KR20220026234A - Cell Culture and Separation Device for Improving Cell Culture and Separation Efficiency and Cell Culture and Separation Method Using the Same - Google Patents

Abstract

A cell culture and separation device with improved cell culture and separation efficiency according to an embodiment of the present invention includes: a plurality of attachment members having a cell attachment surface to which cells are attached, and including a plurality of films for culturing by attaching cells to the cell attachment surface; a culture vessel accommodating a plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and a spacing means provided on at least one attachment member of the plurality of attachment members, and the cell attachment surface of at least one attachment member of the plurality of attachment members is separated from at least a portion of the cell attachment surface of the other attachment member by a predetermined distance.

Description

Cell Culture and Separation Device for Improving Cell Culture and Separation Efficiency and Cell Culture and Separation Method Using the Same

The present invention relates to a cell culture and separation apparatus and a cell culture and separation method using the same, and more particularly, to a cell culture and separation apparatus with improved cell culture and separation efficiency, and a cell culture and separation method using the same.

Among animal cells, tissue-forming cells, such as stem cells, are generally cultured as a single layer because they have strong properties of being adhered to and cultured on a plane. Monolayer culture has the advantage of being able to observe cells in real time under a microscope, so that visual data based on a long history of cell analysis can be utilized. However, monolayer culture has a disadvantage in that it is difficult to obtain a large number of cells because cells are attached to a surface perpendicular to the direction of gravity, such as the inner bottom of a vessel, and grow as a single layer and do not grow into multiple layers.

In order to overcome these shortcomings, the following various methods have been tried in the prior art.

First, when culturing animal cells, there is a suspension culture or spinner culture in which the cells are cultured in a suspended state in a culture medium. Although it is possible to culture specific cells that proliferate in a suspended state, such as blood cells or cancer cells, it is difficult to culture general animal cells.

Second, there is culture by bead-type microcarriers in which cells are attached to and cultured on a plurality of bead-type microcarriers in the culture medium. This has the advantage of being able to utilize the culture space in three dimensions. However, this culture has the disadvantage that it is not easy to observe the cells under a microscope. In addition, in this culture method, it is difficult to use a physical method such as a mechanical method to separate the cultured cells from the microcarrier, so a proteolytic enzyme must be used, but there is a disadvantage that the use of the proteolytic enzyme can affect the cells. Furthermore, when a plurality of microcarriers covered with cultured cells are stacked on top of each other, there is a problem in that the culture medium is not circulated between the microcarriers and the cells die in many cases.

Third, there is culture by plate pieces in which cells are adhered to and cultured on a plurality of plate pieces in a culture medium. Compared to culture using bead-type microcarriers, microscopic observation is easy and it is easy to separate cells from plate pieces by physical methods such as mechanical methods. However, this culture method has a problem in that cells cannot be attached or cultured because the culture medium cannot flow between the plate pieces overlapping each other when there is no space between the plate pieces due to the overlapping of the plurality of plate pieces. In addition, if the plate pieces covered with the cultured cells overlap each other, the cultured cells die due to the pressure of the plate pieces overlapping each other, or there is a problem in that the culture medium is not circulated between the overlapping plate pieces.

Fourth, there is a culture using a sponge-type support in which cells are attached to a porous elastic material such as a sponge and cultured. This can expand the cell culture area, but there is a problem in that it is difficult to use a physical method to separate the cells from the sponge-type support, so that an enzyme or a support made of a soluble material must be used. In order to solve this problem, there is a method of separating cells from the support by temperature control by coating a temperature-sensitive polymer on the sponge-type support, but this has a disadvantage in that it is impossible to recycle the support.

Considering the problems of the conventional cell culture method as described above, in order to obtain a large number of cells through cell culture by the adherent member, the cell culture area in the adherent member is expanded as well as the smooth flow and adhesion of the cell culture solution between the adherent members. There is a need for a culture method capable of easily separating cultured cells from members and culturing cells and repeatedly using the separation member.

Korean Intellectual Property Office Registered Patent Publication No. 10-2035022 Patent Publication

One problem to be solved by the present invention is cell culture and separation efficiency, which can significantly improve cell culture and separation efficiency by actively performing cell culture through expansion of a cell culture area in a plurality of attachment members To provide an improved cell culture and separation apparatus and a cell culture and separation method using the same.

Another problem to be solved by the present invention is to enable cell culture to be actively performed through smooth flow of a cell culture solution between a plurality of attachment members, thereby significantly improving cell culture and separation efficiency. To provide a cell culture and separation device with improved separation efficiency, and a cell culture and separation method using the same.

Another problem to be solved by the present invention is that the culture and separation efficiency of cells can be greatly improved by making it easy to separate cultured cells through easy physical separation of cultured cells from a plurality of attachment members, An object of the present invention is to provide a cell culture and separation device with improved cell culture and separation efficiency, and a cell culture and separation method using the same.

Another problem to be solved by the present invention is cell culture and isolation, which can significantly improve cell culture and isolation efficiency by repeatedly performing cell culture and isolation through repeated use of cell culture and separation members. An object of the present invention is to provide an apparatus for culturing and separating cells with improved efficiency and a method for culturing and separating cells using the same.

The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

A cell culture and separation apparatus with improved cell culture and separation efficiency according to an embodiment of the present invention includes a plurality of films having a cell attachment surface to which cells are attached, and culturing by attaching cells to the cell attachment surface a plurality of attachment members; a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and at least one of the plurality of attachment members, wherein the cell attachment surface of at least one of the plurality of attachment members is spaced apart from at least a portion of the cell attachment surface of the other attachment member by a predetermined distance. contain means of separation.

The spacing means of the present embodiment is such that the cell attachment surface of at least one attachment member among the plurality of attachment members and the cell attachment surface of the other attachment member are spaced apart from each other at a distance sufficient to allow the cells to be adhered and cultured. At least a portion of the curved portion may be a plurality of curved portions formed on the cell attachment surface or a plurality of protrusions formed on the cell attachment surface.

A cell culture and separation apparatus with improved cell culture and separation efficiency according to another embodiment of the present invention includes a cell attachment surface to which cells are attached, and a plurality of films for culturing by attaching cells to the cell attachment surface. a plurality of attachment members including; a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and a plurality of through-holes formed to have a diameter larger than that of cells to allow cells to pass through the cell attachment surface of at least one of the plurality of attachment members.

The plurality of through-holes of the present embodiment includes a plurality of first through-holes formed in at least one attachment member of the plurality of attachment members, and a plurality of second through-holes formed in the other attachment member of the plurality of attachment members. may include

The plurality of first through-holes and the plurality of second through-holes of the present embodiment may be arranged to communicate with each other.

The plurality of attachment members of the present embodiment collide with each other by movement of the culture vessel, thereby physically separating the cultured cells attached to the plurality of attachment members from the plurality of attachment members.

At least one selected from the group consisting of weight, specific gravity, thickness, surface area, and shape of the plurality of attachment members colliding with each other by the movement of the culture vessel of the present embodiment may be different from each other.

The plurality of attachment members colliding with each other by the movement of the culture vessel of the present embodiment may include an attachment member that is restricted in movement and an attachment member that allows movement.

A cell culture and separation device with improved cell culture and separation efficiency according to another embodiment of the present invention is provided with a cell attachment surface to which cells are attached, and a circular lamination in which cells are attached to the cell attachment surface for culturing. a plurality of attachment members including a plurality of films formed thereon; a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and a rotation shaft for rotating at least one of the plurality of stacked attachment members about a central axis thereof.

The plurality of attachment members of the present embodiment includes a first attachment member coupled to a rotation shaft at the center of the circular film and rotated by the rotation of the rotation shaft, and a hole through which the rotation shaft passes in the center of the circular film and stacked adjacent to the first attachment member It may include a second attachment member that is not rotated by the rotation of the rotation shaft by forming the .

A cell culture and separation device with improved cell culture and separation efficiency according to another embodiment of the present invention is provided with a cell attachment surface to which cells are attached, and a stacked plurality of cells are cultured by attaching cells to the cell attachment surface. a plurality of attachment members including films of a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and a culture solution flow means for flowing the cell culture solution between the cell attachment surfaces of the plurality of stacked attachment members.

The culture solution flow means of this embodiment applies pressure partially on the stacked plurality of attachment members, and moves on the stacked plurality of attachment members to flow the cell culture solution between the cell attachment surfaces of the plurality of attachment members, It may be a sphere having a predetermined specific gravity.

The culture medium flow means of the present embodiment may move on the inclined stacked plurality of attachment members by inclining the plurality of stacked attachment members.

The culture medium flow means of this embodiment may move according to a change in the external magnetic field by having a magnetism that responds to the external magnetic field.

The plurality of attachment members of this embodiment may be a plurality of films.

The plurality of attachment members of the present embodiment may be light-transmitting.

The plurality of attachment members of this embodiment are polyethylene (PE), polypropylene (PP), polyethyleneterephthalate (PET), polytetrafluoroethylene (PTFE), polystyrene (PS), Polycarbonate (PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide)acid (PLLA), pteroylglutamic acid acid, PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone (PCL), poly(lactic -co-glycolic acid), PLGA), amide (amide), silicon (silicon) and may include at least one material selected from the group consisting of quartz.

The plurality of attachment members of this embodiment may be electrically conductive.

The cell adhesion surface of the present embodiment may include a predetermined pattern.

The cell attachment surface of the present embodiment may have an area with an average diameter of 5 mm to 20 mm.

The cell adhesion surface of this embodiment may include a protein coating.

The protein of this example is extracellular matrix (ECM), gelatin, lysine, laminin, dopamine, heparin, collagen, leptin, and It may include at least one selected from the group consisting of elastin.

The culture vessel of this embodiment may be light-transmissive.

The culture vessel of this embodiment may be elastic.

The culture vessel of this embodiment may be sealed.

The culture vessel of this embodiment may be sealed to allow the introduction or discharge of the culture solution.

The culture vessel of this embodiment may include at least one gas selected from the group consisting of carbon dioxide, nitrogen, oxygen, nitrous oxide and hydrogen.

The culture vessel of this embodiment may be filled with a gas having a pressure of 0.9 bar to 1.1 bar.

The culture vessel of this embodiment is polyethylene (polyethylene, PE), polypropylene (polypropylene, PP), polyethylene terephthalate (polyethyleneterephthalate, PET), polytetrafluoroethylene (polytetrafluoroethylene, PTFE), polystyrene (polystyrene, PS), polycarbonate (polycarbonate, PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide)acid, PLLA), pteroylglutamic acid, PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone (PCL), poly(lactic-co) -glycolic acid), PLGA), amide (amide), silicon (silicon) and may include at least one material selected from the group consisting of quartz.

The culture vessel of this embodiment may be tilted intermittently at a temperature of 25°C to 40°C to induce the movement of the culture medium by gravity.

The culture vessel of this embodiment may be intermittently discharged or filled with a culture solution at a temperature of 25°C to 40°C.

The culture vessel of the present embodiment may include an integration unit in which cells separated from the plurality of attachment members are accumulated.

A plurality of attachment members may not be arranged in the integrated part of this embodiment.

The integration part of this embodiment may include a filter that passes cells and does not pass foreign substances.

The accumulator of this embodiment may include a discharging means for discharging the accumulated cells to the outside of the culture vessel.

In a cell culture and separation method with improved cell culture and separation efficiency according to another embodiment of the present invention, a culture medium and cells to be cultured in a culture vessel accommodating a plurality of attachment members having a cell attachment surface to which cells are attached to inject; culturing by attaching cells to a plurality of attachment members accommodated in a culture vessel; physically separating the cells cultured in the plurality of attachment members from the plurality of attachment members by moving the culture vessel so that the plurality of attachment members collide with each other; and discharging the cells separated from the plurality of attachment members to the outside of the culture vessel, provided on at least one attachment member of the plurality of attachment members, and attaching cells of at least one attachment member of the plurality of attachment members and spacer means for allowing the surface to be spaced apart from at least a portion of the cell attachment surface of the other attachment member at a predetermined distance.

One effect of the present invention is to improve cell culture and separation efficiency, which can significantly improve cell culture and separation efficiency by actively performing cell culture through expansion of a cell culture region in a plurality of attachment members. It is possible to provide a cell culture and separation apparatus and a cell culture and separation method using the same.

Another effect of the present invention is that cell culture and separation efficiency, which can significantly improve cell culture and separation efficiency, by enabling cell culture to be actively performed through smooth flow of the cell culture medium between the plurality of attachment members. It is possible to provide an improved cell culture and separation apparatus and a cell culture and separation method using the same.

Another effect of the present invention is that the cell culture and separation efficiency of cells can be greatly improved by allowing the cultured cells to be easily separated through the easy physical separation of the cultured cells from the plurality of attachment members. It is possible to provide a cell culture and separation device with improved separation efficiency and a cell culture and separation method using the same.

Another effect of the present invention is to improve cell culture and isolation efficiency, which can significantly improve cell culture and isolation efficiency by repeatedly performing cell culture and isolation through repeated use of cell culture and separation members. It is possible to provide a cell culture and separation apparatus and a cell culture and separation method using the same.

Effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a view showing a cell culture and separation apparatus with improved cell culture and separation efficiency according to an embodiment of the present invention.
2 is a perspective view illustrating an attachment member of a cell culture and separation device with improved cell culture and separation efficiency according to an embodiment of the present invention.
3 is a cross-sectional view illustrating a section III-III of FIG. 2 .
4 is a plan view schematically showing a cell culture and separation apparatus with improved cell culture and separation efficiency according to another embodiment of the present invention.
5 is a diagram schematically showing a cell culture and separation apparatus with improved cell culture and separation efficiency according to another embodiment of the present invention.
Fig. 6 is a plan view showing the first attachment member of Fig. 5;
Fig. 7 is a plan view showing the second attachment member of Fig. 5;
8 is a diagram schematically showing a cell culture and separation apparatus with improved cell culture and separation efficiency according to another embodiment of the present invention.
9 is a flowchart schematically illustrating a cell culture and separation method with improved cell culture and separation efficiency according to another embodiment of the present invention.

Specific contents for carrying out the invention will be described based on examples. These embodiments 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 It is not limited by the following examples.

In addition, the terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

(Example 1)

The cell culture and separation apparatus 100 having improved cell culture and separation efficiency according to an embodiment of the present invention is illustratively as shown in FIG. 1 , a plurality of attachment members 110 , a culture vessel ( 120 ), the sealing member 130 , the spacing means 140 , and a plurality of through-holes 150 .

The cell culture and separation device 100 is a device for culturing and separating cells, and has the effect of efficiently culturing and separating cells. Here, the cells include various cells. For example, the cells may be stem cells that form tissues among animal cells.

As illustrated in FIG. 2 , the plurality of attachment members 110 are members having a cell attachment surface 111 to which cells are attached and culturing by attaching cells to the cell attachment surface 111 . The cell attachment surface 111 may be various surfaces of the attachment member 110 , but may be a surface in the direction of gravity due to the characteristics of the cell.

The plurality of attachment members 110 may have a cell attachment surface 111 to which cells are attached, and may have various structures capable of culturing cells by attaching the cells to the cell attachment surface 111 . For example, the plurality of attachment members 110 may be a plurality of films. In this case, the plurality of attachment members 110 can be accommodated relatively more in the same space, so that the cell attachment surface 111 is greatly enlarged to expand the cell attachment surface 111 . It becomes possible to facilitate adhesion and culture.

The plurality of attachment members 110 may not be light-transmitting, but preferably light-transmitting. When the plurality of attachment members 110 are light-transmitting, it is easy to observe the culture state of the cells attached to the cell attachment surface 111 of the plurality of attachment members 110 and cultured with a microscope. Appropriate measures can be taken to ensure smooth cell culture.

The plurality of attachment members 110 may be made of various materials. The plurality of attachment members 110 may be made of a translucent polymer or a transparent material. For example, the plurality of attachment members 110 may include polyethylene (PE), polypropylene (PP), polyethyleneterephthalate (PET), polytetrafluoroethylene (PTFE), polystyrene (polystyrene, PS), polycarbonate (PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide)acid, PLLA), pteroyl Glutamic acid (pteroylglutamic acid, PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone (PCL), PLG ( It may include at least one material selected from the group consisting of poly(lactic-co-glycolic acid), PLGA), amide, silicon, and quartz.

The plurality of attachment members 110 may have various thicknesses. For example, the plurality of attachment members 110 preferably have a thickness of 0.002 mm to 1.5 mm. When the thickness of the attachment member is less than 0.002 mm, the attachment member 110 moves together when the culture solution flows, so that the culture solution is not substantially supplied to the cells on the attachment member 110, and the thickness of the attachment member 110 is In the case of more than 1.5 mm, it becomes difficult to substantially observe the cultured state of the cells on the attachment member 110 under the attachment member 110 when observed under a microscope. However, when the material of the attachment member 110 is particularly highly permeable so that the cell culture state can be easily observed with a microscope, the thickness of the attachment member 110 may exceed 1.5 mm. More preferably, the thickness of the attachment member 110 is preferably 0.05 mm.

The plurality of attachment members 110 are not necessarily electrically conductive. However, when the plurality of attachment members 110 are electrically conductive, electricity is applied to the plurality of attachment members 110 to form the attachment members 110 . ) to promote cell culture or to measure the number of cells cultured in the attachment members 110 . There may be various methods for making the plurality of attachment members 110 electrically conductive. For example, the plurality of attachment members 110 may be made of a conductive polymer material, or a conductive coating may be applied or conductive wires may be printed on the surfaces of the plurality of attachment members 110 . By applying electricity to the plurality of conductive attachment members 110 directly or indirectly such as magnetic induction to stimulate the cells cultured in the attachment member 110, culture can be promoted, and resistance is measured The number of cells can be measured.

The plurality of attachment members 110 collide with each other by the movement of the culture vessel 120 to physically separate the cultured cells attached to the plurality of attachment members 110 from the plurality of attachment members 110 . there is. As a result, cell culture and separation efficiency can be greatly improved by allowing cultured cells to be easily separated from the plurality of attachment members 110 through easy physical separation of the cultured cells.

The plurality of attachment members 110 collide with each other by the movement of the culture vessel 120 to physically separate the cultured cells attached to the plurality of attachment members 110 from the plurality of attachment members 110 . It can be made in a variety of configurations. For example, when the plurality of attachment members 110 collide with each other by the movement of the culture vessel 120 by being configured to form a sharp end with an acute cross-section of the edge, as illustrated in FIG. 3 , for example, It is possible to smoothly separate cells from the attachment members 110 .

At least one selected from the group consisting of weight, specific gravity, thickness, surface area, and shape of the plurality of attachment members 110 colliding with each other by the movement of the culture vessel 120 may be different from each other. As a result, the plurality of attachment members 110 have different kinetic energies according to the movement of the culture vessel 120 , so that they easily collide with each other, so that cells can be easily separated from the attachment members 110 .

The plurality of attachment members 110 colliding with each other by the movement of the culture vessel 120 may include an attachment member 110 with limited movement and an attachment member 110 allowing movement. As a result, the attachment members 110, which are allowed to move according to the movement of the culture vessel 120, move on the cell attachment surfaces 111 of the attachment members 110, whose movement is restricted, thereby limiting the movement of the attachment members 110. ) can be easily separated from the cell attachment surface 111 of the cell. For example, the attachment members 110 that are allowed to move according to the movement of the culture vessel 120 have an area substantially equal to the size of the inner cross-section of the culture vessel 120 and the attachment members 110 whose movement is restricted are It may have an area smaller than the size of the inner cross-section of the culture vessel 120 .

The cell attachment surface 111 of the attachment member 110 may include a predetermined pattern. Accordingly, it is possible to easily observe the culture state such as the growth state of the cells or the movement state of the cells on the cell adhesion surface 111 , and furthermore, irregularities are formed on the cell adhesion surface 111 so that the cells are attached to the cell adhesion surface 111 . It can be easily attached. The pattern formation of the cell attachment surface 111 may be performed by various methods such as laser printing or powder melt compression printing. The pattern of the cell attachment surface 111 may have a wire diameter of 5 μm to 200 μm. When the pattern of the cell attachment surface 111 is set to a wire diameter smaller than 5 μm, the pattern cannot be technically implemented. it becomes difficult The pattern of the cell attachment surface 111 may have a regular or irregular shape. The pattern of the cell attachment surface 111 is printed by melting and pressing powder having at least one color selected from the group consisting of various colors, for example, black, red, blue, green, purple, brown, sky blue, pink, yellow, etc. can be formed.

The cell attachment surface 111 of the attachment member 110 may have various shapes, such as a polygonal shape, a circular shape, and an oval shape.

The cell attachment surface 111 of the attachment member 110 may have an area of various sizes. For example, the cell attachment surface 111 preferably has an area with an average diameter of 5 mm to 20 mm. When the cell attachment surface 111 has an area smaller than the average diameter of 5 mm, the collision force between the attachment members 110 is very low, and the cell attachment surface 111 has an area larger than the average diameter of 20 mm. In this case, the attachment members 110 overlap each other, making it very difficult to collide.

The cell attachment surface 111 of the attachment member 110 may include a protein coating. Thereby, cells can be easily attached to the cell attachment surface 111 . Here, the protein is extracellular matrix (ECM), gelatin (gelatin), lysine, laminin (laminin), dopamine (dopamine), heparin (heparin), collagen (collagen), leptin (leptin) and elastin (elastin) may include at least one selected from the group consisting of.

The culture vessel 120 is a member for accommodating the plurality of attachment members 110 and culturing the cells attached to the cell attachment surface 111 of the plurality of attachment members 110 .

The culture vessel 120 may further accommodate a culture solution in order to culture the cells attached to the cell attachment surface 111 of the plurality of attachment members 110 in the culture solution.

The culture vessel 120 may have various structures capable of accommodating the plurality of attachment members 110 and culturing cells attached to the cell attachment surface 111 of the plurality of attachment members 110 . For example, the culture vessel 120 may be made of a main body 121 of a rectangular cylindrical shape, as shown in FIG. 1 by way of example. A plurality of attachment members 110 and, if necessary, a culture solution may be accommodated in the body 121 .

The culture vessel 120 may be light-transmissive. As a result, cells attached to the cell attachment surfaces 111 of the plurality of attachment members 110 in the culture container 120 and cultured can be observed with a microscope from the outside of the culture container 120 .

The culture vessel 120 may be elastic. Accordingly, when the internal size of the culture vessel 120 is changed due to gas injection or the like, the volume of the culture vessel 120 can also be changed.

The culture vessel 120 may be made of various materials. For example, the culture vessel 120 is polyethylene (polyethylene, PE), polypropylene (polypropylene, PP), polyethylene terephthalate (polyethyleneterephthalate, PET), polytetrafluoroethylene (polytetrafluoroethylene, PTFE), polystyrene (polystyrene, PS), Polycarbonate (PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide)acid (PLLA), pteroylglutamic acid acid, PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone (PCL), poly(lactic -co-glycolic acid), PLGA), amide (amide), silicon (silicon) and may include at least one material selected from the group consisting of quartz.

The culture vessel 120 may be sealed. For example, the culture vessel 120 may be closed by a sealing member 130 such as a valve or a rubber port. Accordingly, it is possible to block the inflow of foreign substances or pathogens from the outside while the cells are attached to the cell attachment surfaces 111 of the plurality of attachment members 110 in the culture vessel 120 and cultured.

When the culture vessel 120 is sealed, it is preferable that the culture vessel 120 is sealed to allow the inflow or discharge of the culture solution. For example, when the culture vessel 120 is sealed with a rubber port, the injection needle is injected from the outside of the culture vessel 120 to the inside through the rubber port, and the culture solution is introduced into the culture vessel 120 through the injected needle. or the culture solution may be discharged to the outside of the culture vessel 120 .

The culture vessel 120 may include at least one gas selected from the group consisting of carbon dioxide, nitrogen, oxygen, nitrous oxide, and hydrogen. Accordingly, it is possible to facilitate cell culture by providing a dissolved gas necessary for the culture solution according to the characteristics of the cells attached to the plurality of attachment members 110 in the culture vessel 120 to be cultured. However, the gas included in the culture vessel 120 is not limited thereto, and may include various other gases necessary for cell culture or capable of promoting cell culture.

The culture vessel 120 may be filled with a gas having a slight negative or positive pressure, for example, a pressure of 0.9 bar to 1.1 bar. Thereby, it is possible to adjust the concentration of dissolved gas in the culture medium necessary for cell culture or to promote cell culture.

The culture vessel 120 is preferably maintained at an appropriate temperature for cell culture. For example, the culture vessel 120 is preferably maintained at a temperature of 25 ℃ to 40 ℃. When the temperature of the culture vessel 120 is maintained lower than 25°C or higher than 40°C, the efficiency of cell culture is reduced.

The culture vessel 120 is preferably tilted intermittently at a temperature of 25°C to 40°C. Thereby, the temperature in the culture vessel 120 can be uniformly maintained at the temperature at which the cells are cultured, so that the cell culture can be performed smoothly. Intermittently tilting the culture vessel 120 may be performed in various ways, either manually or mechanically.

The culture vessel 120 is preferably discharged or filled with the culture medium intermittently at a temperature of 25 ℃ to 40 ℃. As a result, by replacing the existing culture solution with a new culture solution in the culture container 120, cell culture in the culture container 120 can be performed smoothly. Discharge of the culture solution from the culture vessel 120 to the outside or the filling of the culture solution from the outside into the culture vessel 120 may be accomplished through various methods, for example, a device such as a hose passing through the culture vessel 120 .

The culture vessel 120 may include an accumulation unit 122 in which cells separated from the plurality of attachment members 110 are accumulated. As a result, cells cultured in the culture vessel 120 and separated from the plurality of attachment members 110 can be easily integrated and discharged to the outside.

The accumulation unit 122 may have various structures in which cells separated from the plurality of attachment members 110 by being cultured in the culture vessel 120 may be accumulated.

For example, as shown in FIG. 1 by way of example, the integration part 122 protrudes outward from the rectangular cylindrical body 121 of the culture vessel 120 and may have a smaller internal volume than the body 121 . there is.

In addition, the integration part 122 preferably has a tapered internal space so that the cells separated from the plurality of attachment members 110 can be easily integrated. By naturally precipitating or centrifuging the separated cells in the tapered accumulation unit 122 , the cells can be easily integrated into the accumulation unit 122 .

It is preferable that the plurality of attachment members 110 are not arranged in the integration part 122 . Thereby, it is preferable that the attachment members 110 prevent the cells accumulated in the integration part 122 from being discharged to the outside. There may be various methods for preventing the plurality of attachment members 110 from being arranged in the integrated part 122 . For example, by making the average diameter of the cross section of the internal space of the integrated part 122 smaller than the average diameter of the plurality of attachment members 110 , the plurality of attachment members 110 are structurally moved into the internal space of the integrated part 122 . can be prevented from entering.

The integration part 122 may include a filter (not shown) that passes cells and does not pass foreign substances. Thereby, foreign substances other than the cells separated from the plurality of attachment members 110 are not accumulated in the integration unit 122 , and the degree of cell integration is increased, thereby improving cell separation efficiency.

The accumulation unit 122 may include a discharge means for discharging the integrated cells to the outside of the culture vessel 120 . Accordingly, it is possible to easily discharge the cells accumulated in the accumulation unit 122 to the outside. The discharge means may have various configurations for discharging the cells accumulated in the accumulation unit 122 to the outside of the culture vessel 120 . For example, the discharging means may be a sealing member 130 for sealing an externally communicating hole formed in the integration part 122 . The cells accumulated in the integration part 122 can be easily discharged to the outside of the culture vessel 120 by injecting the injection needle into the integration part 122 or opening the sealing member 130 through the sealing member 130. there will be In this case, the sealing member 130 is a means for sealing the culture vessel 120 and at the same time a means for discharging the cells from the culture vessel 120 to the outside. However, the present invention is not limited thereto, and the discharge means may be formed as a means separate from the sealing member 130 .

The spacer 140 is provided on at least one attachment member among the plurality of attachment members 110 , and the cell attachment surface 111 of the at least one attachment member 110 of the plurality of attachment members 110 is disposed on the cell attachment surface 111 . It is a member that is spaced apart from at least a portion of the cell attachment surface 111 of the other attachment member 110 by a predetermined distance. Thereby, by securing a space between the plurality of attachment members 110 to expand the cell culture area or to allow the cell culture solution to flow smoothly, cell culture is actively performed and cell culture efficiency can be greatly improved.

The spacing means 140 may have various configurations such that the cell attachment surfaces 111 of the attachment member 110 are spaced apart from each other at a predetermined distance. For example, in the spacer 140 , the cell attachment surface 111 of at least one attachment member 110 of the plurality of attachment members 110 and the cell attachment surface 111 of the other attachment member 110 are attached to the cell. It can be made to fall at an interval sufficient to be cultured. As a result, the cells are smoothly attached to the cell attachment surfaces 111 spaced apart from each other at a distance sufficient to allow the cells to be adhered and cultured, thereby enabling the cells to be smoothly cultured.

The spacing means 140 may have various shapes. For example, as illustrated in FIGS. 2 and 3 , the spacer 140 includes a plurality of curved portions 141 formed on the cell attachment surface 111 by bending at least a portion of the attachment member 110 . can As a result, the culture medium flows along the curved portion 141 and supplied to the cell attachment surface 111 , so that the cells attached to the cell attachment surface 111 can be smoothly cultured.

For example, when the two attachment members 110 are in close contact with each other and the cells move to the cell attachment surface 111 between the two attachment members 110 and it is difficult or impossible to attach thereto, the cells between the two attachment members 110 are If the curved portion 141 is formed on the attachment surface 111 , the cell culture area may be expanded by easily moving and attaching cells to the cell attachment surface 111 through the curved portion 141 .

In addition, when the two attachment members 110 are in close contact with each other and the culture solution does not flow smoothly to the cell attachment surface 111 between the two attachment members 110 , the cell attachment surface between the two attachment members 110 ( If the curved portion 141 is formed in the 111), the culture solution is smoothly supplied to the cell attachment surface 111 through the curved portion 141 and thus cell culture can be performed smoothly.

The plurality of curved portions 141 may extend from one end to the other end of the cell attachment surface 111 of the attachment member 110 . As a result, the culture medium flows from one end to the other end of the cell attachment surface 111 along the curved portion 141 and flows across the cell attachment surface 111 to smoothly adhere to the cells attached to the cell attachment surface 111 . can be supplied.

As illustrated in FIG. 2 , the plurality of curved portions 141 may extend to cross each other on one cell attachment surface 111 .

Meanwhile, the spacing means 140 may be a plurality of protrusions 142 formed on the cell attachment surface 111 as illustratively shown in FIGS. 2 and 3 . As a result, the attachment members 110 are separated by a predetermined distance, and the culture solution flows between the attachment members 110 and is supplied to the cell attachment surface 111 so that the cells attached to the cell attachment surface 111 are smoothly cultured. be able to

For example, when the two attachment members 110 are in close contact with each other and the cells move to the cell attachment surface 111 between the two attachment members 110 and it is difficult or impossible to attach thereto, the cells between the two attachment members 110 are If the protrusions 142 are formed on the attachment surface 111 , the cell culture area can be enlarged by easily moving and attaching cells to the cell attachment surface 111 through the gap between the two attachment members 110 . .

In addition, when the two attachment members 110 are in close contact with each other and the culture solution does not flow smoothly to the cell attachment surface 111 between the two attachment members 110 , the cell attachment surface between the two attachment members 110 ( If the protrusions 142 are formed on the 111), the culture solution can be smoothly supplied to the cell attachment surface 111 through the gap between the two attachment members 110, and thus the cell culture can be performed smoothly.

It is preferable that the plurality of protrusions 142 are dispersedly arranged on the cell attachment surface 111 . Thereby, the distance between the attachment members 110 is uniformly separated to induce smooth flow of the culture medium.

The plurality of protrusions 142 preferably have a height greater than the average diameter of the cells. As a result, the cells smoothly flow in the gap between the attachment members 110 to be easily attached to the cell attachment surface 111 .

The plurality of through holes 150 are members formed to have a predetermined diameter through which cells pass through the cell attachment surface 111 of at least one of the plurality of attachment members 110 . As a result, cell culture and cell separation efficiency can be greatly improved by expanding the cell culture region in the plurality of attachment members 110 or actively performing cell culture through smooth flow of the cell culture solution.

The plurality of through-holes 150 preferably have a larger diameter than the cell. As a result, cells can smoothly pass through the plurality of through-holes 150 to be easily attached to the cell attachment surface 111 and cultured.

The plurality of through holes 150 may include a plurality of first through holes 151 formed in at least one attachment member 110 among the plurality of attachment members 110 , and a plurality of first through holes 151 among the plurality of attachment members 110 . A plurality of second through holes 152 formed in the other attachment member 110 ′ may be included.

Here, the plurality of first through-holes 151 and the plurality of second through-holes 152 may be arranged to communicate with each other. Thereby, the cell can pass through the first through-holes 151 and the second through-holes 152 successively. However, the present invention is not limited thereto, and the first through-holes 151 and the second through-holes 152 may be arranged not to communicate with each other.

For example, when the two attachment members 110 and 110' are in close contact with each other and the cells move to the cell attachment surface 111 between the two attachment members 110 and 110', and it is difficult or impossible to attach thereto, the two attachment members If the first through-hole 151 and the second through-hole 152 are respectively formed at 110 and 110', the cell attachment surface 111 through the first through-hole 151 and the second through-hole 152 Cell culture area can be expanded by easily moving and attaching cells to the

In addition, when the two attachment members 110 and 110' are in close contact with each other and the culture solution does not flow smoothly to the cell attachment surface 111 between the two attachment members 110 and 110', the two attachment members 110, If the first through hole 151 and the second through hole 152 are respectively formed in 110 ′), the culture solution is transferred to the cell attachment surface 111 through the first through hole 151 and the second through hole 152 . By smoothly flowing and supplied, cell culture can be performed smoothly.

The operation of the cell culture and separation apparatus with improved cell culture and separation efficiency according to the present Example is as follows.

First, a plurality of attachment members 110 having a cell attachment surface 111 to which cells are attached are injected into the culture vessel 120 together with a culture solution and cells to be cultured. At this time, in a state in which the plurality of attachment members 110 are already injected into the culture vessel 120 , cells to be cultured and a culture solution suitable therefor may be injected into the culture vessel 120 .

Conditions such as temperature are maintained so that the cells are adhered to and cultured on the plurality of attachment members 110 accommodated in the culture vessel 120 . At this time, the adhesion and culture state of the cells can be confirmed by observing under a microscope from the outside of the culture vessel 120 .

As a result of microscopic observation, when it is confirmed that the cells have been sufficiently cultured, the cells cultured in the plurality of attachment members 110 are collected by moving the culture vessel 120 so that the plurality of attachment members 110 collide with each other. physically separated from the attachment members 110 of the Thereby, the cultured cells are easily separated through the easy physical separation of the cultured cells from the plurality of attachment members 110 , thereby greatly improving the cell culture and separation efficiency.

Cells separated from the plurality of attachment members 110 are accumulated in the accumulation unit 122 by natural precipitation or centrifugation, and discharged to the outside of the culture vessel 120 to collect the separated cultured cells.

Spacer means 140 or a plurality of through-holes 150 such as a plurality of curved portions 141 or a plurality of protrusions 142 provided in at least one attachment member 110 among the plurality of attachment members 110 . ), it is possible to significantly improve cell culture and separation efficiency by expanding the cell culture region in the plurality of attachment members 110 or actively performing cell culture through smooth flow of the cell culture solution.

By means of the plurality of through-holes 150 provided in at least one of the plurality of attachment members 110 , the expansion of the cell culture area or the expansion of the cell culture medium from the plurality of attachment members 110 or By actively culturing cells through smooth flow, it is possible to significantly improve cell culture and separation efficiency.

(Example 2)

The cell culture and separation apparatus 200 with improved cell culture and separation efficiency according to another embodiment of the present invention is illustratively shown in FIG. 4 , a plurality of attachment members 210 , a culture vessel 220 , the inflow and outflow means 230 , and a plurality of protrusions 240 and a plurality of through-holes 250 as spaced means.

The present embodiment will be mainly described with respect to the differences compared to the first embodiment.

The plurality of attachment members 210 include a first attachment member 211 and a second attachment member 212 .

The first attachment member 211 is formed of a rectangular film including a plurality of first protrusions 241 and a plurality of first through holes 251 . The first attachment member 211 has an area smaller than a cross-section of the inner space of the culture vessel 220 . One corner portion of the first attachment member 211 is constituted by a first chamfer portion 211a.

The second attachment member 212 has an interval different from the interval between the plurality of second protrusions 242 and the plurality of first through-holes 251 arranged at positions shifted from the positions of the plurality of first protrusions 241 . It is made of a rectangular film including a plurality of second through-holes 252 arranged as The second attachment member 212 has an area substantially the same size as a cross-section of the inner space of the culture vessel 220 . One corner of the second attachment member 212 corresponding to the first chamfer 211a is formed of the second chamfer 212a.

Since the first attachment member 211 has an area smaller than the cross-section of the inner space of the culture container 220, the movement by the culture container 220 is not relatively limited, whereas the second attachment member 212 is cultured. Since it has an area of substantially the same size as the cross-section of the inner space of the vessel 220 , the movement is relatively limited by the culture vessel 220 . As a result, the first attachment member 211 moves relatively larger on the cell attachment surface of the second attachment member 212 according to the movement of the culture vessel 220 , so that it is attached to the cell attachment surface of the second attachment member 212 . The cultured cells are efficiently separated from the cell adhesion surface by the first attachment member 211 .

The cells thus separated are integrated into the accumulator 222 by natural precipitation or centrifugation according to a change in the position of the culture vessel 220 . The integration part 222 is a space formed by the first chamfer 211a of the first attachment member 211 , the second chamfer 212a of the second attachment member 212 , and the culture vessel 220 .

The cells accumulated in the accumulation unit 222 are discharged to the outside through the discharge means 231 connected to the accumulation unit 222 .

The culture vessel 220 is a hexahedral-shaped vessel having a cross section of an inner space corresponding to the first attachment member 211 and the second attachment member 212 , in particular, corresponding to the second attachment member 212 . .

The culture vessel 220 includes an inflow and outflow means 230 including first to fourth inflow and outflow means 231 to 234 for filling or discharging the culture solution or cells to be cultured near the corner. The first inflow and outflow means 231 connected to the accumulator 222 also functions as a means for discharging the separated cells.

In the present embodiment, compared to the first embodiment, the plurality of attachment members 210 including the first attachment member 211 and the second attachment member 212 have a large area, so that the adhesion and culture efficiency of cells is greatly improved. may increase, and when there is a movement of the culture vessel 220 , the first attachment member 211 permits movement and the second attachment member 212 restricts the movement of the second attachment member 211 by the first attachment member 211 . It is advantageous in that the separation efficiency of the cultured cells from the cell adhesion surface on the second attachment member 212 can be greatly increased because the collision with the attachment member 212 occurs entirely.

(Example 3)

The cell culture and separation apparatus 300 having improved cell culture and separation efficiency according to another embodiment of the present invention is illustratively shown in FIGS. 5 to 7 , a plurality of attachment members 310 . ), a culture vessel 320 , a rotation shaft 330 , and a plurality of protrusions 341 and a plurality of through-holes 351 as spaced means.

The present embodiment will be mainly described with respect to the differences compared to the first and second embodiments.

The stacked plurality of attachment members 310 are stacked adjacent to the first attachment member 311 coupled to the rotation shaft 330 at the center of the circular plate shape, and are stacked adjacent to the first attachment member 311 and have a circular plate shape. A second attachment member 312 having a hole 312a through which the rotation shaft 330 passes is included in the central portion.

The first attachment member 311 is rotated by the rotation of the rotation shaft 330 , and the second attachment member 312 is not rotated by the rotation of the rotation shaft 330 . The rotating shaft 330 is rotated by an external driving means 331 .

In this embodiment, the first attachment member 311 and the second attachment member 311 and the second attachment member 311 and the second attachment member 311 are rotated by the rotation shaft 330 and the second attachment member 312 is not rotated by the rotation shaft 330 compared to the first and second embodiments. It is advantageous in that the cell culture efficiency can be significantly increased by smoothly flowing the culture medium between the attachment members 312 .

(Example 4)

The cell culture and separation apparatus 400 with improved cell culture and separation efficiency according to another embodiment of the present invention is illustratively shown in FIG. 8 , a plurality of attachment members 410 and culture It includes a vessel 420 and a culture medium flow means 430 .

The present embodiment will be mainly described with respect to the differences in comparison with Examples 1 to 3.

The culture medium flow means 430 applies a partial pressure on the stacked plurality of attachment members 410 as indicated by an arrow in the vertical direction, and moves on the stacked plurality of attachment members 410 , and the plurality of attachment members It is a sphere having a predetermined specific gravity that flows the cell culture solution between the cell attachment surfaces of the 410 as shown by the arrows in the horizontal direction.

The culture medium flow means 430 may move on the inclined plurality of stacked attachment members 410 by inclining the plurality of stacked attachment members 410 . However, without being limited thereto, the culture medium flow means 430 may move according to a change in the external magnetic field, such as according to the movement of the magnet 431 outside the culture vessel 420 by having a magnetism that responds to the external magnetic field.

In this embodiment, in contrast to Examples 1 to 3, the flow of the culture solution between the cell attachment surfaces by flowing the cell culture solution between the cell attachment surfaces of the plurality of attachment members 410 by the movement of the culture liquid flow means 430 is reduced. It is advantageous in that cell culture efficiency can be significantly increased by being smoothly performed.

(Example 5)

In the cell culture and separation method (S500) with improved cell culture and separation efficiency according to another embodiment of the present invention, as illustratively shown in FIG. 9 , first, the cell adhesion surface to which the cells are attached A culture solution and cells to be cultured are injected into a culture vessel accommodating the provided plurality of attachment members (S510).

Next, the cells are attached to the plurality of attachment members accommodated in the culture vessel and cultured (S520).

Thereafter, the cells cultured in the plurality of attachment members are physically separated from the plurality of attachment members by moving the culture vessel so that the plurality of attachment members collide with each other ( S530 ).

In this way, the cells separated from the plurality of attachment members are discharged to the outside of the culture vessel (S540).

Here, provided on at least one attachment member of the plurality of attachment members, the cell attachment surface of at least one attachment member of the plurality of attachment members is spaced apart from at least a portion of the cell attachment surface of the other attachment member by a predetermined distance The cell culture and separation efficiency can be greatly improved by including a spacer means for actively performing cell culture through expansion of the cell culture region or smooth flow of the cell culture solution in the plurality of attachment members.

The present invention can be applied to a cell culture and separation apparatus and a cell culture and separation method.

100, 200, 300, 400: cell culture and separation device, 110, 110', 210, 310, 410: attachment member, 111: cell attachment surface, 120, 220, 320, 420: culture vessel, 121: main body; 122, 222: integration part, 130: sealing member, 140, 240, 340: spacer means, 141: bent portion, 142, 341: protrusion, 241: first protrusion, 242: second protrusion, 150, 250, 351: penetrating Ball, 151, 251: first through hole, 152, 252: second through hole, 211, 311: first attachment member, 211a: first chamfer, 212, 312: second attachment member, 212a: second chamfer Base, 231, 232, 233, 234: first to fourth inflow means, 330: rotation shaft, 311: 430: culture medium flow means, 312a: rotation shaft through hole, 331: external driving means, 431: magnet

Claims (34)

a plurality of attachment members having a cell attachment surface to which cells are attached, and including a plurality of films for culturing by attaching cells to the cell attachment surface;
a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and
A spacing provided on at least one attachment member of the plurality of attachment members such that the cell attachment surface of at least one attachment member of the plurality of attachment members is spaced apart from at least a portion of the cell attachment surface of the other attachment member by a predetermined distance including means;
A cell culture and separation device with improved cell culture and separation efficiency. The separation means of claim 1, wherein the cell attachment surface of at least one attachment member of the plurality of attachment members and the cell attachment surface of the other attachment member are spaced apart from each other at a distance sufficient to allow the cells to be adhered and cultured. A cell culture and separation apparatus with improved cell culture and separation efficiency, wherein at least a portion is a plurality of curved portions formed on the cell attachment surface or a plurality of protrusions formed on the cell attachment surface. a plurality of attachment members having a cell attachment surface to which cells are attached, and including a plurality of films for culturing by attaching cells to the cell attachment surface;
a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and
A cell attachment surface of at least one attachment member among the plurality of attachment members comprising a plurality of through-holes formed to have a diameter larger than that of the cells to allow cells to pass therethrough,
A cell culture and separation device with improved cell culture and separation efficiency. The plurality of through-holes of claim 3 includes: a plurality of first through-holes formed in at least one attachment member of the plurality of attachment members, and a plurality of second through-holes formed in the other attachment member of the plurality of attachment members A cell culture and separation device with improved cell culture and separation efficiency, including those. A cell culture and separation apparatus with improved cell culture and separation efficiency, in which the plurality of first through-holes and the plurality of second through-holes of claim 4 are arranged to communicate with each other. The plurality of attachment members of claim 1 or 3 are attached to the plurality of attachment members by colliding with each other by movement of the culture vessel to physically separate the cultured cells from the plurality of attachment members, improving cell culture and separation efficiency cell culture and isolation device. A cell culture and separation device with improved cell culture and separation efficiency, wherein at least one selected from the group consisting of weight, specific gravity, thickness, surface area and shape of the plurality of attachment members colliding with each other by the movement of the culture vessel of claim 6 is different . A cell culture and separation apparatus with improved cell culture and separation efficiency, wherein the plurality of attachment members colliding with each other by the movement of the culture vessel of claim 6 includes an attachment member with limited movement and an attachment member allowing movement. a plurality of attachment members having a cell attachment surface to which cells are attached, and including a plurality of circularly stacked films for culturing by attaching cells to the cell attachment surface;
a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and
A rotating shaft for rotating at least one of the plurality of stacked attachment members about a central axis thereof,
A cell culture and separation device with improved cell culture and separation efficiency. The plurality of attachment members of claim 9 include a first attachment member coupled to a rotation shaft at the center of the circular film and rotated by the rotation of the rotation shaft, and a hole stacked adjacent to the first attachment member and through which the rotation shaft passes in the center of the circular film A cell culture and separation device with improved cell culture and separation efficiency, comprising a second attachment member that forms and is not rotated by the rotation of the rotation shaft. a plurality of attachment members having a cell attachment surface to which cells are attached, and including a plurality of stacked films for culturing by attaching cells to the cell attachment surface;
a culture vessel accommodating the plurality of attachment members and culturing cells adhered to the cell attachment surfaces of the plurality of attachment members; and
A culture solution flow means for flowing a cell culture solution between the cell attachment surfaces of the stacked plurality of attachment members, comprising:
A cell culture and separation device with improved cell culture and separation efficiency. The culture solution flow means of claim 11, partially applying pressure on the stacked plurality of attachment members, and moving on the stacked plurality of attachment members to flow the cell culture solution between the cell attachment surfaces of the plurality of attachment members, A cell culture and separation device having a predetermined specific gravity and improved cell culture and separation efficiency. The cell culture and separation device of claim 12, wherein the culture medium flow means has a magnetism that responds to an external magnetic field and moves according to a change in the external magnetic field, improving cell culture and separation efficiency. The cell culture and separation device with improved cell culture and separation efficiency, wherein the plurality of attachment members of any one of claims 1, 3, 9 and 11 are light-transmissive. The plurality of attachment members of any one of claims 1, 3, 9 and 11 are polyethylene (PE), polypropylene (PP), polyethyleneterephthalate (PET), polytetrafluoroethylene ( polytetrafluoroethylene, PTFE), polystyrene (PS), polycarbonate (PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide) )acid, PLLA), pteroylglutamic acid (PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone (polycaprolactone, PCL), poly(lactic-co-glycolic acid, PLGA), amide (amide), silicon (silicon) and cell culture and isolation containing at least one material selected from the group consisting of quartz Cell culture and isolation devices with improved efficiency. The cell culture and separation device with improved cell culture and separation efficiency, wherein the plurality of attachment members of any one of claims 1, 3, 9 and 11 are electrically conductive. The cell culture and separation device with improved cell culture and separation efficiency, wherein the cell adhesion surface of any one of claims 1, 3, 9 and 11 has a predetermined pattern. The cell culture and separation device with improved cell culture and separation efficiency, wherein the cell attachment surface of any one of claims 1, 3, 9 and 11 has an area with an average diameter of 5 mm to 20 mm. The cell culture and separation device of any one of claims 1, 3, 9 and 11, wherein the cell adhesion surface comprises a protein coating, improving cell culture and separation efficiency. The protein of claim 19 is extracellular matrix (ECM), gelatin, lysine, laminin, dopamine, heparin, collagen, leptin and A cell culture and separation device with improved cell culture and separation efficiency, comprising at least one selected from the group consisting of elastin. The cell culture and separation apparatus of any one of claims 1, 3, 9 and 11, wherein the culture vessel is light-transmissive, improving cell culture and separation efficiency. The cell culture and separation apparatus of any one of claims 1, 3, 9 and 11, wherein the culture vessel is elastic, improving cell culture and separation efficiency. The cell culture and separation apparatus of any one of claims 1, 3, 9 and 11, wherein the culture vessel is sealed, and cell culture and separation efficiency is improved. The culture vessel of claim 23 is a cell culture and separation device with improved cell culture and separation efficiency, which is sealed to allow inflow or discharge of the culture solution. The culture vessel of claim 23 is a cell culture and separation device with improved cell culture and separation efficiency, containing at least one gas selected from the group consisting of carbon dioxide, nitrogen, oxygen, nitrous oxide and hydrogen. The cell culture and separation apparatus of claim 23 is filled with a gas having a pressure of 0.9 bar to 1.1 bar, improving cell culture and separation efficiency. The culture vessel of any one of claims 1, 3, 9 and 11 is polyethylene (PE), polypropylene (PP), polyethyleneterephthalate (PET), polytetrafluoroethylene, PTFE), polystyrene (PS), polycarbonate (PC), polyvinylchloride (PVC), cellulose, polyurethane (PU), poly(l-lactide)acid , PLLA), pteroylglutamic acid (PGA), polylactic acid (PLA), alkylbenzene sulfonate (ABS), polydioxanone (PDO), polycaprolactone , PCL), poly(lactic-co-glycolic acid), PLGA), amide, silicone, and at least one material selected from the group consisting of quartz, cell culture and separation efficiency Improved cell culture and isolation devices. The cell culture and separation efficiency of any one of claims 1, 3, 9 and 11, wherein the culture vessel of any one of claims is inclined intermittently at a temperature of 25°C to 40°C to induce movement of the culture medium by gravity. Cultivation and separation devices. The cell culture and separation device of any one of claims 1, 3, 9 and 11, wherein the culture vessel of claim 11 intermittently discharges or fills the culture medium at a temperature of 25°C to 40°C, improving cell culture and separation efficiency. The cell culture and separation apparatus of any one of claims 1, 3, 9, and 11, wherein the culture vessel includes an accumulation unit in which cells separated from a plurality of attachment members are accumulated. The cell culture and separation apparatus of claim 30, wherein a plurality of attachment members are not arranged in the integration part, and cell culture and separation efficiency are improved. The cell culture and separation device with improved cell culture and separation efficiency, including a filter that passes through cells and does not pass through foreign substances in the integration part of claim 30 . The cell culture and separation device with improved cell culture and separation efficiency, including a discharge means for discharging the accumulated cells to the outside of the culture vessel in the integration part of claim 30 . injecting a culture solution and cells to be cultured into a culture vessel accommodating a plurality of attachment members having a cell attachment surface to which cells are attached;
culturing by attaching cells to a plurality of attachment members accommodated in a culture vessel;
physically separating the cells cultured in the plurality of attachment members from the plurality of attachment members by moving the culture vessel to cause the plurality of attachment members to collide with each other; and
Comprising discharging the cells separated from the plurality of attachment members to the outside of the culture vessel,
A spacing provided on at least one attachment member of the plurality of attachment members such that the cell attachment surface of at least one attachment member of the plurality of attachment members is spaced apart from at least a portion of the cell attachment surface of the other attachment member by a predetermined distance including means;
Cell culture and isolation methods with improved cell culture and isolation efficiency.

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