WO2012011705A2

WO2012011705A2 - Centrifuge cell strainer and centrifuge method using same

Info

Publication number: WO2012011705A2
Application number: PCT/KR2011/005266
Authority: WO
Inventors: 정광회; 이광형; 백인걸; 고정재
Original assignee: (주)차바이오메드
Priority date: 2010-07-19
Filing date: 2011-07-18
Publication date: 2012-01-26
Also published as: KR20120008714A; WO2012011705A3

Abstract

According to the invention, a cell strainer is disposed between a centrifuge receiver, which centrifugally separates a target cell from tissue originating from an organism, and a source providing the tissue, and comprises: a housing including an inlet and an outlet, which are connected to the source and to the centrifuge receiver, respectively; a plug or a check valve for shutting the inlet and the outlet off from the outside; and a mesh member disposed between the inlet and the outlet to shut the housing off from the outside. The housing is connected to the source and to the centrifuge receiver in an airtight manner. The mesh member allows the target cell through, and blocks flexible solid cell material.

Description

Centrifugal Separation Method Using Cell Strainer and Cell Strainer

The present invention relates to centrifugation, and more particularly, to a cell strainer for centrifugation and a centrifugation method for separating target cells from isolated tissues derived from living organisms.

Specific target cells extracted from organisms can be used for various purposes. For example, adipose stem cells isolated from adipose tissue can be used in various medical institutions or research institutes for the purpose of regenerating various damaged tissues in the human body, and in particular, hematopoietic cells, nerve cells, skin reconstruction, chondrocytes, cardiomyocytes, and blood vessel cells. It can be used in a variety of fields because it can be differentiated by the back.

The usefulness of adipose stem cells isolated from adipose tissue may be of practical value over stem cells derived from other tissues. Specifically, the adipose stem cells can be separated from the excess adipose tissue in the human body, can be obtained only with a relatively simple treatment. However, when stem cells are separated from bone marrow, umbilical cord blood, peripheral blood, and embryos, it is necessary to overcome ethical problems, pain of the subjects to be collected, and contamination by external factors such as bacteria or viruses during the process.

However, in order to separate adipose stem cells from adipose tissue, in the process of transferring adipose tissue collected from living organisms to a separate container or treating adipose tissue using a reaction substance such as enzymes or chemicals, May be easily exposed to external factors. In addition, the centrifugation process for extracting only the target cells from the mixture of adipose tissue and reactants is carried out again in a separate space, so that the processed materials of each process in the process of extracting the whole adipose stem cells such as being exposed to external factors again There is a problem of being exposed to external factors every time.

Moreover, other tissues other than adipose stem cells, such as the Stromal-vascular fraction (SVF), of the adipose tissue reacted with enzymes or chemicals are in a flexible solid state, such as jelly or collagen. Can be coagulated into cell pellets. In this case, the fat stem cells may be separated by centrifuging the substance in which the fat stem cells and other tissues are separated, but the solid substance described above before centrifugation to separate the fat stem cells at a high concentration. Is simply filtered using a gauze or gauze having a fine mesh as a sieve, and centrifuged by putting the contents of the gauze or gauze through again in a container for centrifugation. Is common.

That is, conventionally, when extracting adipose stem cells from adipose tissue, a process of mixing adipose tissue and reactants, filtering a mixture of adipose tissue and reactants using gauze or gauze, gauze or gauze in the mixture Since the process of centrifuging the processed materials again is performed in different spaces, it is cumbersome to move the processed materials to different spaces for each process, and each time the processed materials are transported. There is a problem that water is exposed to external factors.

The present invention provides a cell strainer and a centrifugal separation method using a cell strainer, which can perform several processes for obtaining target cells from the tissue to be separated, in particular, the tissue to be reacted with adipose tissue. When obtained by reacting a substance, in addition to adipose stem cells, which are target cells, substances of a flexible state such as jelly or collagen are mixed together, such as cell grains of high-density interstitial blood vessels, and cell grains such as jelly or collagen should be filtered. , The cell strainer of the present invention provides a cell strainer capable of moving a target cell directly to a centrifuge bag without exposing blood or serum to the outside.

The present invention is a single-use cell strainer and centrifugal separation method that can recover cells without contamination and without the use of a clean bench or other aseptic facilities, and can easily extract pure target cells without the use of separate filtering equipment. to provide.

According to one exemplary embodiment of the present invention for achieving the above object of the present invention, the cell strainer is a centrifuge receiver and blood for separating the target cells from the separated tissue resulting from the organism using centrifugation An apparatus interposed between a source for providing a separation tissue, the apparatus comprising a housing including an inlet and an outlet connected to the source and the centrifuge receiver respectively, and a mesh member blocking the interior of the housing between the inlet and the outlet, The housing connects the source and the centrifuge receiver in a hermetic coupling, and the mesh member selectively passes through the target cells and blocks the cellular material in a flexible solid state.

In the prior art, blood or serum from exposed sources, such as syringes or tubes, was exposed to the gauze, and the blood or serum passed through the gauze must be moved back to the centrifuge receiver. There may be cases where the tissue to be separated is contaminated or deteriorated from external factors. In order to prevent this, an expensive clean bench or a sterile facility is required, but according to the present invention, the source of the tissue to be separated is directly connected to the centrifuge receiving port, and the cell is centrifuged from the source through a sterile disposable cell strainer. As the blood or serum moves to the separating receptors, the flexible solid cell material can be entangled with each other.

To this end, the mesh member is provided in a structure that spatially blocks between the inlet and the outlet in the housing. Preferably, the mesh membrane in the shape of a bag or a basket may be formed around the inlet, but alternatively, it may be provided in the form of a single or a plurality of membranes to block the inside of the housing. That is, the membrane of the mesh member may be provided in various shapes or structures.

In the present specification, the separated tissue may be a pure cell tissue extracted from an organism, as well as a result of a previous centrifugation or other separation process, and may be composed of a material obtained purely from the body or react with a cell tissue. It can also be provided as a mixture mixture. Reactants may include, for example, enzymes or chemicals for isolating or extracting desired cells from the above-described isolated tissues.

In the case of using the cell strainer of the present invention, it is possible to easily solve several processes for obtaining target cells from the above-mentioned isolated tissues with one cell strainer. For example, when the tissue to be separated is a mixture of adipose tissue and reactants, other substances besides the adipose stem cells, which are the target cells, are reacted by the reactants, for example, the high-density interstitial blood vessels are jelly or collagen. Can be solidified into flexible solid state cell pellets such as Conventionally, a complicated process of separating or extracting a desired target cell by centrifuging the solid substance by using a separate gauze or a gauze and then again passing the gauze or gauze into a container for centrifugation is performed. Going through. In particular, in the process of filtering the separated tissue through gauze or gauze, there is a risk that the separated tissue is exposed to external factors such as bacteria or viruses. However, when the cell strainer according to the present invention is used, the solid substance is filtered out by the mesh member, and the contents passing through the mesh member are directly stored as the centrifuge receiving port and used directly for centrifugation. Therefore, the process of filtering the solid state material and the process of centrifuging the filtered content through the mesh member can be hygienically minimized to expose the tissue to the external factors.

In the present specification, the centrifugal container includes a container or a bag for accommodating and storing the tissue to be separated during centrifugation, and the centrifugal bag such as a centrifuge bag or a tube such as glass or plastic made of a synthetic resin film. And other articles that receive and store the tissue to be separated during.

The mesh member may be made of a material capable of selectively penetrating the target cell, and may be provided using a material having a sieve function such as gauze or gauze using natural or synthetic fabrics. have. More specifically, for example, when the tissue to be separated is a mixture of adipose tissue and reactants, a material having a mesh of tens to hundreds of microns so that the material solidified in a flexible solid state does not easily pass through the mesh member. Can be used.

Conventional centrifuge receivers can be used as they are, and hygienic and simple centrifugation is possible with just one cell strainer.

According to an exemplary embodiment of the present invention, the centrifugation method for separating target cells from the tissue to be separated from the living organism, comprises a housing including an inlet and an outlet, and blocking the inside of the housing between the inlet and the outlet. Providing a cell strainer comprising a mesh member, connecting an outlet of the cell strainer to an inlet of a centrifuge receiver, connecting a source for providing tissue to be connected to an inlet of the housing, and a cell strainer And supplying the tissue to be separated from the source to the receiving port for centrifugation, wherein the target cells of the tissue are passed through the mesh member, and the cellular material in the flexible solid state is blocked by the mesh member. do.

The cell strainer of the present invention can perform several treatments in one space for obtaining target cells from the tissue to be separated. In particular, when the separated tissue is obtained by reacting adipose tissue with a reactant, in addition to the adipose stem cells, which are target cells, flexible solid substances such as jelly or collagen are mixed together, such as cell granules of high-density interstitial blood vessels, Cell granules such as jelly or collagen should be filtered. The cell strainer of the present invention can easily move a target cell directly to a centrifuge receiver without exposing blood or serum to the outside, and thus can simply omit a conventional complicated process. have.

The cell strainer of the present invention is blocked from the outside and the inlet and outlet to prevent contamination without a separate sterile facility, it is possible to inject through a syringe, so it is easy to extract pure target cells at a faster time than cell filtration by gravity can do.

1 to 3 are diagrams for explaining a cell strainer and a centrifugal separation method using a cell strainer according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited or limited by the embodiments. For reference, in the present description, the same numbers refer to substantially the same elements, and may be described by referring to the contents described in the other drawings under these rules, and the contents determined to be obvious to those skilled in the art or repeated may be omitted.

Referring to FIG. 1, a cell strainer 100 for separating target cells from a tissue extracted from a living organism using centrifugation includes a housing 110 and a mesh member 130. The housing 110 includes an inlet 120 and an outlet 125, the inlet 120 being connected with a source 20 of blood to be separated, i.e., blood or serum, the outlet 125 being a centrifuge bag 10 ) May be directly or indirectly connected.

As shown, the source 20 for supplying the tissue to be separated may be a tube or syringe for storing serum or the like, or other centrifuge bag, and in some cases, the body itself. In the present embodiment, the separated tissue includes adipose tissue, and the target cells included in the adipose tissue may be adipose stem cells.

The separated tissue supplied from the source 20 preferentially passes through the inlet 120 to meet the mesh member 130. The mesh member 130 blocks the inside of the housing 110 and specifically blocks the inlet 120 and the outlet 125. Therefore, blood or serum introduced into the housing 110 through the inlet 120 is forced through the mesh member 130 to the outlet 125.

The mesh member 130 includes a mesh frame 132 and a mesh film 134. The mesh frame 132 may be omitted in some cases, but may function to support the mesh membrane 134 around the inlet 120. When the mesh membrane 134 blocks cellular material in a flexible solid state, It is possible to prevent the mesh film 134 from being crushed so as to maintain the maximum area at all times. In addition, even when excessive pressure is applied from a source, the mesh film 134 may be prevented from being torn or opened.

In this embodiment, the mesh member 130 is supported by the mesh frame 132 and maintains a shape like a tent, but the shape of the mesh frame 132 and the mesh member 130 may be variously selected and conical. It may be formed in various shapes such as a cylinder, a polygonal cylinder. In any case, the mesh member 130 may include a mesh frame 132 positioned inside the inlet of the housing 110 and a mesh film 134 attached to the mesh frame 132.

The outlet 125 of the housing 110 may be formed in a nozzle shape, that is, a shape in which the outer surface becomes narrower, and the outlet 125 of the nozzle shape is inserted into the inlet 120 of the centrifugal bag 20. It may be in close contact. As described above, the outlet 125 of the housing 110 and the inlet 120 of the centrifugal bag 20 may be indirectly connected through a hose or the like, but as shown, the outlet of the housing 110 ( 125 may be provided to be directly inserted into or coupled to the inlet 120, at which time it may remain sealed.

In addition, the inlet 120 may be provided with a valve for selectively opening and closing each, it may also be provided in the form of a stopper or a check valve. For reference, such a stopper and check valve type valve may be connected to an external tool using a pointed thin tube such as a cannula, a needle, or the like.

In some cases, the inlet 120 of the housing 110 may be provided with an elastic material through which the needle of the syringe may penetrate, and if inserted with the syringe needle to provide blood or serum, it may block contamination from the outside. Sanitary injection is possible. Of course, the inlet 120 of the housing 110 may be formed in other ways. For example, if the inlet is formed as a check valve to allow only one-way injection, the tissue can be easily injected into the housing even with a syringe or other injection device without a needle.

Referring to FIG. 3, the cell strainer 100 may be used as a medium for moving the separated tissue of the source 20 to the centrifuge bag 20. The cell strainer 100 may be directly connected to the inlet 120 of the centrifuge bag 20, and the outlet 125 may communicate with the inlet 120 through insertion, interference fit, screw coupling, and needle insertion. Can be connected. After the source 20 is connected to the inlet of the housing 110, the separated tissue of the source 20, for example, serum or blood, is applied inside the housing 110 by applying pressure to the source 20 or by using a pump. Can be supplied as Some of the separated tissues supplied into the housing 110 are filtered by the mesh member 130, and the remainder including the target cells passes through the mesh member 130, so that the outlet 125 or centrifugation of the housing 110 is performed. It may be supplied to the solvent bag 20.

The centrifugal bag 20 may be a conventional synthetic resin bag, a plastic or glass tube, and the separation tissue from the source 20 to the centrifuge bag 20 is not exposed to the outside. The flexible solid cell material in the tissue to be separated can be directly separated in the process of passing the tissue to be separated without any filtering. Of course, a tube or other centrifuge receiver may be used instead of the centrifuge bag.

In the present embodiment, the separated tissue may be provided by mixing the adipose tissue and the reactant reacting with the adipose tissue removed from the organism, and the adipose tissue and the reactant are mixed with each other through the stirring process. For reference, the reaction material used may be an enzyme (collagenase type I), it can be reacted for about several minutes to several hours at 37 ° C.

For reference, when the purity of the adipose stem cells obtained through centrifugation is lower than the intended value, the second cell strainer 100 and another centrifugation bag through the inlet and centrifugation again to repeat the process of higher purity It is also possible to obtain the target cells of the cell, even when using a plurality of centrifuge bags connected to each other by placing the cell strainer 100 between each can easily increase the amount of the target cells.

As described above, although described with reference to the preferred embodiment of the present invention, those skilled in the art various modifications and variations of the present invention without departing from the spirit and scope of the invention described in the claims below I can understand that you can.

The cell strainer for centrifugation and the centrifugation method using the cell strainer according to the present invention can be widely used to separate the target cells from the tissue to be separated from the organism.

Claims

In the cell strainer interposed between the centrifugation receiver for separating the target cells from the separated tissue resulting from the organism using centrifugation and the source providing the separated tissue,

A housing including an inlet and an outlet connected to said source and said centrifuge receiver, respectively;

It includes a mesh member for blocking the inside of the housing between the inlet and the outlet,

And said housing connects said source and said centrifuge receiver in a hermetic coupling, said mesh member selectively passes said target cell and blocks cellular material in a flexible solid state.
The method of claim 1,

The mesh member includes a mesh frame positioned inside the inlet of the housing and a mesh membrane attached to the mesh frame to spatially block the inlet and the outlet.
The method of claim 1,

The outlet of the housing is provided in the form of a nozzle that the outer surface is inserted in close contact with the inlet of the centrifugal receiving port, the cell strainer, characterized in that directly connected to the inlet of the centrifugal receiving port.
The method of claim 1,

The outlet and the inlet of the housing is provided with an elastic material that the needle of the syringe penetrates the cell strainer, characterized in that the entry and hygienic injection of the syringe needle.
The method of claim 1,

And said outlet and inlet of said housing are provided as check valves.
The method of claim 1,

The blood separation tissue may be a tissue including adipose tissue, the target cell is a cell strainer, characterized in that the tissue-derived cells including adipose stem cells.
In the centrifugation method for separating the target cells from the tissue to be separated from the organism,

Providing a cell strainer comprising a housing comprising an inlet and an outlet and a mesh member blocking the interior of the housing between the inlet and the outlet;

Connecting the outlet of said cell strainer to the inlet of a centrifuge receiver;

Connecting a source for providing the tissue to be separated to an inlet of the housing; And

And supplying the tissue to be separated from the source to the centrifuge receiver through the cell strainer.

The target cell of the tissue to be separated passes through the mesh member, the cell material in a flexible solid state is blocked by the mesh member.
The method of claim 7, wherein

And providing the outlet of the housing in the form of a nozzle, the outer surface of which is in close contact with the inlet of the centrifugal container, and directly sealingably connected to the inlet of the centrifugal container.
The method of claim 7, wherein

Centrifugal separation method characterized in that the inlet of the housing is provided with an elastic material that the needle of the syringe can penetrate the entrance and hygienic injection of the syringe needle.
The method of claim 7, wherein

The inlet of the housing is provided with a check valve.
The method of claim 7, wherein

The separated tissue comprises adipose tissue, the target cell is a centrifugal separation method, characterized in that the fat stem cells.