310, 311: attachment member
120, 220, 320: living tissue
130, 230, 330: stromal cells
340: culture medium
350: courage
353: cover
354a: first convergence unit
354b: second convergence unit
360: barrier
370a: first filter
370b: second filter
380: culture medium through tube
390: sealing member

Claims (29)

A method for isolating stromal cells from living tissue without using an enzyme, comprising:
(1) finely cutting living tissue;
(2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution;
(3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and
(4) separating the stromal cells moved to the outside of the living tissue from a plurality of attachment members,
When the living tissue has a smaller average specific gravity than the culture solution, the plurality of attachment members have a smaller average specific gravity than the culture solution,
Separation of stromal cells in (4) is achieved by applying a physical force to the stromal cells by colliding with each other a plurality of attachment members due to the turbulent movement of the culture medium,
Any one of the plurality of attachment members comprises a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member. A method for isolating stromal cells from living tissue without using an enzyme, comprising:
(1) finely cutting living tissue;
(2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution;
(3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and
(4) separating the stromal cells moved to the outside of the living tissue from a plurality of attachment members,
When the living tissue has a larger average specific gravity than the culture solution, the plurality of attachment members have a larger average specific gravity than the culture solution,
Separation of stromal cells in (4) is achieved by applying a physical force to the stromal cells by colliding with each other a plurality of attachment members due to the turbulent movement of the culture medium,
Any one of the plurality of attachment members comprises a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member. A method for isolating stromal cells from living tissue without using an enzyme, comprising:
(1) finely cutting living tissue;
(2) attaching the microscopically cut living tissue to a plurality of attachment members to which the living tissue can be attached in a culture solution;
(3) inducing spontaneous migration of stromal cells on a plurality of attachment members to move stromal cells to the outside of living tissue; and
(4) separating the stromal cells moved to the outside of the living tissue from a plurality of attachment members,
When the living tissue has the same average specific gravity as the culture solution, the plurality of attachment members have the same average specific gravity as the culture solution,
Separation of stromal cells in (4) is achieved by applying a physical force to the stromal cells by colliding with each other a plurality of attachment members due to the turbulent movement of the culture medium,
Any one of the plurality of attachment members comprises a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member. 4. The method according to any one of claims 1 to 3,
The method, characterized in that the biological tissue of (1) is finely cut so that at least a portion of the stromal cells are exposed to the outside between the collagen surrounding the stromal cells in the living tissue. delete 4. The method according to any one of claims 1 to 3,
(5) The method characterized in that it further comprises collecting the stromal cells isolated from the living tissue. 4. The method according to any one of claims 1 to 3,
A method characterized in that (2) to (4) are repeated in order. 4. The method according to any one of claims 1 to 3,
Method, characterized in that after replacing at least one selected from the group consisting of a living tissue, a culture medium and a plurality of attachment members, (2) to (4) are repeated in order. 4. The method according to any one of claims 1 to 3,
The biological tissue comprises at least one selected from the group consisting of skin, fat, cartilage, mucous membrane, blood vessel, ligament, heart, brain, placenta, umbilical cord, amniotic membrane, muscle and peripheral nerve. 4. The method according to any one of claims 1 to 3,
The culture medium comprises at least one selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum. As a device for separating stromal cells from living tissue without using enzymes,
A plurality of attachment members for inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in the culture medium to move the stromal cells to the outside of the living tissue,
When the living tissue has a smaller average specific gravity than the culture solution, the plurality of attachment members have a smaller average specific gravity than the culture solution,
Any one of the plurality of attachment members has a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member,
Further comprising a container for accommodating the culture solution, living tissue, and a plurality of attachment members therein,
The vessel is an apparatus, characterized in that it can induce turbulence of the culture solution by forward and reverse rotation. 12. The method of claim 11,
The material of the plurality of attachment members is polypropylene, polyethylene, polyurethane, ECM (Extracellular Matrix), collagen, polydioxanone, polycaprolactone ), PLLA(poly(L-lactide)), PLGA(poly(lactic-co-glycolic acid)), PLA(poly(lactic acid)), PGA(pterolyglutamic acid), hyaluronic acid and silicone Device comprising at least one selected from the group. As a device for separating stromal cells from living tissue without using enzymes,
A plurality of attachment members for inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in the culture medium to move the stromal cells to the outside of the living tissue,
When the living tissue has a larger average specific gravity than the culture solution, the plurality of attachment members have a larger average specific gravity than the culture solution,
Any one of the plurality of attachment members has a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member,
Further comprising a container for accommodating the culture solution, living tissue, and a plurality of attachment members therein,
The vessel is an apparatus, characterized in that it can induce turbulence of the culture solution by forward and reverse rotation. 14. The method of claim 13,
Materials of the plurality of attachment members include Teflon, polycarbonate, polyethylene, phthalate, polystyrene, polyurethane, ECM (Extracellular Matrix), and collagen. , polydioxanone, polycaprolactone, PLLA (poly(L-lactide)), PLGA (poly(lactic-co-glycolic acid)), PLA (poly(lactic acid)), PGA (pterolyglutamic acid), hyaluronic acid (hyaluronic acid) and a device comprising at least one selected from the group consisting of silicone. As a device for separating stromal cells from living tissue without using enzymes,
A plurality of attachment members for inducing spontaneous migration of stromal cells of living tissue by attaching living tissue in the culture medium to move the stromal cells to the outside of the living tissue,
When the living tissue has the same average specific gravity as the culture solution, the plurality of attachment members have the same average specific gravity as the culture solution,
Any one of the plurality of attachment members has a scraping part having a shape capable of scraping the stromal cells arranged in the other attachment member,
Further comprising a container for accommodating the culture solution, living tissue, and a plurality of attachment members therein,
The vessel is an apparatus, characterized in that it can induce turbulence of the culture solution by forward and reverse rotation. 16. The method of any one of claims 11, 13 and 15,
A device characterized in that the plurality of attachment members attach the microscopically cut living tissue to the collagen surrounding the stromal cells in the living tissue so that at least a portion of the stromal cells are exposed to the outside. 16. The method of any one of claims 11, 13 and 15,
The plurality of attachment members adhere to the living tissue in the culture medium to induce the spontaneous migration of the stromal cells of the living tissue to move the stromal cells to the outside of the living tissue and separate the stromal cells from the living tissue. Device characterized in that. 18. The method of claim 17,
The plurality of attachment members are provided with a main body constituting a region in which stromal cells moved to the outside of a living tissue by spontaneous migration are arranged, and a scraping unit extending outward from the main body and having a thickness smaller than the thickness of the main body. device characterized. 19. The method of claim 18,
The device according to claim 1, wherein the angle formed by the corner portion of the scraping section forms an acute angle. delete 16. The method of any one of claims 11, 13 and 15,
The container forms a space in which the culture solution, the living tissue, and a plurality of attachment members are accommodated, and has an inclined portion formed to enable centrifugation by rotation.
delete 16. The method of any one of claims 11, 13 and 15,
The vessel is arranged at a position immersed in the culture medium in a state in which the biological tissue is not permeated but the culture solution is permeated and the vessel is stopped, and further comprises a blocking film that blocks the biological tissue floating in the culture solution from floating in the culture solution. 16. The method of any one of claims 11, 13 and 15,
The container is formed at a position where the centrifugal force is maximum, and has a converging part for converging the stromal cells separated from the living tissue by the centrifugal force. 25. The method of claim 24,
The container is located on the path from the receiving space to the convergence part, allowing the movement of the stromal cells by centrifugal force and further comprising a filter for blocking the movement of the living tissue and the plurality of attachment members. 25. The method of claim 24,
The container further comprises a stromal cell discharge unit formed in the converging portion to discharge the stromal cells converged in the converging portion to the outside. 16. The method of any one of claims 11, 13 and 15,
The container is extended from the outside to the receiving space, the apparatus characterized in that it further comprises a culture medium through-pipe for injecting or discharging the culture solution. 16. The method of any one of claims 11, 13 and 15,
The container further comprises a gas injection unit for injecting a gas for internal disinfection. delete

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