JP2005323534A - Membrane for separating and recovering cell and method for separating and recovering cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a membrane for separating and recovering a cell designed to efficiently separate the hematopoietic stem cell from peripheral blood, cord blood, bone marrow fluid, and the like, and recover the hematopoietic stem cell in high yield and to provide a method for separating and recovering the cell. <P>SOLUTION: The membrane is used for separating and recovering the useful cell from a cell mixture containing the useful cell and cells to be removed. The membrane for separating and recovering the cell is obtained by introducing carboxyl groups into a polyurethane foam. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell separation / recovery membrane capable of separating hematopoietic stem cells among useful cells existing in blood and the like, and a cell separation / recovery method using the membrane, in particular, by modifying a polyurethane membrane with a functional group, More particularly, the present invention relates to a cell separation / recovery membrane and a cell separation / recovery method capable of recovering hematopoietic stem cells in a high yield among useful cells.

In recent years, it has become a major issue in the field of regenerative medicine to regenerate each function of cells, organs, and tissues that have lost their own regenerative ability and have irreversibly failed.
Regenerative medicine is mainly composed of gene therapy, organ creation using ES cells, cell therapy in which stem cells are injected into functionally defective tissues or organs, and organ creation using stem cells.
Among these, when organ creation using stem cells or injection of stem cells into a function-deficient tissue or the like, it is necessary to extract target stem cells or tissue precursor cells with high purity.
In order to develop regenerative medicine, a technology for easily separating useful cells such as high-purity stem cells and tissue progenitor cells from peripheral blood, umbilical cord blood, bone marrow fluid, and the like is required for the development of regenerative medicine. .

Conventionally, a technique for separating each target component from blood or the like is known and used for treatment in a medical field.
For example, many viruses have been reported to selectively infect lymphocytes. Therefore, in order to prevent viral infection due to blood transfusion, whole blood is not transfused, but leukocytes are removed by a leukocyte removal filter, etc., and erythrocyte preparations, platelet preparations, plasma preparations, etc. are produced from the blood from which leukocytes have been removed. These formulations are generally used for therapy.
Thus, until now, research aimed at separating and purifying blood cells has been performed.
However, in recent studies, peripheral blood, umbilical cord blood, and bone marrow fluid may contain useful cells such as various stem cells such as hematopoietic stem cells, vascular endothelial cells, vascular endothelial progenitor cells, and organ progenitor cells. As a result, a technique for separating and collecting these useful cells from peripheral blood, umbilical cord blood, bone marrow fluid and the like has come to be desired.

The importance of hematopoietic stem cells separated from peripheral blood, bone marrow fluid, and umbilical cord blood in the field of regenerative medicine is increasing.
Hematopoietic stem cells are cells that have the ability to self-replicate and have the ability to differentiate into all blood cells. However, in recent years, there have been many reports showing the plasticity of hematopoietic stem cells, suggesting the possibility of being involved in the production of various cells as well as blood cells.
Currently, in the field of regenerative medicine, the hematopoietic stem cells are transplanted and are effectively used for the treatment of intractable diseases such as leukemia. Further, due to the suggestion of the plasticity of hematopoietic stem cells, further effectiveness in regenerative medicine is also expected.
However, the amount of hematopoietic stem cells contained in bone marrow fluid, peripheral blood, and umbilical cord blood is very small, and it is very difficult to secure the amount required in the medical field.
For this reason, a method has been developed in which hematopoietic stem cells are separated from bone marrow fluid, peripheral blood, and umbilical cord blood and propagated together with various cytokines.
However, in order to efficiently proliferate hematopoietic stem cells, it was necessary to separate hematopoietic stem cells from bone marrow fluid, peripheral blood, and umbilical cord blood with high purity and high yield.

Thus, for example, the Ficoll Hypaque method is known as a method for separating hematopoietic stem cells from bone marrow fluid, peripheral blood, and umbilical cord blood.
This is a centrifugal separation method in which a target component is separated by specific gravity.
However, this method has a problem that it is necessary to perform centrifugation many times and a long time is required for the separation operation.
In addition, the operation is complicated, and there is a problem that a skilled technique is necessary to perform the separation operation.

As another method, the Isolex Magnetic Cell Separation System developed by Baxter by applying magnetism is known.
In this method, hematopoietic stem cells are separated by a monoclonal antibody that specifically binds to a surface marker expressed on the surface of the hematopoietic stem cell, and the monoclonal antibody bound to the surface marker is bound to magnetic beads to recover hematopoietic stem cells bound to the antibody. Is the method.
However, this method has a problem that operation is complicated and separation takes a long time.
Furthermore, since mouse-derived antibodies are used, it is feared that viruses and antibodies derived from different species will be mixed in blood products mainly composed of hematopoietic stem cells. Is in debate.

Furthermore, as another method, a hematopoietic stem cell separation system using high-speed FACS (Fluorescence-activated cell sorter) has been developed.
This is a system for selectively separating cells stained with a fluorescent antibody, but has a problem that the separation operation takes a long time.
In addition, there is a problem that a special device is required and high cost is required to perform the separation.
Furthermore, since cell separation cannot be performed in a sterilized state, it is impossible to use the purified hematopoietic stem cells for treatment.
In addition, since mouse-derived antibodies are used, it is feared that viruses and antibodies derived from different species will be mixed in blood products mainly composed of hematopoietic stem cells. Is in debate.

Thus, as a technique for separating hematopoietic stem cells easily and inexpensively, a technique for separating stem cells using a separation membrane has been developed (see, for example, Patent Document 1 and Patent Document 2).
Patent Document 1 discloses a technique for capturing nucleated cells using a filter made of a synthetic polymer, a natural polymer, or the like.
In this technique, the raw cell fluid is pretreated by the Ficoll-Hypaque method (specific gravity centrifugation), and this raw material cell fluid is passed through a filter, so that CD45 antigen positive, CD34 antigen negative, and differentiation antigen negative humans. Undifferentiated hematopoietic stem cells are separated and captured. Then, the undifferentiated hematopoietic stem cells captured on the filter are recovered by introducing a recovery solution from the downstream side of the filter.
Patent Document 2 uses a filter medium in which a plurality of non-woven filters made of a synthetic polymer, a natural polymer, or the like are laminated, and the filter end faces are continuously fused at a high temperature so as to be liquid-tight. A technique for recovering CD34-positive cells captured on a filter by separating and capturing CD34-positive cells from umbilical cord blood and introducing a recovery solution from the downstream side of the filter is disclosed.

JP 2000-166541 A (2nd to 4th pages, FIG. 1)

JP 2001-136955 A (pages 5 to 7, FIG. 1 and FIG. 2)

However, in the technique of the above-mentioned Patent Document 1, a pretreatment by Ficoll-Hypaque specific gravity centrifugation is required for the collected umbilical cord blood in order to obtain a raw material cell solution. As described in the section of the prior art, this pretreatment requires a complicated technique and a special technique. In addition, since it is difficult to perform this pretreatment in a completely closed system, there is a problem that the sterility of the raw material cell solution cannot be sufficiently ensured.
In addition, further improvement that can recover hematopoietic stem cells at a higher recovery rate has been desired.

  In the technique of Patent Document 2, as many as 16 filters are laminated, and a stainless steel plate heated at a high temperature is pressed against the laminated filter end face, and continuously superheated (about 250 ° C.), and the filters are continuously liquid-tightly fused. Therefore, the filter preparation work is complicated, and a special technique for preparing a filter having a certain liquid tightness is required.

Further, the technique of Patent Document 2 is not a method for selectively separating and collecting CD34-positive cells because there is almost no difference between the recovery rate of leukocytes and the recovery rate of CD34-positive cells.
Therefore, development of a method for selectively and efficiently concentrating CD34 positive cells, which are hematopoietic stem cells that play an important role in the field of regenerative medicine, has been desired.

  An object of the present invention is to provide a cell separation and recovery membrane and a cell separation method capable of efficiently separating hematopoietic stem cells from peripheral blood, umbilical cord blood, bone marrow fluid, etc., and recovering them in a high yield. An object of the present invention is to provide a hematopoietic stem cell separation and recovery membrane and a hematopoietic stem cell separation method capable of easily collecting hematopoietic stem cells of high purity.

According to the cell separation / recovery membrane of claim 1 of the present invention, the subject is a cell separation / recovery membrane used for separating and recovering useful cells from a cell mixture containing useful cells and removed cells, The cell separation and recovery membrane is solved by introducing a carboxyl group into a polyurethane foam.
Polyurethane is a general term for synthetic polymers having a urethane bond in the main chain, and polyurethane foam refers to polyurethane that has undergone a foaming reaction.
By using a carboxyl group-introduced polyurethane foam membrane, hematopoietic stem cells, which are useful cells, can be separated and recovered at a higher recovery rate than a polyurethane foam membrane to which no functional group has been introduced simply by filtering the cell mixture. be able to.
Moreover, according to this carboxyl group-introduced polyurethane foam membrane, hematopoietic stem cells, which are useful cells, can be separated and recovered at a high recovery rate without performing a pretreatment such as centrifuging the cell mixture.

In the cell separation and recovery membrane, CD34 antigen-positive hematopoietic stem cells are preferably separated and recovered as the useful cells.
That is, in recent years, in the field of regenerative medicine, CD34 antigen-positive hematopoietic stem cells that are undifferentiated and can be differentiated into all blood cells are useful for treatment, and therefore it is desired to collect them at a high yield.
In the cell separation and recovery membrane according to the present invention, this CD34 antigen-positive hematopoietic stem cell can be easily separated with a higher recovery rate than a polyurethane foam membrane into which a functional group has not been introduced. Can be recovered.

Furthermore, it is preferable that a compound having an epoxy ring is introduced into the cell separation and recovery membrane, and the carboxyl group is introduced through the epoxy ring.
Since epoxy groups are highly reactive with each functional group, various functional groups, including carboxyl groups, can be efficiently introduced into polyurethane foam membranes by introducing a compound having an epoxy ring into the membrane in advance. It becomes.

The cell separation / recovery membrane is provided with a plurality of holes through which the removed cells pass, and the diameter of the holes is preferably 3 μm or more and 100 μm or less.
Since it is constituted in this way, separation and collection of useful cells are efficiently performed in the cell separation and collection membrane.
That is, if the pore diameter is smaller than this range, the filtration time becomes longer and the recovery rate of useful cells also decreases. In addition, if the pore diameter is larger than this range, useful cells that permeate the cell separation and recovery membrane during the useful cell capture operation increase, and the useful cell recovery rate decreases.

In addition, according to the cell separation and recovery method according to claim 5 of the present invention, the above problem is a method for separating and recovering useful cells from a cell mixture containing useful cells and removed cells, wherein a carboxyl group has been introduced. A useful cell capturing step for allowing the cell mixture to pass through the polyurethane foam membrane, and a useful cell recovery step for recovering the useful cells by passing the recovery solution through the carboxyl group-introduced polyurethane foam membrane. It is preferable that
Since it is comprised in this way, it becomes possible to isolate | separate and collect hematopoietic stem cells which are useful cells only by filtering a cell liquid mixture with a carboxyl group introduction polyurethane foam membrane.
In addition, since this separation and recovery method is a filtration operation, it is possible to easily separate and recover high-purity useful cells without requiring expensive equipment and equipment.

Furthermore, in the useful cell recovery step, it is preferable that CD34 antigen-positive hematopoietic stem cells are recovered as the useful cells.
In the field of regenerative medicine, CD34 antigen-positive hematopoietic stem cells that are undifferentiated and can be differentiated into all blood cells are useful for treatment, and therefore it is desired to collect them at a high yield. In the cell separation and recovery method according to the present invention, the CD34 antigen-positive hematopoietic stem cells can be easily and efficiently separated and recovered.

In the useful cell recovery step, it is preferable that any one of physiological saline, buffer solution, physiological saline containing protein, and buffer solution containing protein is used as the recovery solution.
Further, it is preferable that 0.05% by weight or more and 3% by weight or less of an albumin aqueous solution is used as the recovery solution used in the useful cell recovery step.

Furthermore, it is preferable that a pretreatment step of infiltrating the carboxyl group-introduced polyurethane foam membrane with a phosphate buffer solution or an aqueous albumin solution is performed before the useful cell capturing step.
Thus, when the carboxyl group-introduced polyurethane foam membrane is pretreated, useful cells can be recovered at a higher recovery rate than the carboxyl group-introduced polyurethane foam membrane without pretreatment, There is no significant difference in the separation rate.
In addition, the pretreatment of the carboxyl group-introduced polyurethane foam membrane involves only infiltrating the membrane with a phosphate buffer or a human albumin aqueous solution, so that special equipment is required unlike the pretreatment of a sample that is repeatedly centrifuged. It can be implemented simply without doing.

  According to the present invention, by using a polyurethane membrane modified with a carboxyl group as a hematopoietic stem cell separation and recovery membrane, hematopoietic stem cells used in regenerative medicine can be easily and efficiently separated from peripheral blood, umbilical cord blood, bone marrow fluid, and the like. It becomes possible to collect, and labor saving of the work of separating and collecting hematopoietic stem cells is realized.

  In addition, according to the present invention, it is possible to introduce a carboxyl group into a polyurethane foam membrane by a simple operation, and it is possible to easily separate and collect hematopoietic stem cells simply by setting the membrane on an existing filter holder. it can. For this reason, the cost is low and hematopoietic stem cells can be easily separated and recovered.

  In addition, since the amount of hematopoietic stem cells present in peripheral blood, umbilical cord blood, and bone marrow fluid is very small, it is necessary to culture and proliferate hematopoietic stem cells in order to ensure the amount of hematopoietic stem cells required in regenerative medicine . Therefore, it is necessary to separate and collect hematopoietic stem cells present in peripheral blood, umbilical cord blood, and bone marrow fluid with high yield and high purity. In the hematopoietic stem cell separation and recovery membrane and hematopoietic stem cell separation method according to the present invention, Hematopoietic stem cells can be recovered with high purity and high yield.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Note that the members, arrangements, and the like described below do not limit the present invention and can be variously modified within the scope of the gist of the present invention.

FIG. 1 is an explanatory view showing a hematopoietic stem cell separation and recovery apparatus S according to the present embodiment.
In the present specification, hematopoietic stem cells are cells that can be differentiated into all blood cells, express the surface marker CD34, the surface marker CD45 is weakly positive (blast region), and the side scatter is low. Refers to cells that are blast regions with low to moderate forward scatter (hereinafter simply referred to as “CD34 ⁺ hematopoietic stem cells”). That is, according to the International Society for Hematology and Transplantation (ISHAGE guidelines), CD34 positive cells are defined as hematopoietic stem cells, and hereinafter referred to as “CD34 ⁺ hematopoietic stem cells”.
Surface markers, also called surface antigens, refer to molecules that are expressed on the cell surface. It is thought that what kind of molecule is expressed on the cell surface reflects the state, differentiation, and function of the cell.
In this embodiment, human peripheral blood collected from the cubital vein of the human upper arm is used as a sample.
However, the present invention is not limited to human peripheral blood, and CD34 ⁺ hematopoietic stem cells can be separated and collected from umbilical cord blood, bone marrow fluid, and the like in the hematopoietic stem cell separation and collection apparatus S according to this embodiment.
In addition to the hematopoietic stem cell separation and recovery device S, peripheral blood, umbilical cord blood, bone marrow fluid and the like injected into the blood collection bag are allowed to permeate into the polyurethane foam membrane by natural fall, and thereafter, recovery of human albumin aqueous solution and the like It is also possible to separate and recover CD34 ⁺ hematopoietic stem cells by permeating the solution through the polyurethane foam membrane.

The hematopoietic stem cell separation and recovery apparatus S includes a pump 1, a sample filling syringe 2, a pressing syringe 3, a micro syringe filter holder 4, a support base 5, and a filtrate recovery member support holder 6.
The pump 1 is a known peristaltic pump including a water supply unit, a discharge unit, and a pump mechanism. One end of the tube 11a is connected to the water supply side, and one end of the tube 11b is connected to the discharge side.
A water supply member 11c is connected to the other end of the tube 11a and is immersed in a beaker 11d filled with pure water. The water supply member 11c is a filter material and is used to remove impurities mixed in the sucked liquid.
The pump 1 sucks pure water through the tube 11a through the water supply member 11c. The sucked pure water passes through the tube 11a and is quantitatively sent out to the tube 11b by a pump mechanism (not shown). The pure water sent out to the tube 11b is transferred in the drawing Y direction.
The sample filling syringe 2 is a known syringe-type syringe and includes a discharge port 21, a piston 22, and a pressing unit 23. The sample filling syringe 2 is filled with peripheral blood as a sample.
The pressing syringe 3 is a known syringe-type syringe, like the sample filling syringe 2, and includes a discharge port 31, a piston 32, and a pressing portion 33. The pressing syringe 3 is used for receiving pure water fed from the pump 1 and transmitting the force transmitted to the piston 32 to the piston 22 of the sample filling syringe 2.

The microsyringe filter holder 4 is a known filter holder, and a hematopoietic stem cell separation / recovery membrane (not shown) is sandwiched between them.
The support table 5 is a known device support table, and includes handles 51, 52, 53, a base 54, and a support bar 55.
A support bar 55 is fixed to the base 54 in an upright state.
Handles 51, 52, and 53 are fixed to the support bar 55 at predetermined positions by fixtures 55a, 55b, and 55c, respectively. The grips 51, 52, and 53 are integrated with the fixtures 55a, 55b, and 55c, respectively, and can be moved up and down along the support rods 55. The handles 51, 52, and 53 are positioned at predetermined positions by screws provided in the fixtures 55a, 55b, and 55c, respectively. Fixed to.
The handles 51, 52, 53 are fixed at predetermined positions in a state orthogonal to the support rod 55, respectively.
The handle 51 is formed by fixing a clip 51b to the device support side end 51a. The pressing syringe 3 is detachably fixed to the clip 51b. In addition, the discharge port 31 of the pressing syringe 3 is detachably fixed upward.
The handle 52 is formed by fixing a clip 52b to a device support side end 52a. The sample filling syringe 2 is detachably fixed to the clip 52b. The discharge port 21 of the sample-filled syringe 2 is fixed so as to be detachable downward.
The handle 53 is formed by fixing a clip 53b to the device support side end 53a. The micro syringe filter holder 4 is detachably fixed to the clip 53b.
The grips 51 and 52 are fixed to the support rod 55 by fixtures 55a and 55b at a position where the pressing portion 33 of the piston 32 and the pressing portion 23 of the piston 22 are in contact with each other.
The handle 53 is fixed to the support rod 55 by a fixing tool 55 c at a position where the sample introduction port 41 of the microsyringe filter holder 4 is joined to the discharge port 21 of the sample filling syringe 2.
The filtrate collection member support holder 6 is a known test tube holder, and is installed at the bottom of the microsyringe filter holder 4. A test tube 6 a is supported on the filtrate recovery member support holder 6. The test tube 6 a is disposed at a position immediately below the filtrate outlet of the microsyringe filter holder 4.

The other end side of the tube 11 b connected to the discharge port (not shown) of the pump 1 is connected to the discharge port 31 of the pressing syringe 3.
Therefore, when the pump 1 is operated and pure water is transferred in the direction of the drawing Y, water enters the pressing syringe 3 from the discharge port 31 and the piston 32 is pushed down in the direction of the drawing X.
When the piston 32 of the pressing syringe 3 is pushed down in the X direction in the drawing, the pressing portion 33 of the piston 32 and the pressing portion 23 of the sample-filling syringe 2 are in contact with each other. Pushed down.
When the piston 22 is pushed down in the X direction in the drawing, the pressure acting in the X direction becomes the pressing force, and the sample filled in the sample filling syringe 2 is pressure filtered.

  The sample to which pressure is applied is introduced into the microsyringe filter holder 4 from the discharge port 21 and is filtered under pressure through a hematopoietic stem cell separation / recovery membrane (not shown) sandwiched between the microsyringe filter holder 4.

Here, the hematopoietic stem cell separation and recovery membrane sandwiched between the microsyringe filter holder 4 will be described.
The hematopoietic stem cell separation / recovery membrane according to this embodiment is a polyurethane foam membrane chemically modified with a carboxyl group.
The hematopoietic stem cell separation and recovery membrane according to the present embodiment uses a polyurethane foam membrane into which glycidyl methacrylate (hereinafter simply referred to as “GMA”) is introduced as a basic structure.
GMA has an epoxy ring in its structure, and membrane modification is performed by introducing a functional group (carboxyl group) into this epoxy ring.
GMA introduction into the polyurethane foam film is performed by graft polymerization using plasma irradiation.

Next, a method for introducing a carboxyl group into the polyurethane foam film will be described.
A glycine solution is prepared by dissolving glycine in an aqueous sodium hydroxide solution. Next, the polyurethane foam film and the glycine solution are placed in a weighing bottle and immersed in a thermostatic bath at 80 ° C. for 24 hours to prepare a carboxyl group-introduced polyurethane foam film (hereinafter referred to as “PU-COOH film”).
After the reaction is completed, the PU-COOH membrane is taken out of the weighing bottle and washed with shaking with ultrapure water to obtain a hematopoietic stem cell separation and recovery membrane.
When storing the hematopoietic stem cell separation and recovery membrane, the membrane is stored refrigerated while immersed in ultrapure water.
In the hematopoietic stem cell separation and recovery membrane, a plurality of pores are formed in order to separate useful cells from the cell mixture. This hole has a hole diameter of 3 μm to 100 μm, and preferably has a hole diameter of 5 μm to 100 μm. This is a pore size selected based on the relationship between the size and adhesion of hematopoietic stem cells.
Moreover, after introducing a carboxyl group into a polyurethane foam film, a pretreatment for infiltrating a human albumin aqueous solution or a phosphate buffer into the PU-COOH film may be performed. In addition, regarding albumin, it is not restricted to human albumin, You may use albumin from which origins, such as bovine serum albumin, differ.
By this pretreatment, the recovery rate of hematopoietic stem cells is further increased.

Next, an example of a hematopoietic stem cell separation and recovery method using a PU-COOH membrane as a hematopoietic stem cell separation and recovery membrane will be described.
First, peripheral blood is collected.
Peripheral blood is collected from the elbow vein of a human upper arm using a plastic vacuum blood collection tube containing a blood anticoagulant (EDTA2Na), and after blood collection, the anticoagulant and blood are mixed slowly.

The end of the tube 11 is set in the pump 1, and the other end of the tube 11 is connected to the discharge port 31 of the pressing syringe 3 fixed to the handle 51 of the support 5 so that air does not enter the pressing syringe 3. .
The hematopoietic stem cell separation and recovery membrane is set in the microsyringe filter holder 4, and the sample introduction port 41 is connected to the discharge port 21 of the sample filling cylinder 2. A plastic centrifuge tube for collecting the filtrate is installed under the microsyringe filter holder 4.

Peripheral blood collected in advance and mixed with an anticoagulant is filled into the sample-filled syringe 2, and the discharge port 21 is connected to the sample inlet 41 of the microsyringe filter holder 4.
The positions of the grips 51, 52, 53 of the support 5 are adjusted, and the pressing syringe 3, the sample filling syringe 2, and the microfilter are arranged at positions where the pressing portion 33 of the pressing syringe 3 and the pressing portion 23 of the sample filling syringe 2 are in contact with each other. Each holder 4 is fixed.

First, a step of separating hematopoietic stem cells present in peripheral blood from the peripheral blood is performed.
When the installation of the hematopoietic stem cell separation and recovery device S is completed, the pump 1 is turned on to start filtering peripheral blood. When the pump 1 is operated, the pressure from the pump 1 is conducted to the piston 22 of the sample filling syringe 2 via the piston 32 of the pressing syringe 3, and the peripheral blood filled in the sample filling syringe 2 is pressed. Peripheral blood can be filtered well.
At this stage, CD34 ⁺ hematopoietic stem cells are captured by the filter.
However, the method is not limited to this method, and a method of allowing peripheral blood, umbilical cord blood, bone marrow fluid, and the like injected into a blood collection bag to permeate through the PU-COOH membrane by natural fall is also an efficient method.

Next, a step of collecting CD34 ⁺ hematopoietic stem cells captured by the filter is performed.
When the filtration of the peripheral blood filled in the sample-filled syringe 2 is completed, the pump 1 is turned off, and the microsyringe filter holder 4 is turned upside down and fixed with the handle 53.
Next, an aqueous human albumin solution is prepared as a cell recovery solution, filled into a new sample-filled syringe 2, and the pressure syringe 3 and the sample-filled syringe 2 are fixed at predetermined positions in the same manner as when filtering peripheral blood. If it is confirmed that each device is fixed, the pump 1 is operated and the human albumin aqueous solution is passed through the microsyringe filter holder 4.
Not only this method but also a method of allowing the human albumin aqueous solution injected into the blood collection bag to permeate through the PU-COOH membrane by natural fall is also an efficient method, as in the separation step.
Further, not only the method of inverting the membrane, but the recovery liquid may be introduced from the downstream direction of the membrane. For example, it is possible to connect the tube and the syringe filled with the recovery liquid in the downstream direction of the membrane and introduce the recovery liquid from the syringe to the downstream side of the membrane. The downstream side of the membrane refers to the surface opposite to the membrane surface on the side where peripheral blood is introduced in the separation step.
The concentration of the human albumin aqueous solution may be 0.05 to 3% by weight, preferably 0.5% by weight.
When an aqueous human albumin solution in this concentration range is used as a recovery solution, hematopoietic stem cells can be efficiently recovered as shown in the experimental results described later.
However, the cell recovery solution is not limited to a human albumin aqueous solution in which human albumin is dissolved in pure water, but physiological saline, various buffers, physiological saline containing other proteins of human albumin, various buffers Any liquid or the like that does not adversely affect the cells may be used.
In addition, regarding albumin, it is not restricted to human albumin, You may use albumin from which origins, such as bovine serum albumin, differ.
The solution collected from the microsyringe filter holder 4 is collected in a plastic centrifuge tube. In the solution collected at this time, CD34 ⁺ hematopoietic stem cells captured by the filter in the separation step are eluted.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
1. Preparation of hematopoietic stem cell separation and recovery membrane (1) Introduction of GMA into polyurethane foam membrane (Preparation of PU-GMA membrane)
The PU-GMA film is a film in which GMA (glycidyl methacrylate) is introduced into a polyurethane foam film by graft polymerization using plasma irradiation.
GMA has an epoxy ring in its structure, and membrane modification is performed by introducing a functional group (carboxyl group) into this epoxy ring.
An ampoule tube (10 mm (diameter) × 170 mm (long) manufactured by Tsukubarika Seiki) containing an appropriate amount of GMA (Methacrylic Acid Glycidyl Ester manufactured by Tokyo Chemical Industry Co., Ltd.) was filled with nitrogen gas and bubbled for 20 minutes.
Next, the ampule tube was frozen in liquid nitrogen with the vacuum line attached and the valve connecting the vacuum line and the ampule tube closed.
After confirming that the GMA was completely frozen, the valve was opened to release oxygen and the valve was closed. Thereafter, the GMA was melted in a water bath.
This series of freeze-thaw operations was repeated three times to completely degas oxygen in GMA.
Next, the polyurethane foam film is fixed to the SUS mesh, placed in a reaction container (110 mm (diameter) × 240 mm (long) made by Tsukubarika Seiki), attached to a vacuum line, until 10 ⁻¹ to 10 ⁻² Pa. The pressure was reduced. Next, Ar gas was introduced (about 3.8 to 5.7 × 10 ² Pa), and then vacuumed to 10 ⁻¹ Pa.
This series of vacuum operations was repeated three times, and was evacuated until finally reaching 0.65 Pa or less.
The pressure was maintained at 26.6 Pa in an Ar gas stream, and a high frequency power supply (AX-300 manufactured by Adtec Co., Ltd.) was connected to the reaction vessel to perform plasma irradiation (output 200 W, 30 seconds). After completion of the plasma irradiation, the introduction of Ar gas was stopped, and the vacuum line system was evacuated for 90 seconds (pressure≈6.5 × 10 ⁻¹ Pa).
Next, with the reaction system maintained in a vacuum state, the valve connecting the vacuum pump and the vacuum line was closed, the valve between the degassed GMA and the reaction vessel was opened, and graft polymerization was performed at room temperature for 5 minutes.
When the graft polymerization was completed, the valve connecting the vacuum pump and the vacuum line was opened, and the GMA monomer in the reaction vessel was removed in vacuo for 2 minutes.
Next, the membrane after graft polymerization was washed with shaking for 30 minutes (twice) with a mixed solution of water and methanol (mixed in the same volume), and then washed with ultrapure water for 30 minutes.
The GMA introduced into the GMA-introduced polyurethane foam membrane (PU-GMA membrane) by this operation was 0.61%.
The GMA introduction rate was determined by the following formula.
Plasma treatment, then drying (vacuum drying 80 ° C. 24 hours treatment) film weight (X g) after introduction of GMA is the drying (vacuum drying 80 ° C. 24 hours treatment) membrane weight (Y g) before plasma treatment. The divided value was defined as the GMA introduction rate.
GMA introduction rate (%) = (X / Y) × 100
Moreover, it is possible to change the GMA introduction rate by changing the plasma output and the irradiation time. However, there was no change in the recovery rate of CD34 ⁺ hematopoietic stem cells in the GMA introduction rate range of 0.3% to 0.7%.
(2) Preparation of carboxyl group-introduced polyurethane foam film A GMA-introduced polyurethane foam film (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm (pore diameter: 5 μm).
A 0.4 M glycine aqueous solution was prepared by dissolving 0.6 g of glycine (special grade Glycine manufactured by Wako Pure Chemical Industries, Ltd.) in an aqueous solution of sodium hydroxide (0.1 M sodium hydroxide solution manufactured by Wako Pure Chemical Industries, Ltd.).
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared glycine solution was added. Then, it was immersed in a thermostat at 80 ° C. for 24 hours to prepare a carboxyl group-introduced polyurethane foam film (PU-COOH film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
(3) Preparation of hematopoietic stem cell separation and recovery membrane used in Comparative Example a. Preparation of sulfonic acid group-introduced polyurethane foam membrane A GMA-introduced polyurethane foam membrane (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm in the same manner as the PU-COOH membrane.
Isopropyl alcohol (Wako Pure Chemical Industries, Ltd. Isopropyl Alcohol) and ultrapure water were prepared so that the ratio of ultrapure water: isopropyl alcohol was 75:15 (weight ratio), and this was used as an IPA aqueous solution.
Using this IPA aqueous solution as a solvent, sodium sulfite (Sodium Sulfite Anhydrous manufactured by Wako Pure Chemical Industries, Ltd.) was prepared to be 10% by weight.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared solution was added. Then, it was immersed in a thermostat bath at 80 ° C. for 6 hours to prepare a sulfonic acid group-introduced polyurethane foam membrane (PU-SO ₃ H membrane).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
b. Preparation of hydroxyl group-introduced polyurethane foam membrane A GMA-introduced polyurethane foam membrane (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm in the same manner as the PU-COOH membrane.
Concentrated sulfuric acid (Sulfuric Acid, manufactured by Wako Pure Chemical Industries, Ltd.) (0.98 g) was added to 19 ml of ultrapure water and diluted to obtain a 0.5 M aqueous solution.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared aqueous solution was added. Then, it was immersed in a thermostat bath at 80 ° C. for 2 hours to prepare a hydroxyl group-introduced polyurethane foam film (PU-OH film).
The membrane after the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and stored refrigerated in ultrapure water until use. .
c. Preparation of Amino Group-Introduced Polyurethane Foam Membrane Similar to the PU-COOH membrane, a GMA-introduced polyurethane foam membrane (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
1 ml of an aqueous ammonia solution (first grade Ammonia Solution, manufactured by Kokusan Chemical Co., Ltd.) was dissolved in 34.23 ml of an aqueous sodium hydroxide solution (0.1M Sodium hydroxide Solution, manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a 0.5 M aqueous ammonia solution.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared aqueous ammonia solution was added. Then, by immersing for six hours at 30 ° C. in a constant temperature bath, amino group-introduced polyurethane foam layer (PU-NH ₂ film) was prepared.
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
d. Preparation of Amine (—N (C ₂ H ₅ ) ₂ ) -Introduced Polyurethane Foam Membrane Similar to the PU-COOH membrane, the GMA-introduced polyurethane foam membrane (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
Diethylamine (Diethylamine manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ultrapure water to prepare a 50% by volume diethylamine aqueous solution.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared diethylamine aqueous solution was added. Then, it was immersed in a thermostat bath at 30 ° C. for 3 hours to prepare a —N (C ₂ H ₅ ) ₂ -introduced polyurethane foam film (PU-N (C ₂ H ₅ ) ₂ film).
The membrane after the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and stored refrigerated in ultrapure water until use. .
e. Preparation of Amine Derivative (—NHC ₂ H ₄ OH) Introduced Polyurethane Foam Membrane Similar to the PU-COOH membrane, the GMA-introduced polyurethane foam membrane (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of a 100% aminoethanol (2-Aminoethanol manufactured by Wako Pure Chemical Industries, Ltd.) solution was added. Then, it was immersed in a thermostat at 30 ° C. for 6 hours to prepare a —NHC ₂ H ₄ OH introduced polyurethane foam film (PU-NHC ₂ H ₄ OH film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .

2. Sample collection Using a 7ml plastic vacuum blood collection tube (Terumo Co., Ltd. Benogect 2 vacuum blood collection tube) filled with 10.5mg of blood anticoagulant (EDTA2Na), sample peripheral blood as a sample from the elbow vein of the human upper arm. did. After the collection, the anticoagulant and the collected blood were mixed.
Next, a plastic dropper is cut in half and attached to the discharge port 21 of a 10 ml sample-filled syringe 2 (SS-10ESZ manufactured by Terumo Corporation), and this dropper portion is inserted into a vacuum blood collection tube. The sample-filled syringe 2 was aspirated.

3. Hematopoietic stem cell separation operation The hematopoietic stem cell separation and recovery apparatus S shown in FIG. 1 was assembled.
Three hematopoietic stem cell separation and recovery membranes to be used were stacked and set in the microsyringe filter holder 4.
Remove the dropper from the discharge port 21 of the sample-filled syringe 2 filled with 6 ml of peripheral blood, connect the discharge port 21 to the sample introduction port 41 of the microsyringe filter holder 4 (MILLIPORE 25 mm), 53 and fixed in place.
The pressing syringe 3 was fixed by the handle 51 of the support 5 at a position where the pressing portion 33 of the 30 ml pressing syringe 3 (SS-30ESZ manufactured by Terumo Corporation) was in contact with the pressing portion 23 of the sample-filled syringe filled with peripheral blood.
The end of the Tygon tube 11 was set in a peristaltic pump 1 (MICRO TUBE POMP MP-3 manufactured by TOKYO AIKAKIKAI), and the other end of the Tygon tube 11 was connected to the discharge port 31 of the pressing syringe 3.
A 15 ml plastic centrifuge tube (Centrifuge Tubes 2325-015 manufactured by IWAKI) was set at the bottom of the microsyringe filter holder 4 in order to collect the filtrate.
After the assembly of the hematopoietic stem cell separation and recovery apparatus S was completed, the switch of the peristaltic pump 1 was turned on. The peristaltic pump 1 was adjusted so that the membrane permeation flow rate of peripheral blood as a sample was 1 ml / min (Flux: 3.1342 × 10 ⁻⁷ (m / sec)).
After confirming that filtration of 6 ml of peripheral blood as a sample was completed, the peristaltic pump 1 was turned off.

4). Hematopoietic stem cell recovery procedure Prepare a 0.5% by weight aqueous solution of human albumin (Wako Pure Chemical Industries, Ltd., Albumin from Human Serum) as a recovery liquid, and the same type of syringe as the sample-filled syringe 2 (hereinafter referred to as “recovered liquid-filled syringe”) 6 ml.
The sample-filled syringe 2 containing the peripheral blood was removed, and the microsyringe filter holder 4 was turned upside down. A recovery liquid-filled syringe was connected to the microsyringe filter holder 4, and the hematopoietic stem cell separation and recovery apparatus S was assembled in the same manner as in the hematopoietic stem cell separation operation.
A 15 ml plastic centrifuge tube (Centrifuge Tubes 2325-015 manufactured by IWAKI) was set at the bottom of the microsyringe filter holder 4 in order to collect the filtrate.
After the assembly of the hematopoietic stem cell separation and recovery apparatus S was completed, the switch of the peristaltic pump 1 was turned on. The peristaltic pump 1 was adjusted so that the membrane permeation flow rate of the recovered liquid was 1 ml / min (Flux: 3.1342 × 10 ⁻⁷ (m / sec)).
After confirming that filtration of 6 ml of the recovered liquid was completed, the switch of the peristaltic pump 1 was turned off.
In this way, a filtrate that was considered to contain CD34 ⁺ hematopoietic stem cells was collected in a 15 ml plastic centrifuge tube.

5). Analysis (1) Analysis of introduction of each functional group The membrane into which each functional group was introduced was dried for 24 hours, and subjected to electron spectroscopic analysis by XPS (ESCA-3400 manufactured by Shimadzu Corporation).
(2) Sample Preparation and Staining of Hematopoietic Stem Cell Surface Markers Analysis of CD34 ⁺ hematopoietic stem cells was performed using a commercially available Stem-Kit (manufactured by Beckmancoulter) according to its manual (International Blood Therapy and Transplantation Society ISHAGE Guidelines).
That is, prepare the same number of sample tubes (Beckmancoulter) as the samples, and add 20 μl of anti-CD34 antibody (CD45-FITC / CD34-PE from Beckmancoulter) to each sample tube using a micropipette (20 μl manufactured by Nichiyo Co., Ltd.). It was. 100 μl of each sample was added to each sample tube and stirred well. Then, 20 μl of a dye for removing dead cells (7-ADD Viability Dye manufactured by Beckmancoulter) was added, and incubated at room temperature in the dark for 15 minutes.
Then, 500 μl of hemolytic agent (OptiLyseC) was added to each sample tube, stirred well, and incubated at room temperature for 10 minutes.
Next, 9.6 g of PBS (-) powder (manufactured by Nissui Pharmaceutical Co., Ltd.) is dissolved in 1 l of ultrapure water to prepare a PBS buffer solution, and 500 μl of this PBS buffer solution is added to each sample tube and stirred well. And incubated for 10 minutes at room temperature.
Samples thus prepared were analyzed using flow cytometry (EPICS XL from Beckmancoulter).
By analyzing in advance the number of CD34 ⁺ hematopoietic stem cells contained in peripheral blood collected as a sample and analyzing the filtrate collected in the centrifuge tube by the hematopoietic stem cell separation operation and the collected solution collected in the centrifuge tube by the hematopoietic stem cell collection operation CD34 ⁺ hematopoietic stem cell permeability and recovery can be calculated. The transmittance and the recovery rate are obtained by the following calculation.
Transmittance (%) = (hematopoietic stem cell separation number of CD34 ⁺ hematopoietic stem cells contained in the peripheral blood collected as a CD34 ⁺ hematopoietic stem cells / sample in the filtrate collected into a centrifuge tube in operation) × 100
Recovery rate (%) = (CD34 ⁺ number of hematopoietic stem cells in collected liquid collected in centrifuge tube by hematopoietic stem cell collecting operation / CD34 ⁺ number of hematopoietic stem cells contained in peripheral blood collected as sample) × 100

未修飾のＰＵ膜と比較して、ＧＭＡ導入後の膜のＮの量が減少していることがわかる。ＧＭＡにはＮが入っていないため、この結果よりＧＭＡが導入されていることがわかる。
また、ＧＭＡ導入後の膜とＰＵ−ＳＯＨ_３膜を比較すると、ＰＵ−ＳＯＨ_３膜にＳが０．８３％入っていることから、スルホン酸基が導入されていることがわかる。
更に、ＧＭＡ導入後の膜とＰＵ−ＣＯＯＨ膜を比較すると、ＰＵ−ＣＯＯＨ膜のＮの量が増加した。これは、カルボキシル基を導入する際にグリシンを用いているためにＮが増加したと考えられる。よって、カルボキシル基が導入されていることがわかる。
また、ＰＵ−ＮＨ_２膜、ＰＵ−Ｎ（Ｃ_２Ｈ_５）_２膜、ＰＵ−ＮＨ_２Ｃ_２Ｈ_４ＯＨ膜のアミン系統の膜は、ＧＭＡ導入後の膜と比較して、Ｎが増加していることから、アミン系統の置換基が導入されていることがわかる。
なお、全ての膜にみられたＳｉは、ポリウレタン発泡体膜を発泡させる際に混入したものであると考えられる。
（２）ＣＤ３４^＋造血幹細胞の透過率及び回収率
結果を表２及び図２のグラフに示す。
なお、本明細書に添付するグラフは、ｎ＝４の独立した測定の平均値をプロットしたものであり、縦のバーは標準偏差を示す。

官能基未修飾のＰＵ膜、ＰＵ−ＳＯ_３Ｈ膜、ＰＵ−ＯＨ膜、ＰＵ−ＣＯＯＨ膜、ＰＵ−ＮＨ_２膜共に、造血幹細胞の透過は、ほとんどなかった。
このことより、試料の末梢血に含まれるＣＤ３４^＋造血幹細胞は、造血幹細胞分離操作において、造血幹細胞分離回収膜を透過することなく、全て膜に捕捉されていることがわかる。
このことより、官能基の修飾に関係なく、ポリウレタン発泡体膜には、ＣＤ３４^＋造血幹細胞を捕捉することができると考えられる。
次いで、ＣＤ３４^＋造血幹細胞の回収率を比較すると、ＰＵ−ＣＯＯＨ膜での回収率が約７５％と、一番高い回収率を示した。
その他、ＰＵ−ＳＯ_３Ｈ膜及びＰＵ−ＯＨ膜では、約２０％前後の回収率であり、ＰＵ−ＮＨ_２膜では、ＣＤ３４^＋造血幹細胞をほとんど回収できなかった。
また、ＰＵ−Ｎ（Ｃ_２Ｈ_５）_２膜及びＰＵ−ＮＨ_２Ｃ_２Ｈ_４ＯＨ膜においても、ＣＤ３４^＋造血幹細胞はほとんど回収できず、未修飾のポリウレタン発泡体膜においては、約５％程度の回収率しか示さなかった。
これらの結果より、カルボキシル基を導入したポリウレタン発泡体膜におけるＣＤ３４^＋造血幹細胞の回収率が最も高いことが示された。 6). Results (1) Surface analysis results of the film into which each functional group was introduced Table 1 shows the results of the surface analysis.
In addition, all the numerical values in the table | surface described in this specification are shown by the average value +/- standard deviation.
The population is n = 4 and is an independent measurement.

It can be seen that the amount of N in the membrane after introduction of GMA is reduced as compared with the unmodified PU membrane. Since GMA does not contain N, this result shows that GMA is introduced.
In addition, when the membrane after introduction of GMA and the PU-SOH ₃ membrane are compared, S is contained in the PU-SOH ₃ membrane, which indicates that sulfonic acid groups are introduced.
Furthermore, when the film after introduction of GMA and the PU-COOH film were compared, the amount of N in the PU-COOH film increased. This is thought to be due to the increase in N due to the use of glycine when introducing carboxyl groups. Therefore, it can be seen that a carboxyl group is introduced.
In addition, the amine-based films of PU-NH ₂ film, PU-N (C ₂ H ₅ ) ₂ film, and PU-NH ₂ C ₂ H ₄ OH film have increased N compared to the film after GMA introduction. From this, it can be seen that an amine-based substituent has been introduced.
In addition, it is thought that Si seen in all the films is mixed when foaming the polyurethane foam film.
(2) CD34 ⁺ hematopoietic stem cell permeability and recovery rate The results are shown in Table 2 and the graph of FIG.
In addition, the graph attached to this specification plots the average value of the independent measurement of n = 4, and a vertical bar shows a standard deviation.

The functional group-unmodified PU membrane, PU-SO ₃ H membrane, PU-OH membrane, PU-COOH membrane, and PU-NH ₂ membrane showed almost no permeation of hematopoietic stem cells.
This shows that all CD34 ⁺ hematopoietic stem cells contained in the peripheral blood of the sample are trapped in the membrane without permeating the hematopoietic stem cell separation and recovery membrane in the hematopoietic stem cell separation operation.
From this, it is considered that CD34 ⁺ hematopoietic stem cells can be captured by the polyurethane foam film regardless of the modification of the functional group.
Next, when the recovery rate of CD34 ⁺ hematopoietic stem cells was compared, the recovery rate with the PU-COOH membrane was about 75%, showing the highest recovery rate.
In addition, the recovery rate was about 20% in the PU-SO ₃ H membrane and the PU-OH membrane, and almost no CD34 ⁺ hematopoietic stem cells could be recovered in the PU-NH ₂ membrane.
In addition, almost no CD34 ⁺ hematopoietic stem cells can be recovered in the PU-N (C ₂ H ₅ ) ₂ membrane and the PU-NH ₂ C ₂ H ₄ OH membrane, and about 5% in the unmodified polyurethane foam membrane. Only a moderate recovery was shown.
From these results, it was shown that the recovery rate of CD34 ⁺ hematopoietic stem cells in the polyurethane foam membrane into which carboxyl groups were introduced was the highest.

According to Example 1, it was found that the recovery rate of CD34 ⁺ hematopoietic stem cells in a polyurethane foam membrane into which a carboxyl group was introduced using glycine was high, so using membranes whose surfaces were modified with amino acids having various structures, The separation and recovery of CD34 ⁺ hematopoietic stem cells was examined.
1. Preparation of hematopoietic stem cell separation and recovery membrane (1) Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared and stored in the same manner as in Example 1.
(2) Preparation of hematopoietic stem cell separation and recovery membrane used in Comparative Example a. Preparation of Aspartic Acid-Introduced Polyurethane Foam Film A GMA-introduced polyurethane foam film (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
Aspartic acid (L-Aspartic Acid, manufactured by Wako Pure Chemical Industries, Ltd.) 0.227 g was dissolved in an aqueous sodium hydroxide solution (0.1N Sodium hydroxide Solution, manufactured by Wako Pure Chemical Industries, Ltd.), and 0.1 M aspartic acid was dissolved. An aqueous solution was prepared.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared aspartic acid aqueous solution was added. Then, it was immersed in a thermostat at 80 ° C. for 24 hours to prepare an aspartic acid-introduced polyurethane foam film (PU-Asp acid film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
b. Preparation of lysine-introduced polyurethane foam film A GMA-introduced polyurethane foam film (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
1.169 g of L (+)-lysine (L (+)-Lysine manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an aqueous sodium hydroxide solution (0.1N Sodium hydroxide Solution manufactured by Wako Pure Chemical Industries, Ltd.). A 0.4 M lysine aqueous solution was prepared.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared lysine aqueous solution was added. Then, it was immersed in a thermostat at 80 ° C. for 24 hours to prepare a lysine-introduced polyurethane foam film (PU-Lys film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
c. Preparation of Arginine-Introduced Polyurethane Foam Film A GMA-introduced polyurethane foam film (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
0.5324 g of L (+)-arginine (L (+)-Arginine manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an aqueous sodium hydroxide solution (0.1N Sodium hydroxide Solution manufactured by Wako Pure Chemical Industries, Ltd.). A 0.4 M arginine aqueous solution was prepared.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared arginine aqueous solution was added. Then, it was immersed in a constant temperature bath at 80 ° C. for 24 hours to prepare an arginine-introduced polyurethane foam film (PU-Arg film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .
d. Preparation of Asparagine-Introduced Polyurethane Foam Film A GMA-introduced polyurethane foam film (PU-GMA) was cut out with a circular cutter having a diameter of 25 mm.
L-Asparagine (L-Asparagine, manufactured by Wako Pure Chemical Industries, Ltd.) (0.6005 g) was dissolved in a sodium hydroxide aqueous solution (0.1N Sodium hydroxide Solution, manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a 0.4M asparagine aqueous solution. Was prepared.
Three PU-GMA membranes cut out in a weighing bottle were placed, and 20 ml of the prepared asparagine aqueous solution was added. Then, it was immersed in a thermostat at 80 ° C. for 24 hours to prepare an asparagine-introduced polyurethane foam film (PU-Asp film).
The membrane after completion of the reaction was taken out from the weighing bottle, washed twice at 25 ° C. for 10 minutes while shaking in 10 ml of ultrapure water, and refrigerated and stored in ultrapure water until use. .

2. Sample collection, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, analysis Sample collection method, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, analysis method are the same as in Example 1.

グリシンを導入したＰＵ膜（ＰＵ−Ｇｌｙ膜）、アスパラギン酸を導入したＰＵ膜（ＰＵ−Ａｓｐ ａｃｉｄ膜）、リシンを導入したＰＵ膜（ＰＵ−Ｌｙｓ膜）、アルギニンを導入したＰＵ膜（ＰＵ−Ａｒｇ膜）、アスパラギンを導入したＰＵ膜（ＰＵ−Ａｓｐ）共に、造血幹細胞の透過は、ほとんどなかった。
このことより、試料の末梢血に含まれるＣＤ３４＋造血幹細胞は、造血幹細胞分離操作において、造血幹細胞分離回収膜を透過することなく、ほとんど全てが膜に捕捉されていることがわかる。
このことより、官能基の修飾に関係なく、ポリウレタン発泡体膜には、ＣＤ３４^＋造血幹細胞を捕捉することができると考えられる。
次いで、ＣＤ３４^＋造血幹細胞の回収率を比較すると、ＰＵ−Ｇｌｙ膜での回収率が約７０％と、一番高い回収率を示した。
その他、ＰＵ−Ａｓｐ ａｃｉｄ膜及びＰＵ−Ａｓｐ膜のＣＤ３４^＋造血幹細胞回収率は、約３０％程度であり、ＰＵ−Ｌｙｓ膜及びＰＵ−Ａｒｇ膜のＣＤ３４^＋造血幹細胞の回収率は、約２０％前後であった。
これらの結果より、グリシンを用いてカルボキシル基を導入した膜のＣＤ３４^＋造血幹細胞回収率が最も高いことが示された。 3. Results The results are shown in Table 3.

PU membrane introduced with glycine (PU-Gly membrane), PU membrane introduced with aspartic acid (PU-Asp acid membrane), PU membrane introduced with lysine (PU-Lys membrane), PU membrane introduced with arginine (PU- There was almost no permeation of hematopoietic stem cells in both the Arg membrane) and the PU membrane into which asparagine was introduced (PU-Asp).
This shows that almost all CD34 + hematopoietic stem cells contained in the peripheral blood of the sample are trapped in the membrane without permeating the hematopoietic stem cell separation and recovery membrane in the hematopoietic stem cell separation operation.
From this, it is considered that CD34 ⁺ hematopoietic stem cells can be captured by the polyurethane foam film regardless of the modification of the functional group.
Subsequently, when the recovery rate of CD34 ⁺ hematopoietic stem cells was compared, the recovery rate with the PU-Gly membrane was about 70%, which was the highest recovery rate.
Other, CD34 ⁺ hematopoietic stem cells recovery of PU-Asp acid film and PU-Asp film is about 30% recovery of CD34 ⁺ hematopoietic stem cells PU-Lys film and PU-Arg film is about 20% Before and after.
From these results, it was shown that the CD34 ⁺ hematopoietic stem cell recovery rate of the membrane into which the carboxyl group was introduced using glycine was the highest.

Next, separation and collection of CD34 ⁺ hematopoietic stem cells by the number of stacked hematopoietic stem cell separation and recovery membranes were examined.
1. Preparation of hematopoietic stem cell separation and recovery membrane Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared and stored in the same manner as in Example 1.

2. Sample collection, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, analysis method The sample collection method and analysis method are the same as in Example 1.
Regarding the hematopoietic stem cell separation operation and the hematopoietic stem cell collection operation, as a comparative example, one PU-COOH membrane was set in the microsyringe filter holder 4. Other operations are the same as those in the first embodiment.

ＰＵ−ＣＯＯＨ膜を３枚積層して使用した場合には、ＣＤ３４^＋造血幹細胞の透過はなく、ＰＵ−ＣＯＯＨ膜にほとんど全てのＣＤ３４^＋造血幹細胞が捕捉されていると考えられる。また、回収率も約７０％と高い回収率を示した。
一方、ＰＵ−ＣＯＯＨ膜を１枚使用した場合には、約４５％のＣＤ３４^＋造血幹細胞がＰＵ−ＣＯＯＨ膜を通過し、回収率も約４５％程度にとどまった。
このことより、ＰＵ−ＣＯＯＨ膜は複数毎積層して使用することが望ましいと考えられる。 3. Results The results are shown in Table 4.

When three PU-COOH membranes are stacked and used, CD34 ⁺ hematopoietic stem cells do not permeate, and it is considered that almost all CD34 ⁺ hematopoietic stem cells are captured by the PU-COOH membrane. Also, the recovery rate was as high as about 70%.
On the other hand, when one PU-COOH membrane was used, about 45% of CD34 ⁺ hematopoietic stem cells passed through the PU-COOH membrane, and the recovery rate was only about 45%.
From this, it is considered that it is desirable to use a plurality of PU-COOH films laminated.

Next, the recovery rate of CD34 ⁺ hematopoietic stem cells according to the concentration of the recovery solution was examined.
1. Preparation of hematopoietic stem cell separation and recovery membrane Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared and stored in the same manner as in Example 1.

2. Sample collection, hematopoietic stem cell separation operation, hematopoietic stem cell recovery operation, analysis method The sample collection method, hematopoietic stem cell separation operation, and analysis method are the same as in Example 1.
Regarding the hematopoietic stem cell recovery operation, as an example, a 0.5% by weight human albumin aqueous solution was used as a recovery solution, and as a comparative example, PBS (phosphate buffer), 0.05% by weight human albumin aqueous solution, 3% by weight The recovery operation was performed using a% human albumin aqueous solution as the recovery solution.

０．０５重量％ヒトアルブミン水溶液及び３重量％ヒトアルブミン水溶液では、ＣＤ３４^＋造血幹細胞の回収率は、約６０％であった。
一方、０．５重量％ヒトアルブミン水溶液では、ＣＤ３４^＋造血幹細胞回収率は、約７０％と高い回収率を示した。
このことより、回収液としては、０．５重量％のヒトアルブミン水溶液を使用することが望ましいと考えられる。 3. Results The results are shown in Table 5.

In the 0.05 wt% human albumin aqueous solution and the 3 wt% human albumin aqueous solution, the recovery rate of CD34 ⁺ hematopoietic stem cells was about 60%.
On the other hand, in the 0.5 wt% human albumin aqueous solution, the recovery rate of CD34 ⁺ hematopoietic stem cells was as high as about 70%.
From this, it is considered desirable to use a 0.5% by weight aqueous human albumin solution as the recovered liquid.

Next, the recovery rate of CD34 ⁺ hematopoietic stem cells by fractionation of the recovered solution was examined.
1. Preparation of hematopoietic stem cell separation and recovery membrane Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared and stored in the same manner as in Example 1.

2. Sample collection, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, analysis method The sample collection method and analysis method are the same as in Example 1.
The hematopoietic stem cell separation operation and the hematopoietic stem cell collection operation are the same as in Example 1 except that the sample is filtered every 2 ml and the filtrate is collected every 2 ml of filtration.

どの濾液分画においても、ＣＤ３４^＋造血幹細胞の透過は認められなかった。
回収率に関しては、０〜２ｍｌ分画では約５０％の回収率を示し、２〜４ｍｌ分画では約２０％の回収率を示した。一方、４〜６ｍｌ分画では、回収率は０％であった。
このことから、ＰＵ−ＣＯＯＨ膜に捕捉されたＣＤ３４^＋造血幹細胞は、初めの４ｍｌでほぼ全て回収できると考えられる。 3. Results The results are shown in Table 6.

No permeation of CD34 ⁺ hematopoietic stem cells was observed in any filtrate fraction.
Regarding the recovery rate, the 0-2 ml fraction showed about 50% recovery, and the 2-4 ml fraction showed about 20% recovery. On the other hand, in the 4-6 ml fraction, the recovery rate was 0%.
From this, it is considered that almost all of the CD34 ⁺ hematopoietic stem cells captured by the PU-COOH membrane can be recovered in the first 4 ml.

Next, the separation and recovery of CD34 ⁺ hematopoietic stem cells by pretreatment of the membrane was examined.
1. Preparation of hematopoietic stem cell separation / recovery membrane Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared in the same manner as in Example 1.
The PU-COOH membrane is infiltrated with water, a 0.5 wt% human albumin aqueous solution (hereinafter referred to as “HSA aqueous solution”), and a phosphate buffer (hereinafter referred to as “PBS”, pH 7.2) before use. A pretreatment was performed.

2. Sample collection, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, analysis method The sample collection method, hematopoietic stem cell separation operation, hematopoietic stem cell collection operation, and analysis method are the same as in Example 1.

ＰＢＳで前処理したＰＵ−ＣＯＯＨ膜では、約２０％程度のＣＤ３４^＋造血幹細胞が分離操作で、透過していた。しかし、水で前処理したＰＵ−ＣＯＯＨ膜では、ＣＤ３４^＋造血幹細胞の透過は認められず、ＰＢＳで前処理したＰＵ−ＣＯＯＨ膜では約１０％程度のＣＤ３４^＋造血幹細胞が透過したのみであった。
回収率に関しては、水で前処理したＰＵ−ＣＯＯＨ膜では約７０％、０．５重量％ＨＳＡ水溶液で前処理したＰＵ−ＣＯＯＨ膜では約４０％、ＰＢＳ水溶液で前処理したＰＵ−ＣＯＯＨ膜では約８０％であった。
このことから、ＰＢＳで前処理したＰＵ−ＣＯＯＨ膜を使用することが望ましいと考えられる。 3. Results The results are shown in Table 7.

In the PU-COOH membrane pretreated with PBS, about 20% of CD34 ⁺ hematopoietic stem cells permeated through the separation operation. However, permeation of CD34 ⁺ hematopoietic stem cells was not observed in the PU-COOH membrane pretreated with water, and only about 10% of CD34 ⁺ hematopoietic stem cells permeated through the PU-COOH membrane pretreated with PBS. .
Regarding the recovery rate, the PU-COOH membrane pretreated with water is about 70%, the PU-COOH membrane pretreated with the 0.5 wt% HSA aqueous solution is about 40%, and the PU-COOH membrane pretreated with the PBS aqueous solution is about 70%. About 80%.
From this, it is considered desirable to use a PU-COOH membrane pretreated with PBS.

Next, the permeability and recovery rate of each cell in the blood in the PU-COOH membrane was examined.
The cells as the contrast were red blood cells, platelets, T cells, B cells, and mononuclear cells.
1. Preparation of hematopoietic stem cell separation and recovery membrane Preparation of glycine-introduced polyurethane foam membrane A PU-COOH membrane (PU-Gly) was prepared and stored in the same manner as in Example 1.

2. Sample collection, separation operation of each cell, collection operation of each cell The sample collection method, the separation operation of each cell, and the collection operation of each cell are the same as in Example 1.

3. Analysis method (1) Analysis of CD34 ⁺ hematopoietic stem cells
The method for analyzing the number of CD34 ⁺ hematopoietic stem cells is the same as in Example 1.
(2) Analysis of erythrocytes and platelets For erythrocyte count, glycophorin A was analyzed as a cell surface marker, and for platelet count, CD41 was analyzed as a cell surface marker.
Peripheral blood, filtrate, and recovered solution were diluted 10-fold.
The same number of sample tubes (Beckmancoulter) as the samples were prepared, and 20 μl of anti-glycophorin A antibody and 20 μl of anti-CD41 antibody were added to each sample tube.
100 μl of the diluted sample was added to each sample tube, stirred, and incubated for 20 minutes in the dark at room temperature. The sample was again diluted 100 times.
Next, 100 μl of Flow-Count Beads Solution was added to 500 μl of the diluted sample to prepare a sample.
Each sample thus prepared was analyzed using flow cytometry (EPICS XL manufactured by Beckmancoulter).
(3) Analysis of T cells and B cells For the number of T cells, CD3 was analyzed as a cell surface marker, and for the number of B cells, CD19 was analyzed as a cell surface marker.
Peripheral blood, filtrate, and recovered solution are used as samples without dilution.
The same number of sample tubes (Beckmancoulter) as the samples were prepared, and 10 μl of anti-CD3 antibody and 10 μl of anti-CD41 antibody were added to each sample tube.
100 μl of each sample was added to each sample tube, stirred, and incubated for 15 minutes in the dark at room temperature.
Subsequently, 500 μl of phosphate buffered saline (pH 7.4) was added, stirred, and incubated again in the dark at room temperature for 15 minutes.
Each sample thus prepared was analyzed using flow cytometry (EPICS XL manufactured by Beckmancoulter).
(4) Analysis of mononuclear spheres The number of mononuclear spheres was estimated from the size and shape of cells by forward and side scatter analysis of laser light (argon laser 488 nm).
(5) Calculation of permeability and recovery rate The permeability and recovery rate of each cell are calculated by analyzing the number of cells in the collected peripheral blood, the number of cells in the filtrate, and the number of cells in the recovery solution. Is possible. The transmittance and the recovery rate are obtained by the following calculation.
Permeability (%) = (number of cells in filtrate collected in centrifuge tube by separation operation / number of cells contained in collected peripheral blood) × 100
Recovery rate (%) = (number of cells in the collected solution collected in the centrifuge tube by the collection operation / number of cells contained in the collected peripheral blood) × 100

透過率については、赤血球、血小板、Ｔ細胞、Ｂ細胞、単核球ともに、１０〜２０％程度の細胞がＰＵ−ＣＯＯＨ膜を透過していることがわかる。しかし、ＣＤ３４^＋造血幹細胞は、ＰＵ−ＣＯＯＨ膜を通過せず、ＰＵ−ＣＯＯＨ膜に完全に捕捉される。
また、回収率に関しては、ＣＤ３４^＋造血幹細胞は約７０％もの高収率で回収されており、他の細胞の回収率は２０％以下でしかない。この結果より、本発明にかかるＰＵ−ＣＯＯＨ膜では、末梢血に前処理を施すことなく、ＣＤ３４^＋造血幹細胞を選択的に高収率で回収することができることがわかる。 3. Results The results are shown in Table 8 and the graph of FIG.

About the transmittance | permeability, it turns out that about 10-20% of cells permeate | transmit the PU-COOH film | membrane with respect to erythrocytes, platelets, T cells, B cells, and mononuclear cells. However, CD34 ⁺ hematopoietic stem cells do not pass through the PU-COOH membrane and are completely trapped in the PU-COOH membrane.
As for the recovery rate, CD34 ⁺ hematopoietic stem cells are recovered with a high yield of about 70%, and the recovery rate of other cells is only 20% or less. From this result, it can be seen that with the PU-COOH membrane according to the present invention, CD34 ⁺ hematopoietic stem cells can be selectively recovered with high yield without pretreatment of peripheral blood.

It is a figure which shows the hematopoietic stem cell isolation | separation collection apparatus S which concerns on a present Example. It is a graph which shows the permeability | transmittance and collection | recovery rate of CD34 ⁺ hematopoietic stem cell based on Example 1. FIG. It is a graph which shows the transmittance | permeability and collection | recovery rate of each cell which concern on Example 7. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Pump, 2 Sample filling syringe, 3 Pressing syringe, 4 Micro syringe filter holder, 5 Support stand, 11 Tube, 21, 31 Discharge port, 22, 32 Piston, 23, 33 Press part, 41 Sample introduction port, S Hematopoietic stem cell Separation and recovery device

Claims (9)

A cell separation and recovery membrane used for separating and collecting useful cells from a cell mixture containing useful cells and removed cells,
The cell separation / recovery membrane is characterized in that a carboxyl group is introduced into a polyurethane foam. The cell separation / recovery membrane according to claim 1, wherein CD34 antigen-positive hematopoietic stem cells are separated and collected as the useful cells. The cell separation / recovery membrane according to claim 1, wherein a compound having an epoxy ring is introduced into the cell separation / recovery membrane, and the carboxyl group is introduced via the epoxy ring. 4. The cell separation / recovery membrane is provided with a plurality of holes through which the removed cells pass, and the diameter of the holes is 3 μm or more and 100 μm or less. The cell separation and recovery membrane according to one. A method for separating and recovering useful cells from a cell mixture containing useful cells and removed cells,
A useful cell capturing step of passing the cell mixture through a polyurethane foam film having a carboxyl group introduced;
And a useful cell recovery step of recovering the useful cells by allowing a recovery solution to pass through the carboxyl group-introduced polyurethane foam membrane. 6. The cell separation and recovery method according to claim 5, wherein in the useful cell recovery step, CD34 antigen-positive hematopoietic stem cells are recovered as the useful cells. 6. The useful cell recovery step uses at least one of a physiological saline, a buffer, a physiological saline containing a protein, and a buffer containing a protein as the collection liquid. The cell separation and recovery method according to 1. 6. The cell separation and recovery method according to claim 5, wherein in the useful cell recovery step, an albumin aqueous solution having a concentration of 0.05% by weight to 3% by weight is used as the recovery solution. The cell separation and recovery method according to claim 6, further comprising a pretreatment step of infiltrating the carboxyl group-introduced polyurethane foam membrane with a phosphate buffer solution or an albumin aqueous solution before performing the useful cell capturing step. .

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