JP2016163550A - Cell separation membrane and cell separation device comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separation membrane which can restrain nonspecific adsorption while specifically adsorbing target cells, and which can improve quality of separated cells.SOLUTION: A cell separation membrane 10 comprises a support 1, higher molecular parts 2a, 2b, and 2c which are fixed on the support 1 and change the structures by temperature, cell adsorption parts 3a, 3b, and 3c which are binding to the higher molecular parts 2a, 2b, and 2c, can be exposed on an outside surface, and can specifically adsorb target cells 20 when contacting with liquid to be treated comprising the target cells 20, and hydrophilic parts 4a, 4b, and 4c which are exposed on the outside surface and are contacted with the liquid to be treated, in which the cell adsorption parts 3a, 3b, and 3c, two or more kinds of membrane proteins of the target cells 20 can be adsorbed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell separation membrane that collects only cells of a target type and a technology of a cell separation device including the cell separation membrane.

  In regenerative medicine, cells (stem cells) that are the source of skin, nerves, bones, blood vessels, etc. are used to recover the function of tissues and organs that have failed due to illness or injury. The types of stem cells are mainly (1) somatic stem cells, (2) ES cells, and (3) iPS cells, each having the following properties.

(1) Somatic stem cells Somatic stem cells are immature cells contained in trace amounts in bone marrow, fat, blood and the like. Somatic stem cells are found in the bone marrow and fat of the body and differentiate into certain types of cells. Those that become blood cells when grown are called hematopoietic stem cells, those that become bone cells or adipocytes are called mesenchymal stem cells, and those that become nerves in the brain are called neural stem cells. Since somatic stem cells are collected from the patient himself, regenerative medicine using somatic stem cells has the advantage that rejection does not occur.

(2) ES cells ES cells are made from a part of a fertilized egg and have a function of becoming any cell in the body.

(3) iPS cells are produced by inserting genes into cells such as skin. iPS cells are considered to be any cells as well as ES cells.

For example, in regenerative medicine, a cell sheet prepared by proliferating and culturing undifferentiated corneal cells or oral cell stem cells collected from the body and appropriately inducing stratification and / or differentiation can be used for treatment. .
Since all the above stem cells are collected in small amounts, it is necessary to proliferate the collected cells to a sufficient amount by culture and induce differentiation into appropriate cells. May induce differentiation into cells different from the target cells. In particular, in cells used for medical purposes, contamination of cells other than the purpose is a major issue.

  In view of these circumstances, the technique described in Patent Document 1 is known. Patent Document 1 discloses a treatment unit having a nonwoven fabric to which a polymer that exhibits hydrophobicity at a temperature higher than a predetermined temperature and is hydrophilic at a temperature lower than the predetermined temperature is bonded, and the physiological at a temperature higher than the predetermined temperature. In a state where the active substance is hydrophobically bonded to the polymer, a liquid introduction part for introducing a liquid containing cells into the treatment part, a liquid temperature adjustment part for adjusting the liquid temperature of the treatment part, and a liquid from the treatment part A liquid discharge part for discharging, wherein the polymer is capable of hydrophobic binding to a physiologically active substance that binds to a target cell, and the physiologically active substance is an antibody that specifically binds to the cell, The treatment unit captures the cells with the physiologically active substance and separates the cells from the liquid, and hydrophilizes the polymer to a temperature lower than the predetermined temperature, whereby the captured cells and the physiologically active substance are separated from the polymer. An apparatus for separating and collecting cells is disclosed, wherein the liquid discharge unit is configured to collect the detached cells, and the polymer is poly (N-isopropylacrylamide). .

Japanese Patent No. 4288352

  In the technique described in Patent Document 1, a physiologically active substance (antibody) that specifically adsorbs to a target cell is bound to a polymer. And the target cell in a solution adsorb | sucks specifically to a bioactive substance by making the solution containing the target cell contact this surface. After the target cells are adsorbed to the physiologically active substance, the polymer is dissolved in water to make it hydrophilic, thereby detaching the target cells and the physiologically active substance from the polymer. Separated and recovered.

  However, the technique described in Patent Document 1 separates and recovers target cells from the solution by separating them from the polymer in a state where the target cells and physiologically active substance are specifically adsorbed from the solution. For this reason, the target cells in a separated and recovered state cannot be used as they are. That is, it is necessary to separate a physiologically active substance (antibody) from the target cell.

  The present invention has been made in view of such a background, and an object of the present invention is to improve the quality (purity) of cells to be separated.

  As a result of intensive studies to solve the above problems, the present inventors have found that the support, the polymer portion fixed to the support, the structure of which changes with temperature, and the outer surface bonded to the polymer portion. And a cell adsorption site capable of specifically adsorbing the target cell when it is exposed to the liquid to be processed containing the target cell, and can be exposed to the outer surface, and is in contact with the liquid to be processed It has been found that the above-mentioned problem can be solved by providing a hydrophilic site, and allowing the cell adsorption site to adsorb two or more types of membrane proteins of the target cell.

  According to the present invention, the quality of cells to be separated can be improved.

It is a schematic diagram explaining the effect | action of the cell separation membrane of this embodiment. It is a perspective view of the cell separation membrane of this embodiment. It is a schematic diagram explaining the relationship between the differentiation induction of a cell and the expressed membrane protein. It is a flowchart of the separation and collection method of a cell using the cell separation membrane of this embodiment. It is a schematic diagram explaining the effect | action of the cell separation membrane of this embodiment, and is a schematic diagram explaining the relationship between an antibody and a membrane protein (the 1). It is a schematic diagram explaining the effect | action of the cell separation membrane of this embodiment, and is a schematic diagram explaining the relationship between an antibody and a membrane protein (the 2). It is a schematic diagram explaining the effect | action of the cell separation membrane of this embodiment, and is a schematic diagram explaining the relationship between an antibody and a membrane protein (the 3). It is a schematic diagram explaining the cell separation membrane of the modification of this embodiment, (a) is a cell separation membrane in which a part of cell adsorption site does not exist, and (b) is a cell separation provided with another hydrophilic part. (C) is a cell separation membrane in which a part of the hydrophilic portion does not exist. It is a block diagram of the culture apparatus comprised using the cell separation membrane of this invention. It is a flowchart of the separation and collection method of the cell performed in the culture apparatus comprised using the cell separation membrane of this invention. It is a photograph explaining the adsorption and detachment | desorption of the cell separation membrane of this embodiment, and a cell, (a) is a microscope picture when a cell is made to adsorb | suck to a cell separation membrane, (b) collect | recovers the adsorbed cell. It is the microscope picture which image | photographed the cell separation membrane after having performed, (c) is the figure which showed the photograph of (a) typically, (d) is the figure which showed the photograph of (b) typically. It is the photograph explaining the relationship between a cell separation membrane and cell sorting, (a) is a microscope picture which image | photographed the cell adsorb | sucked to the cell separation membrane of a comparative example, (b), (c) is the cell separation of this embodiment It is the microscope picture which image | photographed the cell adsorb | sucked to the film | membrane (the 1). It is the photograph explaining the relationship between a cell separation membrane and cell sorting, (a) is a microscope picture which image | photographed the cell adsorb | sucked to the cell separation membrane of a comparative example, (b), (c) is the cell separation of this embodiment It is the microscope picture which image | photographed the cell adsorb | sucked to the film | membrane (the 2).

  Next, modes for carrying out the present invention (hereinafter referred to as “embodiments” as appropriate) will be described in detail with reference to the drawings as appropriate.

<< First Embodiment >>
The cell separation membrane of this embodiment is comprised including the support body 1 (nonwoven fabric) and the thin film 5 couple | bonded with the support body 1, as shown in FIG. 1, FIG. The thin film 5 includes antibodies 31 to 33 (LCST type antibody, UCST type antibody).

<Support 1>
A support (nonwoven fabric) 1 constituting the cell separation membrane fixes a polymer site 2 and the like which will be described later. Such a support 1 may be of any type, but is preferably one that can chemically fix the temperature-responsive polymer part 2 (hydrophobic polymer part) and the like. Specifically, the support 1 is preferably configured to include at least one of a polymer material and a glass material.

  By using such a material, the surface treatment for fixing the polymer part 2 and the like is particularly easy, and there is an advantage that the durability when pressurized for the surface treatment is excellent.

There are no limitations on the material of the support 1 as long as it can fix the polymer part 2 of the antibody.
Examples of the polymer material constituting the support 1 include polystyrene, polymethyl methacrylate, surface-modified agarose gel, and the like. Further, as a material constituting the support 1, a metal material such as a single metal or a metal oxide can be used.

  The shape of the surface of the support 1 may be any shape as long as the high molecular part 2 of the antibody can be fixed, and can be, for example, a flat shape. In addition, the shape of the surface of the support 1 may be a pillar structure, a spherical structure, a porous shape, or the like.

<Antibodies 31-33>
The antibodies 31 to 33 constituting the cell separation membrane are composed of temperature-responsive polymer site 2 (2a, 2b, 2c, hereinafter referred to as polymer site 2 as appropriate), cell adsorption site 3 (3a, 3b, 3c), A hydrophilic portion 4 is included.

(Temperature responsive polymer part 2)
The structure of the temperature-responsive polymer portion 2 fixed to the support 1 changes depending on the temperature. Specifically, for example, the molecular structure is maintained at room temperature, but the molecular structure is destroyed at a predetermined temperature higher than room temperature.
Although details will be described later due to such a structural change, the cell adsorption site 3 bonded to the polymer site 2 can be buried in the hydrophilic site 4 and the target cell can be detached from the cell separation membrane. It becomes easy.
In the present application, a temperature at which a structural change occurs is called a phase transition temperature.

The specific structure of the temperature-responsive polymer portion 2 is not particularly limited as long as the structure changes depending on the temperature. The polymer portion 2 preferably has a helical structure at least partially.
By setting it as the high molecular part which has such a structure, the hydrogen bond which controls the said helical structure can be cut | disconnected by temperature, and it can make it easy to produce a structural change (phase transition).

  The temperature-responsive polymer part 2 may be fixed to the support 1 by any method. For example, methods such as chemical bonding (covalent bonding, etc.) and physical adsorption (adsorption using antigen-antibody reaction, etc.) can be mentioned.

  A preferred specific example of the temperature-responsive polymer portion 2 includes a polyamino acid. A polyamino acid is formed by polymerizing two or more amino acids. The amino acids constituting the polyamino acid are usually polymerized in a straight chain. Polyamino acids have a helical structure due to the occurrence of hydrogen bonds within the molecule. Therefore, the use of a polyamino acid has an advantage that the structural change is easily caused.

  In addition, since polyamino acids are usually linear, it is easy to control the orientation of the polymer sites. For this reason, the hydrophobic part can be easily fixed to a desired position on the support 1. Furthermore, there is an advantage that it becomes easy to bind the cell adsorption site 3 described later to the polymer site 2.

The polyamino acid may be composed of only one amino acid (homopolymerization), or two or more amino acids may be composed of any ratio and combination (copolymerization).
The amino acid constituting the polyamino acid is not particularly limited, and examples thereof include glutamic acid, aspartic acid, asparagine, lysine, glutamine, cysteine, alanine, leucine and the like.

  The number average molecular weight of the polyamino acid is not particularly limited, but is usually 5 kDa or more, preferably 10 kDa or more, more preferably 50 kDa or more. When the number average molecular weight of the polyamino acid is within this range, it becomes easier to fix the polyamino acid to the support 1.

(Cell adsorption site 3)
The cell adsorption site 3 can specifically adsorb the target cell 20 when it is brought into contact with the liquid to be treated containing the target cell 20. Therefore, the cell adsorption site 3 may be appropriately determined and used depending on the target cell to be separated and collected. Among them, it is preferable to use an antibody having the target cell 20 as an antigen as the cell adsorption site 3. By using an antibody, an antigen-antibody reaction, which is a biological reaction, can be used for a cell that is a living tissue, and the target cell 20 can be specifically adsorbed and desorption can be controlled more easily. .
Two or more types of cell adsorption sites 3 are preferable and can be appropriately selected according to the number of separations, the number of adsorptions, cell culture conditions, and the like.

  Examples of antibodies that can be used in the present embodiment include anti-CD29 antibody, anti-CD40 antibody, anti-CD73 antibody, anti-CD90 antibody, and anti-CD105 antibody as antibodies used for selection in osteoblast differentiation of mesenchymal stem cells. Anti-CD166 antibody. These can be bonded to the UCST type temperature-responsive polymer portion described later.

  In inducing differentiation of mesenchymal stem cells, the mesenchymal stem cells are differentiated in the order of osteoprogenitor cells, preosteoblasts, and osteoblasts, and mature osteoblasts are finally transformed into bone cells. Therefore, antibodies that specifically bind to each can also be applied in this embodiment. Examples of antibodies that specifically bind to these that can be applied in the present embodiment include alkaline phosphatase (ALP) and osteopontin (OPN) before and during osteoblast differentiation, and bone sialoprotein in addition to OCN at the later stage of differentiation. And an antibody against (bone sialo-protein: BSP). These can be bonded to the LCST type temperature-responsive polymer portion described later.

  In addition, since mesenchymal stem cells are differentiated in the order of preadipocytes and mature adipocytes, and mature adipocytes are finally transformed into adipocytes, antibodies that specifically bind to each of these are also present. It is applicable in the embodiment. Examples of antibodies that specifically bind to those applicable in the present embodiment include antibodies against CD105 and LPA4 receptors in the pre- and middle stages of adipocyte differentiation, and antibodies against adiponectin and FABP4 (Fatty acid binding protein 4) in the late stages of differentiation. Can be mentioned. These can also be bonded to the LCST type temperature-responsive polymer portion described later.

  Further, the cell adsorption site 3 is bonded to the polymer site 2. The cell adsorption site 3 and the polymer site 2 may be directly bonded, but are preferably bonded via a linker.

  By binding the cell adsorption site 3 to the hydrophobic polymer site 2 via a linker, the cell adsorption site 3 more reliably protrudes outward from the surface of the cell separation membrane 10 (ie, exposed to the outer surface). Form). Thereby, when the cell separation membrane 10 is brought into contact with the liquid to be treated containing the target cell 20, the cell adsorption site 3 can be more specifically adsorbed to the target cell 20.

  A preferred linker includes biotin-avidin. By using biotin-avidin, the cell adsorption site 3 (for example, antibody) and the hydrophobic polymer site 2 (for example, polyamino acid) can be more easily combined. Specifically, the cell adsorption site 3 and the polymer site 2 can be easily combined via avidin by biotinylating the cell adsorption site 3 and biotinylating the polymer site 2.

  Furthermore, a domain constituting protein A or protein G may be used as the linker. Specifically, protein A or protein G may be used as an antigen, and the polymer site 2 and the cell adsorption site 3 may be bound thereto. Thereby, the high molecular part 2 and the cell adsorption part 3 can be combined via IgG (immunoglobulin) antibody, and the same effect as the case where biotin-avidin is used is acquired.

  The specific binding position of the cell adsorption site 3 in the temperature-responsive polymer site 2 is not particularly limited. From the viewpoint of more reliably exposing the cell adsorption site 3 to the outer surface of the cell separation membrane 10, it is preferable that the cell adsorption site 3 is bonded to the end of the polymer constituting the temperature-responsive polymer site 2. For example, when the polymer constituting the temperature-responsive polymer portion 2 is linear, it is a linear terminal, and when the polymer is a network, it is a network terminal. By binding the cell adsorption site 3 to these positions, the cell adsorption site 3 can be more reliably exposed to the outer surface of the cell separation membrane 10.

(Hydrophilic part 4)
The hydrophilic portion 4 is a portion that is exposed on the outer surface of the cell separation membrane 10 and that comes into contact with the treatment liquid when it is brought into contact (for example, impregnated) with the treatment liquid containing the target cells 20. The hydrophilic part 4 is provided at a part other than the temperature-responsive polymer part 2 (that is, the hydrophobic polymer part) and the cell adsorption part 3 so as to be exposed on the outer surface. Non-specific adsorption can be further suppressed.

Examples of the hydrophilic portion 4 include those having a hydrophilic functional group, specifically, a hydroxyl group, a carboxyl group, or the like. More specific examples of the hydrophilic portion 4 include ethylene glycol and a polymer thereof (polyethylene glycol), a molecule having a phospholipid structure, phosphoric acid and a polymer thereof (polyphosphoric acid), and the like.
Among these, from the viewpoint of good hydrophilicity and ease of binding to the temperature-responsive polymer part 2 (hydrophobic polymer part), the hydrophilic part 4 is a polyethylene glycol in which two or more ethylene glycols are polymerized. It is preferable to contain.

  Further, the hydrophilic portion 4 may be provided independently of the temperature-responsive polymer portion 2 (hydrophobic polymer portion). However, from the viewpoint of further promoting the structural change of the polymer part 2, it is preferable that the hydrophilic part 4 is bonded to the temperature-responsive polymer part (2 hydrophobic polymer part). That is, the hydrophilic part 4 is preferably integrated with the temperature-responsive polymer part 2 (hydrophobic polymer part).

  Since the hydrophilic part 4 is bonded to the temperature-responsive polymer part 2 (hydrophobic polymer part), the structural change of the temperature-responsive polymer part 2 (hydrophobic polymer part) accompanying the temperature change is further increased. Can be urged. However, it is not necessary that all the hydrophilic sites 4 provided in the cell separation membrane 10 are bonded to the temperature-responsive polymer site 2 (hydrophobic polymer site). The temperature responsiveness of the responsive polymer part 2 (hydrophobic polymer part) can be controlled.

  The bonding position of the hydrophilic part 4 to the temperature-responsive polymer part 2 (hydrophobic polymer part) is not particularly limited. When the temperature-responsive polymer part 2 (hydrophobic polymer part) is linear, the hydrophilic part 4 is bonded to the side chain of the temperature-responsive polymer part 2 (hydrophobic polymer part). Preferably it is. Thereby, in the cell separation membrane 10, the hydrophilic site | part 4 can be easily arrange | positioned in parts other than the temperature-responsive polymer part 2 (hydrophobic polymer part). Accordingly, the hydrophilic portion 4 can be more reliably exposed on the outer surface of the cell separation membrane 10.

  The temperature-responsive polymer part 2 (hydrophobic polymer part) is preferably a polyamino acid, and the hydrophilic part 4 preferably contains polyethylene glycol. Further, the hydrophilic portion 4 is preferably bonded to the temperature-responsive polymer portion 2 (hydrophobic polymer portion). As a material suitable as the temperature-responsive polymer part 2 (hydrophobic polymer part) and the hydrophilic part 4 satisfying these, for example, a compound represented by the following formula (1) may be mentioned.

* In formula (1), n and x are each independently an integer of 2 or more.

<Action>
The configuration and operation of the cell separation membrane 10 of the present embodiment will be described with reference to the drawings.
FIG. 2 is a perspective view of the cell separation membrane 10 of the present embodiment. When the cell separation membrane 10 is observed from a macro viewpoint, the thin film 5 is formed on the surface of the support 1. As shown in FIG. 1, the thin film 5 has a temperature-responsive polymer part 2 (2a, 2b, 2c), a cell adsorption part 3 (3a, 3b, 3c), and a hydrophilic part 4 (4a, 4b, 4c). ) Etc. are included.

  The antibody constituting the cell separation membrane 10 of the present embodiment includes a temperature-responsive polymer portion 2. The temperature-responsive polymer portion 2 is UCST type (upper critical soluble temperature) that exhibits hydrophobicity at a temperature higher than a predetermined temperature and exhibits hydrophilicity at a temperature lower than the predetermined temperature, or hydrophilic at a temperature higher than the predetermined temperature. And can be classified into LCST type (lower critical soluble temperature) that exhibits hydrophobicity at a temperature lower than the predetermined temperature.

  The cell separation membrane 10 of the present embodiment preferably includes at least one type of antibody including a UCST-type temperature-responsive polymer site and one type of antibody including an LCST-type temperature-responsive polymer site. It is preferable that the phase transition temperature of the temperature-responsive polymer part 2 (hydrophobic polymer part) or the hydrophilic part 4 of each antibody is different. The three-dimensional structure of the temperature-responsive polymer part 2 (hydrophobic polymer part) or the hydrophilic part 4 of each antibody preferably changes thermoreversibly.

  Various types of membrane proteins are expressed in the cell membrane 21 of the cell 20. By inducing differentiation during cell culture, the type of cell changes, and the type of membrane protein expressed changes accordingly (see FIG. 3 etc.).

  Here, the antibody that binds to the membrane protein A on the surface of the cell 20 (22 shown in FIG. 3) is the antibody A (31 shown in FIG. 1), and similarly, the membrane protein B on the cell surface (23 shown in FIG. 3). The antibody that binds to is antibody B (32 shown in FIG. 1), and the antibody that binds to the cell surface membrane protein C (24 shown in FIG. 3) is antibody C (33 shown in FIG. 1).

  Antibody A includes a UCST-type temperature-responsive polymer portion 2a that exhibits hydrophobicity at a temperature higher than a predetermined temperature and exhibits hydrophilicity at a temperature lower than the predetermined temperature. Antibodies B and C are higher than the predetermined temperature. The antibody will be described as comprising an LCST-type temperature-responsive polymer moiety 2b, 2c that is hydrophilic at temperature and hydrophobic at a temperature lower than the predetermined temperature. The phase transition temperatures of antibody A, antibody B, and antibody C are different.

  Note that the cell separation membrane of this embodiment may be configured to include two or more types of antibodies including a UCST-type temperature-responsive polymer site and three or more types of antibodies including an LCST-type temperature-responsive polymer site.

  Membrane protein A is constantly expressed in the cell membrane 21 of the target cell 20 (see FIG. 3 and the like). Membrane proteins B and C are expressed in the cell membrane 21 of the target cell 20 according to the progress of differentiation. Under the conditions for growing cells (for example, 37 ° C.), protein A and antibody A bind to each other (FIG. 4S1, FIG. 5), so that the cells bind on the non-woven fabric (culture vessel surface) and can grow on that (FIG. 4S2). ).

  In the process of proliferation, a cell different from the target cell 20 may be generated (see FIG. 3B, etc.). In order to remove it, by reducing the culture temperature (for example, when the phase transition temperature of antibody B is 33 ° C., the culture temperature is lowered to a predetermined temperature), the binding between antibody A and membrane protein A is eliminated, and the antibody B and membrane protein B can be bound (FIG. 4S3, FIG. 6).

If it is the target cell 20 (target cell), it remains fixed on the non-woven fabric (culture vessel surface) through the binding of membrane protein B-antibody B. However, when it is not a target cell (target cell) (a cell in which membrane protein B is not expressed), the cell floats in the culture solution.
By exchanging the culture solution, cells that are not the target cells 20 (target cells) can be removed (S4 in FIG. 4). Thereafter, in order to continue the growth culture, the culture conditions are restored (in this example, 37 ° C.) (FIG. 4S5).

  Furthermore, as differentiation induction proceeds, the target cell expresses membrane protein C (see FIG. 3 (d), FIG. 4S6, etc.). Similarly, when the culture temperature is lowered (for example, when the phase transition temperature of antibody C is 25 ° C., the culture temperature is lowered to a predetermined temperature), the binding between antibody A and membrane protein A is eliminated, and antibody C and Membrane protein C can be bound (FIG. 4S7, FIG. 7).

If it is the target cell (target cell), it remains fixed on the non-woven fabric (culture vessel surface) through the binding of membrane protein C-antibody C. However, when it is not the target cell 20 (target cell) (a cell in which the membrane protein C is not expressed), the cell floats in the culture solution.
By exchanging the culture solution, cells that are not the target cells 20 (target cells) can be removed (S8 in FIG. 4).

By using more types of antibodies having different phase transition temperatures in accordance with the membrane protein 22, 23, 24 of the target cell 20 (target cell), it is possible to remove unnecessary cells with a single cell separation membrane 10. It can be performed efficiently.
The method for adsorbing the target cells 20 is not limited to immersion, and the culture solution may be flowed over the cell separation membrane 10 or may be appropriately selected as long as the purpose is achieved.

The operation of the cell separation membrane 10 of this embodiment will be described in more detail.
FIG. 1 is a view for explaining the operation of the cell separation membrane 10 of the present embodiment, and is a view showing a state before heating. 5-7 is a figure which shows the state after the heating of the cell separation membrane 10. FIG. In the present embodiment, the structure of the temperature-responsive polymer part 2 (hydrophobic polymer part) is changed (destroyed) by heating the cell separation membrane 10.

  As shown in FIG. 5, in the present embodiment, the thin film 5 has a spiral temperature-responsive polymer portion (hydrophobic polymer portion) 2a and a cell-adsorbed portion bound to the temperature-responsive polymer portion 2a. 3a and a hydrophilic portion 4a bonded to the temperature-responsive polymer portion 2a. Cell adsorption sites 3a, 3b, and 3c are bonded to the temperature-responsive polymer sites 2a, 2b, and 2c (three in the illustrated example), respectively. Further, on the surface of the support 1, the temperature-responsive polymer portions 2a, 2b, and 2c and the hydrophilic portions 4a, 4b, and 4c are alternately arranged. That is, the surface of the support 1 is covered with the hydrophilic portions 4a, 4b, and 4c except for the temperature-responsive polymer portions 2a, 2b, and 2c covered with the cell adsorption portions 3a, 3b, and 3c. It will be.

  And in this embodiment, after the target cell (target cell) 20 adsorb | sucks to the cell adsorption site | part 3a, 3b, 3c (FIG. 5), the cell-responsive membrane part 2a is heated by heating the cell separation membrane 10. The target cell adsorbed on the cell adsorption site 3a is detached (FIG. 6).

  In the illustrated example, for the sake of convenience, one target cell 20 is adsorbed to one cell adsorption site 3a. However, one target cell 20 is adsorbed to two or more target cell sites 3. It may be.

The operation when changing from the state of FIG. 5 to the state of FIG. 6 will be described.
A helical structure is maintained in the temperature-responsive polymer portion 2a before heating. Further, the target cell 20 is adsorbed to the cell adsorption site 3a (FIG. 5). When the cell separation membrane 10 is heated in this state, the helical structure of the temperature-responsive polymer portion 2a is broken by heat energy to change to a random structure, and the cell adsorption portion 3a exposed on the outer surface becomes hydrophilic. It is buried in the part 4a (FIG. 6).

  Thereby, adsorption | suction of the cell adsorption part 3a and the target cell 20 will be interrupted | blocked by the steric hindrance of the temperature-responsive polymer part 2a changed into the random structure. Further, when the cell adsorption site 3a is buried in the hydrophilic site 4a, the target cell 20 is placed on the support 1 covered with the hydrophilic site 4a. If it does so, the target cell 20 which is hydrophobic will be in the state which can be collect | recovered easily from the hydrophilic region 4a.

The temperature (phase transition temperature) that changes from the state of FIG. 5 to the state of FIG. 6 can be controlled by the structure of the temperature-responsive polymer portion 2a.
For example, when the temperature-responsive polymer portion 2a has a long helical structure, the number of hydrogen bonds increases, so that the heat energy necessary for cutting them to change the structure also increases. Therefore, the temperature for changing the structure also tends to increase. On the other hand, when the included helical structure is short, the number of hydrogen bonds is also small. Therefore, the temperature for changing the structure tends to be low. Furthermore, when the hydrophilic part 4a has couple | bonded with the temperature-responsive polymer part 2a, it is controllable also by the structure of the hydrophilic part 4a.

  In the above description, the spiral structure changes to a random structure at a predetermined temperature or higher. However, the spiral structure may be a random structure at a predetermined temperature or lower, but may be a spiral structure at a predetermined temperature or higher. In this case, structural change occurs due to cooling, and the target cells 20 can be collected.

(Modification)
The cell separation membrane 10 of the present embodiment is not limited to the illustrated content or the content of the above description. For example, as shown in FIG. 8 (a), it is not necessary that the cell adsorption sites 3 are bonded to all the temperature responsive polymer sites 2, and the cell adsorption sites only on some of the temperature responsive polymer sites 2. Alternatively, a cell separation membrane 10A to which 3 is bonded may be used. In this way, even when the number of target cells contained in the liquid to be treated is small, the target cells can be efficiently recovered.

In addition to the hydrophilic part 4 bonded to the temperature-responsive polymer part 2, another hydrophilic part 4d is provided in the part between the adjacent hydrophilic parts 4 where the support 1 is exposed. The cell separation membrane 10B may be used (see FIG. 8B). By doing in this way, nonspecific adsorption | suction can be prevented more reliably.
Further, although the hydrophilic part 4 is not bonded to the temperature-responsive polymer part 2, it may be a cell separation membrane 10C in which another hydrophilic part 4d is provided at a position adjacent to the temperature-responsive polymer part 2 ( (Refer FIG.8 (c)). Even if it does in this way, nonspecific adsorption | suction can be prevented more reliably.

<< Second Embodiment >>
Next, a method for manufacturing the cell separation membrane 10 will be described. In this method, a case where one type of LCST type antibody is used will be described. A plurality of types of antibodies including LCST type temperature responsive polymer site 2 and an antibody containing UCST type temperature responsive polymer site are separated into cell separation membranes. It can be performed in the same way when it is fixed on the top 10.

There is no restriction | limiting in particular in the manufacturing method of the cell separation membrane 10 of this embodiment. For example, the cell separation membrane 10 can be manufactured by the following method.
In the following description, as an example of a method for manufacturing the cell separation membrane 10, a case where the cell separation membrane 10 in which the hydrophilic portion 4 is bonded to the temperature-responsive polymer portion 2 will be described.

  First, the temperature-responsive polymer portion 2 (which can be prepared by any method) to which the hydrophilic portion 4 is bonded is applied to the surface of the support 1 by, for example, spin coating, dipping method, vapor deposition method or the like. Fixed. The fixing method is preferably a chemical bond in which the temperature-responsive polymer portion 2 itself is not detached by a reversible reaction. Examples of the chemical bond in which the thermoresponsive polymer part 2 itself is not eliminated by the reversible reaction include, for example, an amide bond, an ester bond, a silanol bond, a maleimide, a thioester bond, a chemical bond by a Diels-Alder reaction, a chemical bond by a click reaction, Or a gold-thiol bond, an imine bond, etc. are mentioned depending on a use.

Here, when the temperature-responsive polymer part 2 is fixed, the hydrophilic part 4 may or may not be fixed to the support 1.
However, if the temperature-responsive polymer part 2 is fixed to the support 1, the hydrophilic part 4 bonded to the side chain can be sufficiently arranged on the surface of the support 1 without fixing. Thereafter, the temperature-responsive polymer portion 2 that has not been fixed is removed with an appropriate solvent.

Next, the cell adsorption site 3 is bonded to the end of the fixed temperature-responsive polymer site 2 by chemical bonding. At this time, the cell adsorption site 3 is not necessarily bound to the end of the temperature-responsive polymer site 2. Therefore, as long as the cell adsorption site 3 is exposed on the outer surface of the cell separation membrane 10, it may be bonded to any part of the temperature-responsive polymer site 2.
By doing in this way, the cell separation membrane which exposed the hydrophilic part 4 on the surface can be manufactured.

  The temperature-responsive polymer part 2 is bonded to the support 1 by dispersing the temperature-responsive polymer part 2 in a solvent as necessary, for example, spin coating, spray coating, dip coating, roll coating, bead coating. You may apply | coat to the surface of the support body 1 by various methods, such as. Also by such a method, the temperature-responsive polymer portion 2 can be disposed on the support 1.

<< Third Embodiment >>
Next, the use of the cell separation membrane 10 will be described with reference to the drawings. The cell separation membrane 10 of the present invention can be applied to any application. As a suitable application of the cell separation membrane 10, the cell culture apparatus 100 will be described as an example.

  The cell culture device 100 of the present embodiment (hereinafter simply referred to as “culture device 100” as appropriate) includes the cell separation membrane 10. The culture apparatus 100 separates and collects the target cells 20 contained in the liquid to be treated. Hereinafter, a specific apparatus configuration of the culture apparatus 100 and a method for separating and collecting the cells 20 using the culture apparatus 100 will be described.

  FIG. 9 is a block diagram of a culture apparatus 100 configured using the cell separation membrane 10 of the present invention. The culture device 100 includes a cell separation membrane 10, an impregnation device 102 b (contact device) for impregnating (contacting) the cell separation membrane 10 with a liquid to be treated containing target cells 20, and a heating device 104 for heating the cell separation membrane 10. (Heating and cooling device) and a cell recovery device 105 for recovering target cells adsorbed on the cell separation membrane 10 are provided.

  In addition to these, the culture apparatus 100 stores a washing solution 100b for washing cells non-specifically adsorbed on the cell separation membrane, a medium reagent 100c for culturing target cells, and a medium reagent 100a. A cool box 100 a and a liquid feeding system 101 that supplies the cleaning liquid 100 b and the medium reagent 100 c to the temperature-controlled room 102 are provided.

  Furthermore, the culture apparatus 100 is provided with a temperature-controlled room 102, and the cell separation membrane 10 is impregnated with a culture solution containing the target cells 20 in the culture apparatus 102a in the temperature-controlled room 102. Further, the atmosphere in the culture apparatus 102 a is controlled by the gas exchange apparatus 103.

A method for separating and collecting the cells 20 using the culture apparatus 100 will be described with reference to FIG.
In the following description, the cells 20 are collected after being adsorbed on the cell separation membrane and cultured. However, the cells 20 may be collected immediately after the adsorption without being cultured.

  First, the to-be-processed liquid containing the target cell 20 is supplied to the culture apparatus 102a (supply process S11). Thereafter, the impregnation apparatus 102b impregnates (contacts) the cell separation membrane 10 with the liquid to be treated containing the target cells 20, and adsorbs the target cells 20 in the liquid to be treated to the cell separation membrane 10 (contacting step S12). ). Thereafter, the cell separation membrane 10 is washed with the washing solution 100b to wash away non-specifically adsorbed cells (washing step S13), and the specifically adsorbed target cells 20 are cultured (culturing step S14). By doing in this way, the target cell 20 can be cultured efficiently.

  Next, the impregnation device 102b takes out the impregnated cell separation membrane 10 from the liquid to be treated (extraction step S15), and then the cell separation membrane 10 is heated by the heating device 104 (heating and cooling step S16). As a result, the structure of the temperature-responsive polymer portion 2 of the cell separation membrane 10 changes, and the specifically adsorbed target cells 20 (cultured target cells 20) can be easily detached. And the cell collection | recovery apparatus 105 supplies the cell collection | recovery liquid 105a with respect to the surface of the cell separation membrane 10 of this state (peeling collection process S17). Thereby, the cultured target cells 20 are peeled off from the surface of the cell separation membrane 10. The peeled target cells are collected by the cell collection device 105.

  According to the above method, it becomes possible to suppress damage to the cells 20 due to peeling that has occurred in the past when using a normal culture medium or a conventional culture sheet, and the use of chemicals during recovery. Thereby, the fall of the collection rate of the target cell 20 can be suppressed.

When there are a plurality of antibodies 31 to 33 including temperature-responsive polymer sites 2 having different phase transition temperatures, the above steps may be repeated by changing the culture temperature.
The cell separation membrane 10 may be used in the form of a flat sheet or in the form of a tube (tubular).

  In the above example, the target cell 20 is detached by heating, but the target cell 20 is detached by cooling by changing the structure of the temperature-responsive polymer portion 2 or the like. Also good. In this case, a cooling device that cools the cell separation membrane 10 may be applied instead of the heating device 104.

  Hereinafter, the present invention will be described in more detail with reference to Production Examples 1 to 3.

<Production Example 1>
(Preparation of PEG-modified polylysine)
First, 1-ethyl-3-carbodiimide (WSC), N-hydroxysuccinimide (NHS) and carboxylic acid-terminated polyethylene glycol (x = 3 in the formula (1)) were dissolved in pure water, and this solution was dissolved at 4 ° C. While cooling and stirring, polylysine (mixed with various molecular weights) was added. And it stirred at normal temperature for 2 hours, and obtained the PEG modification polylysine which polyethyleneglycol (hydrophilic part 4) couple | bonded with the side chain of polylysine (hydrophobic polymer polymer part 2).

  Subsequently, the mixture was further stirred for 24 hours using a dialysis membrane gel filtration chromatography method to remove unreacted low molecular weight substances. PEG-modified polylysine (PEG1) having a number average molecular weight of 5 kDa or more, PEG-modified polylysine (PEG2) having a number average molecular weight of 120 kDa or more, PEG-modified polylysine having a number average molecular weight of 2550 kDa or more (depending on the type of dialysis membrane) Three types of PEG3) were separated. Hereinafter, unless otherwise specified, the description of “polylysine” represents the prepared “PEG-modified polylysine”.

(Immobilization of polylysine to the support 1)
The synthesized polylysine (PEG 1 to 3) was covalently bonded according to the following procedure on a resin plate (polystyrene support) whose surface was treated with an amino group. 1-ethyl-3-carbodiimide (WSC) and N-hydroxysuccinimide (NHS) were added to a 1 mol% polylysine aqueous solution, and each plate was immersed.
Then, it was made to react at room temperature for 2 hours. As a result, a support 1 to which polylysine was bound was obtained.
Thereafter, each plate was washed three times with pure water.

(Binding of protein A to polylysine)
Three kinds of plates to which the synthesized polylysine (PEG 1 to 3) was bound were immersed in 2% glutaraldehyde diluted with a carbonate buffer and allowed to stand at 37 ° C. for 2 hours. Each plate after the reaction was washed three times with a phosphate buffer (PBS). Then, a protein A aqueous solution having a concentration of 0.1 wt% (using a PBS buffer solution of pH 7.4) was prepared, and each plate was immersed in the protein A solution to bind protein A to polylysine.

(Binding of antibody CD40)
Subsequently, according to the following procedure, antibody CD40 was bound to protein A (antigen) immobilized on each plate. Antibody CD40 is an antibody that can specifically bind to Chinese hamster ovary cells. First, each plate was washed 3 times with a washing buffer (200 μl), and then 50 μl of immunoglobulin G (IgG) was added and incubated overnight at 4 ° C. As a result, IgG was bound to protein A.

Thereafter, each plate was washed three times using a washing buffer (200 μl) while maintaining at 4 ° C.
Thereafter, each plate was immersed in a solution of antibody CD40 to bind antibody CD40 to IgG.
By the above operation, the cell separation membrane 10 (test bodies 1 to 3) containing the synthesized polylysine (PEG 1 to 3) was produced.

(Evaluation of cell adsorption selectivity)
A buffer aqueous solution containing Chinese hamster ovary cells (CHO cells) and mesenchymal stem cells (MSC) in a one-to-one relationship was dropped onto the cell separation membrane 10 (test bodies 1 to 3) at 4 ° C. and incubated. MSC is not adsorbed to antibody CD40 of cell separation membrane 10, and CHO cells are adsorbed specifically.

Thereafter, each plate was washed three times with an aqueous buffer solution to wash away non-specifically adsorbed MSC. The washed-out MSC was collected, and the number of cells was confirmed with a flow cytometer.
Note that the CHO cells and the MSC cells in the flow cytometer were distinguished from each other by fluorescently labeling the MSC cells with a FITC-labeled anti-CD105 antibody.

  Next, each plate was heated to 37 ° C. in a buffered aqueous solution to change the structure of polylysine, thereby desorbing specifically adsorbed CHO cells. The detached CHO cells were collected using a buffered aqueous solution. And the cell number of the collect | recovered CHO cell was confirmed with the flow cytometer.

Based on the number of collected MSC cells and the number of CHO cells, the cell selectivity was calculated using the following formula (2).

(Selection rate of cells) = (Number of cells recovered by cell separation membrane 10) / (Number of cells before recovery) × 100 [%] (2)

A cell separation membrane 10 (test bodies 1 to 3) was prepared in the same manner as in test bodies 1 to 3 except that a buffered aqueous solution containing CHO cells and MSCs was used 1: 1 (test body 4), and the selectivity was evaluated.
Note that MSC is not adsorbed to the antibody CD40 of the cell separation membrane 10, and B cells are adsorbed specifically.

A cell separation membrane 10 was prepared in the same manner as in Specimens 1 to 3 except that a buffered aqueous solution containing circulating tumor cells (CTC) and MSCs in a 1: 1 ratio was used (Test Specimen 5), and the selectivity was evaluated. did.
Note that MSC is not adsorbed to the antibody CD40 of the cell separation membrane 10, and CTC is adsorbed specifically.

<Comparative Example 1>
Except that “polyethylene glycol at the carboxylic acid terminal” was not used (that is, the hydrophilic part 4 was not included) and that the number average molecular weight of polylysine (polylysine not including the hydrophilic part 4) was 7 kDa. A cell separation membrane was prepared in the same manner as in test bodies 1 to 3 (test body 21). And this cell separation membrane evaluated the selectivity similarly to the test bodies 1-3.

<Evaluation result 1>
The selectivity of the test bodies 1 to 5 and the test body 21 is shown in Table 1 below.

As shown in Table 1, in the cell separation membrane 10 (test bodies 1 to 5) including the hydrophilic portion 4, both are good choices regardless of the number average molecular weight (size) and the type of the target cell 20. Showed the rate. Therefore, it was found that by including the hydrophilic portion 4 in the cell separation membrane 10, nonspecific adsorption of cells is suppressed, and the target cells 20 can be efficiently separated and recovered.

  On the other hand, in the test body 21 that does not include the hydrophilic part 4, the number average molecular weight and the type of the target cell 20 are almost the same as those of the test bodies 1 to 5, but the selectivity is about the same as that of Example 1. It was half. Therefore, it was found that when a cell separation membrane not containing the hydrophilic site 4 is used, nonspecific adsorption increases and the selectivity is remarkably lowered.

  Moreover, the microscope picture about the surface after making a cell adsorb | suck with respect to the cell separation membrane 10 of Example 1 and the surface after carrying out separation collection | recovery (desorption) is shown in FIG.

  FIG. 11A is a photomicrograph when the cells 20 are adsorbed on the cell separation membrane 10, and FIG. 11B is a photomicrograph of the cell separation membrane 10 after the adsorbed cells are collected. (C) is the figure which showed the photograph of (a) typically, (d) is the figure which showed the photograph of (b) typically.

  As shown in FIG. 11 (a), the cells 20 are adsorbed on the surface of the cell separation membrane 10, but by heating the cell separation membrane 10, the surface of the cell separation membrane 10 as shown in FIG. 8 (b). From the results, it was found that the cells 20 were completely detached.

<Production Example 2>
The difference in cell selectivity was confirmed by changing the type of antibody constituting the cell separation membrane 10. Specifically, the difference in the selectivity of the cells 20 was confirmed when no antibody was used, when one antibody was used, and when two antibodies were used.

In Production Example 2, the following three types were prepared.
-Normal 6-well plate for adherent cells (Comparative Example: Specimen 25)
6 anti-CD29 antibody (phase transition temperature 30 ° C.) bound to LCST type temperature responsive polymer site and anti-bone sialoprotein antibody (phase transition temperature 25 ° C.) bound to LCST type temperature responsive polymer site Plate physically adsorbed on well (Example: Specimen 11)
An anti-CD29 antibody (phase transition temperature 30 ° C.) bound to an LCST-type temperature-responsive polymer site, an anti-bone sialoprotein antibody (phase transition temperature 25 ° C.) bound to an LCST-type temperature-responsive polymer site, and an LCST A plate (Example: Specimen 12) in which an anti-alkaline phosphatase antibody (phase transition temperature 15 ° C.) bound to a type temperature-responsive polymer site is physically adsorbed on 6 wells

(Preparation of cells)
Mesenchymal stem cells (hMSC) were cultured using bovine serum-containing MSCGM medium until they became confluent (cells were buried in the culture bed without any gaps). Cells that have reached 90% confluency are washed 3 times with phosphate buffered saline (PBS), 0.02% trypsin is added, and in the incubator (37 ° C., 5% CO ₂ ,> 90% humidity) ) For about 5 minutes. After confirming the detachment of the cells 20 under a microscope, a trypsin inhibitor was added and centrifuged at 600 × g for 5 minutes to sediment the cells 20. The supernatant containing trypsin and trypsin inhibitor is removed by an aspirator, a medium is added so as to achieve the target cell density, seeded in the above three 6-well plates, and in an incubator (37 ° C., 5% CO ₂ ,> 90% Humidity).

(Induction of differentiation into osteoblasts)
Cells cultured in a serum-free medium were cultured for 2 weeks in an osteoblast induction medium (containing the differentiation-inducing factors dexamethasone, ascorbic acid, and Glycerophosphate into osteoblasts), and the rate of differentiation into osteoblasts was evaluated. The osteoblast induction medium was changed every 3-4 days. Alkaline phosphatase staining was used for confirmation of osteoblasts.

The test body 25 was cultured at a constant 37 ° C. during the culture period.
With respect to the test body 11, the temperature was allowed to stand at 20 ° C. for 3 hours in the first week of culture, and after changing the medium, the temperature was returned to 37 ° C., and then cultured at a constant temperature.
With respect to the test body 12, the temperature was allowed to stand at 20 ° C. for 3 hours at the first week of culture, and after changing the medium, the temperature was returned to 37 ° C., and then cultured at a constant temperature. Was allowed to stand at 10 ° C. for 3 hours, and after changing the medium, the temperature was returned to 37 ° C. and then cultured at a constant temperature.

<Evaluation result 2>
The results are shown in FIG. Bone cells are stained black by staining.
Regarding the test body 25, it can be seen that the stained cells are sparse and there are many cells that are not bone cells. It can be seen that the specimen 11 is differentiated into bone cells at a higher rate than the plate 1. In the test body 12, it can be seen that all the cells are bone cells.
Thus, high purity cells can be obtained by selecting cells in the differentiation induction process.

<Production Example 3>
In the same manner as in Production Example 2, selection of mesenchymal stem cells in adipocyte differentiation was examined.

Production Example 3 prepared the following three types.
-Normal 6-well plate for adherent cells (Comparative Example: Specimen 25)
6 wells of anti-CD29 antibody (phase transition temperature 30 ° C.) bound to UCST type temperature responsive polymer site and anti-CD105 antibody (phase transition temperature 15 ° C.) bound to LCST type temperature responsive polymer site Plate physically adsorbed on the surface (Example: Specimen 13)
Anti-CD29 antibody (phase transition temperature 30 ° C.) bound to the UCST-type temperature-responsive polymer site, anti-CD105 antibody (phase transition temperature 15 ° C.) bound to the LCST-type temperature-responsive polymer site, and LCST-type temperature Plate in which anti-adiponectin (phase transition temperature 25 ° C.) bound to a responsive polymer site is physically adsorbed on 6 wells (Example: Specimen 14)

(Preparation of cells)
Mesenchymal stem cells (hMSC) were cultured using bovine serum-containing MSCGM medium until they became confluent (cells were buried in the culture bed without any gaps). Cells that have reached 90% confluency are washed 3 times with phosphate buffered saline (PBS), 0.02% trypsin is added, and in the incubator (37 ° C., 5% CO ₂ ,> 90% humidity) ) For about 5 minutes. After confirming cell detachment under a microscope, trypsin inhibitor was added and centrifuged at 600 × g for 5 minutes to sediment the cells. The supernatant containing trypsin and trypsin inhibitor is removed by an aspirator, a medium is added to achieve the desired cell density, seeded in the three 6-well plates, and incubated in an incubator (37 ° C., 5% CO ₂ ,> 90% Humidity).

(Induction of differentiation into adipocytes)
HMSCs were cultured in a medium of each condition for 2 weeks on a 96-well plate. Thereafter, the cultured medium was changed to an adipocyte differentiation-inducing medium (containing a differentiation-inducing factor IBMX (3-isobuthyl-1-methyl xanthine), dexamethasone, insulin and indomethacin). After culturing for 3 days, the medium was changed to an adipocyte differentiation maintenance medium and cultured for 3 days. Thereafter, the medium was again replaced with an adipocyte differentiation induction medium. Thus, differentiation induction was performed for 2 weeks while exchanging the adipocyte differentiation induction medium and the adipocyte differentiation maintenance medium every three days. Oil red O staining was used for confirmation of adipocyte differentiation.

The test body 25 was cultured at a constant 37 ° C. during the culture period.
With respect to the test body 13, the temperature was allowed to stand at 20 ° C. for 3 hours in the first week of culture, and after changing the medium, the temperature was returned to 37 ° C., and then cultured at a constant temperature.
With respect to the test body 14, the temperature was allowed to stand at 20 ° C. for 3 hours at the first week of culturing, and after changing the medium, the temperature was returned to 37 ° C., and then cultured at a constant temperature. Was allowed to stand at 10 ° C. for 3 hours, and after changing the medium, the temperature was returned to 37 ° C. and then cultured at a constant temperature.

<Evaluation result 3>
The results are shown in FIG. Adipocytes are stained red by staining. Regarding the test body 25, it can be seen that the stained cells are sparse and there are many cells that are not fat cells. It can be seen that the test body 13 is differentiated into fat cells at a higher rate than the test body 25. In the test body 14, it can be seen that almost all cells are fat cells. Thus, it was confirmed that cells having high purity were obtained by selecting cells during the differentiation induction process.

  As described above, the cell separation membrane according to the present invention and the cell separation apparatus including the same have been described in detail with reference to the best mode and examples. Needless to say, the contents of the present invention are not limited to the above-described embodiments and examples, and can be appropriately modified or changed without departing from the spirit of the present invention.

1 Support 2, 2a, 2b, 2c Temperature-responsive polymer part (hydrophobic polymer part)
3, 3a, 3b, 3c Cell adsorption site 4, 4a, 4b, 4c Hydrophilic site 5 Thin film 10 Cell separation membrane 20 Cell (target cell)
21 Cell membrane 22 Membrane protein A
23 Membrane protein B
24 Membrane protein C
25 Membrane protein X
26 Membrane protein Y
31 Antibody A
32 Antibody B
33 Antibody C
DESCRIPTION OF SYMBOLS 100 Culture apparatus 102b Impregnation apparatus 104 Heating apparatus 105 Cell recovery apparatus 105a Cell recovery liquid

Claims (10)

A support;
A polymer site fixed to the support and having a structure that changes with temperature;
A cell adsorption site that is bound to the polymer site and can be exposed to the outer surface, and can specifically adsorb the target cell when contacted with a liquid to be treated containing the target cell;
A hydrophilic portion exposed on the outer surface and in contact with the liquid to be treated,
The cell separation membrane, wherein the cell adsorption site can adsorb two or more types of membrane proteins of the target cell.   The cell separation membrane according to claim 1, wherein the polymer portion exhibits hydrophobicity at a temperature higher than a predetermined temperature, and exhibits hydrophilicity at a temperature lower than the predetermined temperature.   The cell separation membrane according to claim 1 or 2, wherein the polymer portion exhibits hydrophilicity at a temperature higher than a predetermined temperature, and exhibits hydrophobicity at a temperature lower than the predetermined temperature.   The cell separation membrane according to any one of claims 1 to 3, wherein the polymer site has a different phase transition temperature for each type of the cell adsorption site bound thereto.   5. The cell adsorption site according to claim 1, wherein the cell adsorption site can be in any one of a state exposed on an outer surface and a state buried in the hydrophilic site. As described in any one   The cell separation membrane according to any one of claims 1 to 5, wherein the polymer portion includes a polyamino acid.   The cell separation membrane according to any one of claims 1 to 6, wherein the polymer portion has a helical structure at least in part.   The cell separation membrane according to any one of claims 1 to 7, wherein the hydrophilic portion includes polyethylene glycol.   The cell separation membrane according to any one of claims 1 to 8, wherein the support includes at least one of a polymer material and a glass material.   A cell separation device comprising the cell separation membrane according to any one of claims 1 to 9 as a cell separation means.