JPH08108069A - Separation of stem cell - Google Patents

Abstract

PURPOSE: To efficiently and accurately separate desired cells by using a stem cell separating material formed by immobilizing antibodies to antigens on the membrane surfaces of stem cells on a carrier formed by introducing carboxyl groups and hydrophilic chains into a nonwoven fabric consisting of fibers at the time of separating the stem cells. CONSTITUTION: The stem cell separating material formed by immobilizing the antibodies to the antigens on the membrane surfaces of the stem cells on the carrier formed by introducing the carboxyl groups and hydrophilic chains into the nonwoven fabric consisting of the fibers at the time of separating the stem cells is used. A stem sell separating column formed by immobilizing the anti-CD34 antibodies to the nonwoven fabric introduced with the carboxyl groups and hydrophilic chains, then packing these nonwoven fabrics into the column in such a manner that the fiber packing density attains 0.03 to 0.80g/cm<3> is used. Further, the CD34 positive cells are adsorbed from the myelofluid, etc., and thereafter, the unnecessary cells are removed by washing and only the necessary cells are desorbed by vibration. The stem cell separating column is disposed with caps 3, 3' and filters 4, 4' respectively above and below a main body 1 delineating a space 2 for packing the separating material therein and is disposed with an inflow port 5 and outflow port 6 for the treating liquid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for selectively separating stem cells from bone marrow fluid or peripheral blood.
More specifically, the present invention relates to a method for separating stem cells, which comprises using a stem cell separating material in which an antibody against an antigen on the membrane surface of stem cells is immobilized on a nonwoven fabric type carrier into which a carboxyl group and a hydrophilic chain have been introduced.

High-dose chemotherapy for hematopoietic malignant tumors and various solid cancers also deprives the hematopoietic function. In recent years, a therapy for autologous transplantation of peripheral blood stem cells to recover the hematopoietic function has been vigorously studied. Technology is making great progress.
For peripheral blood stem cell autologous transplantation, it is important to efficiently collect only 0.01% of the nucleated cells in the peripheral blood. By using the method for separating stem cells of the present invention, it becomes possible to efficiently separate and concentrate stem cells, and by returning this to the patient, it is possible to recover the reduced hematopoietic function. Therefore, high-dose chemotherapy and radiotherapy are possible, and the survival rate of cancer patients can be improved.

[0003]

2. Description of the Related Art Stem cell separation methods currently used include (1) centrifugation (2) method of killing cells other than desired cells (3) fluorescent antibody-labeled cell separation method (hereinafter abbreviated as FACS) (4) Separation method using magnetic beads (5) Immunoadsorption method, etc.

[0004]

The centrifugal separation method (1) is a method of separating cells by the difference in cell size and specific gravity, and when there is a large difference in physical properties such as white blood cells, red blood cells, and platelets. However, when the difference in specific gravity between stem cells and other nucleated cells is small, it is very difficult to efficiently separate only stem cells.

Various methods have been studied and developed for the method of killing cells other than the target cells mentioned in (2), but it is actually used in the autologous bone marrow prior to transplantation. Limited to the extent that drugs or monoclonal antibodies are used to kill the cancer cells. Although 4-hydroperoxycyclophosphamide is generally used as the drug in this case, it has been revealed that even normal bone marrow cells are damaged by this drug. Antibodies that react with cancer cells and T cells have been used to kill cells other than the desired cells, but in this case, these antibodies alone cannot kill the cells, so other drugs ( It must be used in combination with complement and toxin. However, because the reactivity of the antibody with the cancer cells is likely to change, it is often impossible to completely kill the cancer cells. Furthermore, the complement and toxin that must be used together with the antibody also have a side effect on normal cells, and thus damage the target cells.

The FACS (3) is a method for collecting target cells by the following method. First, the cell mixture is incubated with a fluorescently labeled monoclonal antibody that recognizes the membrane antigen of the target cell, and then irradiated with laser light.
Fluorescent antibody-bound cells are separated by utilizing the fact that only cells to which antibodies are bound emit fluorescence upon irradiation with laser light. Although this FACS method is an effective method for separating small numbers of cells for laboratory level studies,
Since the treatment of cells at a rate of 0,000 cells / hour is the limit, it is not suitable in time to separate a large amount of cells required for treatment. For example, using a FACS machine, 1000 nucleated cells
It takes several hours to process 15 to 20 ml of blood containing 10 to 20 million cells, and several weeks to separate 1 to 2 billion stem cells generally required for transplantation. Requires. Also, FACS devices have the drawbacks of being very expensive, requiring highly skilled techniques to use the devices, and expensive to maintain.

In the magnetic bead method (4), the cell mixture is first incubated with the antibody-bound magnetic beads to bind the target cells to the magnetic beads. Then, the cells bound to the magnetic beads are separated using a magnetic device. This technique has been applied to clinically remove cancer cells from the bone marrow fluid of patients. However, in order to efficiently bind the magnetic beads to the target cells, long-time incubation is required, which causes non-specific adsorption of cells other than the target cells, resulting in a decrease in the purity of the target cells. In addition, it is very difficult to collect the target cells adsorbed on the small magnetic beads.

The immunoadsorption method (5) can be further classified into several methods. PCT release N
o. The method disclosed in WO87 / 04628 is a so-called panning method, and this patent describes two types of stem cell separation methods. The first method is a method in which a monoclonal antibody against an antigen on the surface of a membrane of a stem cell is directly immobilized on the surface of a separation device, and cell separation is carried out by comparing the monoclonal antibody immobilized on a carrier or a device with antigen-positive cells. It is done by direct binding. The second method is a cell separation apparatus in which a cell mixture is first incubated with a monoclonal antibody that binds to a membrane antigen of stem cells, and then a ligand such as an anti-immunoglobulin antibody that binds to this antibody is immobilized. It is a method of processing. In these panning methods, cell separation is performed on a plastic dish on which antibodies are immobilized. This method is performed in the following procedure. First, the cell mixture,
The antibody is poured onto an immobilized plastic dish and incubated to bind the antibody to the antigen of the target cell membrane. After incubation, the plastic dishes are washed to remove unbound cells and separation is performed. Thus, the operation of the panning method is very simple, but it has some fatal drawbacks. Due to the weak binding of antibody to antigen, it is necessary to incubate the cells on a plastic dish for an extended period of time to effect effective binding of cells to the antibody. Therefore, non-target cells are non-specifically adsorbed,
The purity of the target cells decreases. Moreover, the weak binding of antibody to cells necessitates the immobilization of large amounts of antibody on plastic dishes, resulting in a very expensive device. Moreover, in order to prevent non-specific adsorption of red blood cells, this method must be used after removing the red blood cells from the cell mixture.

A method using an immunoadsorption column is also generally known. In this method, a ligand such as an antibody against the membrane antigen of the target cell is immobilized on the bead surface, and this is packed in a column for cell separation. Also in this case, since the binding force between the cell membrane antigen and the antibody is weak as in the panning method or the magnetic bead method, incubation is required to bind the cells to the antibody on the bead surface. Therefore, nonspecific adsorption of cells other than the intended cells occurs, and the purity of the obtained objective cells decreases. Furthermore, since the antigen-antibody binding strength is weak, it is necessary to immobilize a large amount of antibody, which makes the column very expensive, and therefore it is applicable to the therapeutic field requiring a large amount of cell separation. Have difficulty.

PCT Publication No. WO91 / 16116 discloses a method utilizing the interaction between avidin and biotin. The technique of this patent is as follows. First, a biotinylated antibody (antibody against stem cell membrane antigen) is added to the cell mixture and incubated to form a cell-antibody-biotin complex. This mixed solution is passed through a column in which avidin-immobilized beads are packed in a porous acrylamide gel, and the strong binding force of biotin-avidin is used to adsorb and separate target cells into the column. This method utilizes the strength of the binding force of biotin-avidin. Compared with the above-mentioned various methods, it is improved in processing speed and purity, and it has reached a level where clinical application is possible. I can say. However, there are some drawbacks to this method as well. Biotin-
Since avidin has a very strong binding force, once all of the avidin on the beads has bound biotin after being used for cell separation, it is difficult to break this binding and regenerate the column. That is, it is not possible to adopt a switching method in which the columns are arranged in parallel, and when one column is saturated, the other column is switched to, while the saturated column is regenerated, the column capacity is increased and the priming capacity is increased. Will increase. Furthermore, since avidin is directly bound to the polyacrylamide beads, avidin does not act effectively, and the cell-antibody-biotin adsorption rate and adsorption efficiency are reduced.

The present invention solves the above-mentioned drawbacks of the prior art, enables the target cells to be separated efficiently and at high speed, and the obtained target cells have a high purity, which may lead to a decline in the function of the separated cells. The present invention provides a method for separating cells that does not exist.

[0012]

Means for Solving the Problems The method for separating stem cells of the present invention is a method in which an antibody against an antigen on the membrane surface of a stem cell is added to a carrier constituted by introducing a carboxyl group and a hydrophilic chain using a non-woven fabric as a support. It is characterized by using an immobilized stem cell separating material.

The method for separating stem cells of the present invention is characterized in that the antibody against the antigen on the membrane surface of the stem cells is an anti-CD34 antibody. In the method for separating stem cells of the present invention, a fiber packing density is 0.03 to 0.80 g / after immobilizing an anti-CD34 antibody on a nonwoven fabric into which a carboxyl group and a hydrophilic chain have been introduced.
It is characterized by using a stem cell separation column packed so as to have a cm ³ . The stem cell separation method of the present invention uses a stem cell separation column to extract CD34 from bone marrow fluid or peripheral blood.
After adsorbing the positive cells, unnecessary cells are removed by washing, and the adsorbed CD34-positive cells are desorbed by shaking. The method for separating stem cells of the present invention,
C from bone marrow fluid or peripheral blood using a stem cell separation column
After adsorbing the D34-positive cells, unnecessary cells are removed by washing, and the adsorbed CD34-positive cells are released by stopped flow. In the present invention, "introduction" of "introduction of a carboxyl group and a hydrophilic chain into the nonwoven fabric" is not limited to the meaning of being bonded to the nonwoven fabric through a chemical bond, and the carboxyl group and the hydrophilic chain are easily introduced. If it is not desorbed, it can be used. The fibers forming the non-woven fabric are used as the support, and the carboxyl groups and hydrophilic chain components are coated in a sheath shape around the support. Even if the support and this coating layer are not chemically bonded, the coating layer can be easily formed. Unless desorbed, they are called “introduced” and “introduced”. Here, "not easily desorbed" means that the eluate test described below is carried out, and as a result, the amount of eluate is small and the required standard is satisfied.

Method of Performing Eluate Test and Required Standards 1.0 g of a sample is accurately weighed, cut into a size of about 2 cm × 2 cm, placed in a container, and 100 ml of distilled water is added. After heating at 70 ° C. for 1 hour and cooling, the sample is removed and the remaining liquid is used as a test liquid. In addition, distilled water that was similarly heated without adding a sample is used as a blank. The eluate test examines the following 5 items. Appearance Applies as standard when almost no color is visible and no foreign matter is visible. pH 1.0 g of potassium chloride is dissolved in distilled water to make 1 l (hereinafter abbreviated as KCl solution). Take 20 ml of the test solution, add 1.0 ml of KCl solution, and measure the pH with a pH meter (F-12 manufactured by Horiba Ltd.). Similarly, measure the pH of the blank and record the difference from the test solution. When this value is 1.5 or less, the reference is passed. UV absorption spectrum 200 nm-400 n of the test solution using the blank as a control
Absorbance of m is spectrophotometer (Hitachi U-3210)
The maximum absorbance from 220 nm to 350 nm is recorded. When this value is 0.1 or less, the reference is passed. Potassium permanganate reducing substance 10.0 ml of the test solution is placed in a ground-in stopper Erlenmeyer flask, and then 0.0
Add 20.0 ml of 1N aqueous potassium permanganate solution and 1.0 ml of dilute sulfuric acid and boil for 3 minutes. After cooling, 0.10 g of potassium iodide was added thereto, the container was tightly capped, shaken and left for 10 minutes, and then titrated with an aqueous 0.01 N sodium thiosulfate solution (indicator: starch solution). The blank is also titrated in the same manner, and the difference in the sodium thiosulfate solution required for the titration is recorded. The amount of sodium thiosulfate required for titration corresponds to the amount of potassium permanganate remaining.Therefore, when a large amount of potassium permanganate reducing substance is contained in the test solution, sodium thiosulfate required for titration is used. Will be less. Therefore, when the titration is completed with an amount of sodium thiosulfate that is aml less than the blank, the difference in potassium permanganate consumption is recorded as + aml. When the difference in the consumption amount of potassium permanganate is 1.0 ml or less, the standard passage is performed. Evaporation residue 20 ml of the test solution is placed in a glass container, evaporated to dryness by heating, and the residue is dried at 105 ° C. Record the weight of this residue. When this value is 1.0 mg or less, the reference is passed.

In the present invention, the hydrophilic chain includes linear hydrophilic substances such as polyethylene glycol chain, polypropylene glycol chain, polyacrylamide and the like, as well as cross-linked substances. Furthermore, the use of hydrophilic monomers such as acrylamide monomers and 2-hydroxyethylmethacrylate monomers is also within the scope of the present invention.

CD34, Thy1 and the like are known as antigens expressed in stem cells and progenitor cells. In the present invention, it is preferable to use antibodies against these antigens as ligands, but it is particularly preferable to use anti-CD34 antibody. In the present invention, the stem cells include all cells in which CD34 is expressed as a cell membrane surface antigen, including undifferentiated stem cells and cells which have been differentiated to some extent.

A method for producing a monoclonal antibody generally uses a fused cell (hybridoma) of mouse lymphocyte and mouse myeloma cell.
Rat-mouse, human-human hybridoma may be used, and any method may be used, such as a method of incorporating a gene for producing an antibody into a human type monoclonal producing cell, Escherichia coli, yeast or the like using genetic manipulation, or the like. May be. In addition, antibody production technology that will be developed in the future may be used.

In the present invention, the material of the non-woven fabric introducing the carboxyl group and the hydrophilic chain is polyester,
Polyamide, cellulose, rayon, cellulose acetate, cellulose triacetate, polyacrylonitrile, polyethylene, polypropylene, polystyrene, polyvinyl alcohol, or copolymers thereof, which can form a fiber form, are synthetic, semi-synthetic, natural, or regenerated polymers. Although usable, polyester, polyamide, cellulose, polyacrylonitrile, polyethylene, polypropylene and polystyrene are preferable, and among them, polyester, cellulose and polystyrene are particularly preferable in terms of easiness of modification and stability.

The fiber diameter of the non-woven fabric is 5 to 150 μm, preferably 8 to 100 μm, more preferably 12 to 50 μm.
μm. If the fiber diameter is smaller than this range, nonspecific adsorption of cells other than the target cells, especially leukocytes, is increased, which is not preferable. On the other hand, when the amount is larger than the above range, the surface area per weight is reduced, which leads to a decrease in the amount of bound antibody and, in turn, a decrease in the stem cell adsorption capacity, which is not preferable.
The basis weight of the non-woven fabric is 30 to 400 g / m ² , preferably 4
0-300 g / m ² , more preferably 50-250 g
/ M ² . When the fiber density is lower than this range, the surface area of the fibers packed in the column becomes small, and a sufficient amount of antibody cannot be immobilized. On the other hand, if it is larger than the above range, the fiber gap becomes small, resulting in a large pressure loss when passing the cell suspension, and inconveniences such as nonspecific capture of cells due to the filtration effect. .

The introduction of the carboxyl group and the hydrophilic chain into the nonwoven fabric can be carried out, for example, by the following method when cellulose is selected as the material. (1) reacting monochloroacetic acid in the presence of an alkali,
Method of introducing —CH ₂ COOH (2) Glucose ring is reacted with carboxyl group-containing vinyl compound (acrylic acid, methacrylic acid, etc.) and hydrophilic vinyl compound (acrylamide, hydroxyethyl methacrylate, etc.) in the presence of cerium ion. However, in general, there are many reactions under severe conditions using various toxic reagents, and there are problems such as residual toxic substances and fiber deterioration. is there.

As a reforming method which solves these problems, a method using electron beam (hereinafter abbreviated as EB) irradiation is most suitable. In this method, specifically, the compound of Chemical formula 1, the compound of Chemical formula 2, and an equivalent amount of amine (for example, ethylenediamine, triethylamine, etc.) to Chemical compound 1 are dissolved in a volatile solvent, and the non-woven fabric is dipped in the solvent to apply the solvent. Volatilize the EB
The compound of Chemical formula 1 and Chemical formula 2 is introduced by irradiating with. In this method, since the compound of Chemical formula 1 and the amine form a salt, the compound of Chemical formula 1 does not volatilize when the solvent is volatilized, and the required amount of the carboxyl group can be efficiently introduced. After the completion of the introduction reaction by EB irradiation, the amine can be removed by treating with dilute hydrochloric acid.

CH ₂ ═C (R ¹ ) —COOH (Chemical Formula 1)

CH ₂ = C (R ² ) -CO (OCH ₂ C
H ₂ ) _n OCO-C (R ³ ) = CH ₂ (Chemical Formula 2) In Chemical Formula 1 and Chemical Formula 2, n represents an integer of 1 to 5, R ¹ ,
R ² and R ³ represent a hydrogen atom or a methyl group, and may be the same or different.

The carboxyl group content introduced by the above method is 0.01 to 3.00 meq / g-fiber,
It is preferably 0.03 to 2.00 meq / g-fiber, and more preferably 0.04 to 1.50 meq / g. When the carboxyl group is less than this range, the amount of antibody immobilized becomes small, and a sufficient amount of stem cell adsorption capacity cannot be obtained. The amount of the carboxyl group, which also has an effect of suppressing non-specific adsorption of leukocytes and platelets, is insufficient, and adhesion of these cells increases, which is not preferable. If the carboxyl group is more than the above range, the free carboxyl groups that are not involved in the immobilization of the antibody are too large, which causes non-specific adsorption of cells other than the target cells, and the increase of the adsorption of useful proteins. Occurs. In the present invention, the hydrophilic chain content refers to the weight increase rate before and after the introduction of the carboxyl group and hydrophilic chain into the nonwoven fabric. The hydrophilic chain content (weight increase rate) is 0.1 to 30% by weight based on the fiber,
Preferably 0.5 to 20% by weight, more preferably 1 to
15% by weight. If the weight increase rate is less than this range, the excluded volume effect of the hydrophilic chains is not sufficiently exerted, and nonspecific adsorption of cells or proteins increases. On the other hand, if the weight increase rate is larger than the above range, the stability of the introduced hydrophilic chain layer decreases, the amount of eluate increases, and problems such as peeling of the hydrophilic chain layer occur, which is not preferable.

As a method for immobilizing the antibody to the carboxyl group introduced into the nonwoven fabric, the following methods can be mentioned. (1) A method of reacting a carboxyl group with epichlorohydrin to introduce a glycidyl ester group and immobilizing it by reacting with an amino group of an antibody (2) Reacting a carboxyl group with thionyl chloride to form an acid chloride and then an amino group of the antibody (3) A method of reacting a carboxyl group with N-hydroxysuccinimide to introduce an active ester and immobilizing the same by reacting with an amino group of the antibody (4) ) Using carbodiimide to form an amide bond with the amino group of the antibody to immobilize it Since the reaction using the glycidyl group is usually carried out at room temperature or higher, it may lead to inactivation of the antibody. Immobilization efficiency is usually not very high. The acid chloride method has good reaction efficiency, but the reaction must be carried out in a non-aqueous system, the antibody must be dissolved in a non-aqueous solvent, and there is the possibility of denaturation, and the conditions for forming acid halides are severe, so the nonwoven fabric Is deteriorated, which is not preferable. The active ester method can be immobilized under relatively mild conditions, but usually the immobilization efficiency is not very high.
Compared to these methods, the method of using carbodiimide is a water-soluble carbodiimide such as 1-ethyl-3-
By using (3-dimethylaminopropyl) -carbodiimide hydrochloride (hereinafter abbreviated as EDC.HCl), relatively efficient immobilization is possible under mild conditions of an aqueous system.

When the antibody is immobilized using a water-soluble carbodiimide, the reaction is carried out at pH 4 to 8, preferably pH 5 to 5.
It is preferable to carry out the reaction in the buffer solution of 7.5. The buffer solution used in this case is particularly preferable because morpholinoethanesulfonic acid (hereinafter abbreviated as MES) or morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS) is used as the buffer solution so that the immobilization reaction proceeds efficiently. The amount of antibody immobilized by the above method and conditions is 0.01 to 10.
00 mg / g-nonwoven fabric, preferably 0.02-5.00
mg / g-nonwoven fabric, more preferably 0.03 to 3.0
0 mg / g-nonwoven fabric. If the antibody immobilization amount is less than this range, a sufficient stem cell adsorption capacity cannot be obtained, and if the antibody immobilization amount is greater than the above range, the adsorption capacity improves as the amount of antibody used increases. Not only is the waste of expensive antibodies wasted, but the possibility of elution during use is also increased, which is not preferable.

The non-woven fabric on which the antibody is immobilized by the above method,
The packing density in the column is 0.03 to 0.80 g / cm ³ ,
It is preferably 0.05 to 0.60 g / cm ³ , and more preferably 0.08 to 0.40 g / cm ³ . If the packing density is less than this range, the fiber surface area in the column is insufficient, so a sufficient stem cell adsorption capacity cannot be obtained,
If the amount is larger than this, the fiber gap becomes small, the pressure loss becomes large when passing through the cell suspension, and nonspecific capture of cells by the filtration effect is caused, which is not preferable. 1 to 4 are illustrated as the shape of the column for filling the antibody-immobilized nonwoven fabric, and FIG. 5 and FIG. 6 are illustrated as the method for filling the antibody-immobilized nonwoven fabric. The filling method is not limited to these. The method of filling the nonwoven fabric shown in FIGS. 5 and 6 is a cylindrical column (such as those illustrated in FIGS. 1 and 2).
Is a preferred example. In addition to these filling methods, a method in which the antibody-immobilized nonwoven fabric is finely cut and packed in a column, a method in which antibody-immobilized nonwoven fabrics that are cut out according to the shape of the column cross-section are laminated, and the like can be adopted.

After the bone marrow fluid or peripheral blood is passed through the column prepared as described above to capture the stem cells, the cells other than the target cells are washed out by washing with an albumin solution or the like, followed by antigen-antibody binding. The stem cells captured by are collected. As a method for recovering the captured stem cells, any one of (1) a method in which the column is vibrated by a vibrator or the like while flowing the wash solution to allow the stem cells to flow out (2) a method in which the wash solution is intermittently and vigorously flowed (stopped flow) is recovered Is preferred. As another recovery method, a method of treating with an enzyme such as chymopapain is known, but this method is not preferable because it may impair the cell function.

The present invention will be specifically described below with reference to examples. <Example 1> (1) Modification of non-woven fabric Fiber diameter 12 μm (1.35 d), basis weight 68 g / m ²
The non-woven fabric made of polyethylene terephthalate (hereinafter abbreviated as PET) was cut into 15 cm × 12 cm. This PET
The non-woven fabric was thoroughly washed with acetone before modification. Acrylic acid (compound of R ¹ = H in Chemical formula ¹ , hereinafter AA
Abbreviated. ) 0.86 g, ethylenediamine (hereinafter e)
Abbreviated as n. ) 0.36 g, the compound of Chemical formula 2 (n = 1
4, abbreviated as EG _14- A below. ) 8 g of methanol 5
It melt | dissolved in 00 ml, PET nonwoven fabric was immersed in this solution, and each reagent was apply | coated. After sufficiently drying it (30
An electron beam (hereinafter abbreviated as EB) was irradiated at a dose of 5 Mrad on one side for 6 hours at 6 ° C. to introduce a carboxyl group and a hydrophilic chain onto the surface of the PET nonwoven fabric.

The surface-modified PET non-woven fabric obtained by the above operation was thoroughly washed with running deionized water. Then, it was immersed in boiling ion-exchanged water and washed by boiling for 20 minutes. After repeating this boiling cleaning three times, the non-woven fabric was immersed in methanol, the container was immersed in the ultrasonic cleaner for 15 minutes, and the ultrasonic cleaning was repeated three times, and then sufficiently dried in a vacuum dryer (60). C, 18 hours). Thus, AA-introduced PET nonwoven fabric PET-AA-1 was obtained. The content of the carboxyl group introduced into PET-AA-1 was quantified, the weight increase rate (hydrophilic chain content) was calculated, and the eluate test was carried out. The results are shown in Table 1 below. The weight increase rate was calculated from the weight of the non-woven fabric before and after the modification, and the eluate test was performed according to the method described above. The quantification of the carboxyl group content was carried out by the method shown below.

Quantitative Method for Carboxyl Group Content About 0.2 g of finely chopped PET-AA-1 was accurately weighed (this amount is Wg), and 10 ml of 0.1N sodium hydroxide (titer is F). ) Was added and the suspension was diluted with dioxane / water (1/1 volume ratio) to give a total volume of about 60.
ml. After stirring this for about 30 minutes, it was titrated with a 0.1 hydrochloric acid aqueous solution (titer is F ′) using an automatic titrator (COMITITE 101 manufactured by Hiranuma Sangyo). When the amount of 0.1 N hydrochloric acid aqueous solution required for neutralization was Vml, the carboxyl group content of PET-AA-1 (X ₁ me
q / g) was obtained by the following formula. W × X + 0.1 × F ′ × V = 0.1 × F × 10 X = (F−0.1 × F ′ × V) / W

[0032]

[Table 1] In the table, the functional group content is the amount of carboxyl group introduced, pH is the pH difference from the blank, UV is the UV absorption spectrum, and K is the
MnO ₄ is potassium permanganate consumption (difference from blank), residue is evaporation residue, IN is within the evaluation standard, OUT is above the evaluation standard range, ND
Indicates that it is below the measurement limit.

(2) Immobilization of Ligand Subsequently, the anti-CD34 antibody was immobilized on the PET-AA-1. 0.1 mol / l MOPS aqueous solution and 0.1.
A 1 mol / l sodium hydroxide aqueous solution was appropriately mixed to prepare a solution having a pH adjusted to 6.5 (hereinafter, referred to as pH
Abbreviated as 6.5-MOPS. ). EDC in a glass bottle
Take 1495 mg of HCl, pH 6.5-MOPS30
0 ml was added and dissolved. 10 g of PET-AA-1 obtained by the above operation was weighed and immersed in this solution, and the solution was shaken in an ice bath for 20 minutes to activate the carboxyl group.
To this, 10 ml of the anti-CD34 antibody solution (1 mg / ml) was added, and the mixture was shaken at 25 ° C for 18 hours to give anti-CD34 to the nonwoven fabric.
Antibody immobilization is performed and antibody-immobilized non-woven fabric PET-AA-
ab-1 was obtained.

The amount of antibody immobilized on PET-AA-ab-1 was calculated and is shown in Table 2. The amount of antibody immobilized is BC
The total amount of protein in the antibody solution before and after the reaction was quantified by Method A, and it was calculated that the reduced amount was considered to be immobilized on the nonwoven fabric.

[0035]

[Table 2]

(3) Adhesion Behavior of Blood Cell Components A module for evaluation was prepared using the above PET-AA-ab-1, and leukocyte adhesion behavior to the antibody-immobilized nonwoven fabric was evaluated. The evaluation module has a non-woven fabric packing density of about 0.1.
The amount of non-woven fabric used was adjusted so as to be ³ g / cm ³ .
FIG. 7 shows the form of the evaluation module. After the nonwoven fabric is laminated on the space 2 of the main body 1 having the body fluid inlet 5, and the lid portion 3 ′ having the body fluid outlet 6 is attached,
By filling the concave portion of the lid with a silicone adhesive, the main body and the lid are bonded together. The volume of this module is 5.7 cm ³ . 5 ml / mi of citrated cow blood
The module was passed continuously for 10 minutes at a flow rate of n. Blood that has passed through the module is taken into a test tube every 1 minute, and the white blood cell concentration is measured by Toa Medical Electronic Blood Cell Counter S
It was measured by ysmex F-800. The leukocyte permeability after contact with the non-woven fabric was calculated from this leukocyte concentration. The results are shown in Fig. 8. The white blood cells in the blood that come into contact with the non-woven fabric are
It means that the larger the transmittance, the less likely it is to stick to the nonwoven fabric.

(4) Adsorption / Collection of CD34-Positive Cells Bone marrow fluid was centrifuged at 240 g for 15 minutes to remove plasma, and then the resulting buffy coat cells were again 240 g.
Red blood cells were removed by centrifugation for 15 minutes. The buffy coat cells were washed by centrifugation at 280 g for 10 minutes using a phosphate buffer solution (hereinafter abbreviated as PBS). After the washing operation was completed, the cells contained PB containing 1% bovine serum albumin (hereinafter abbreviated as BSA).
S (hereinafter abbreviated as BSA / PBS) to 2 × 10 ⁷
Dispersion was performed so that the number of particles / ml was set. The antibody-immobilized nonwoven fabric PET-AA-ab-1 obtained by the above operation was 0.13 g /
It was packed in the same column as that used in the blood cell adhesion behavior investigation so that the packing density was cm ³ . The cell suspension obtained above was continuously passed through this module at a flow rate of 1 ml / min for 16 minutes. Blood passing through the module was sampled into a test tube every 2 minutes. C in each sample after passing through the column by fluorescence labeled flow cytometry
The concentration of D34 positive cells was measured, and the transmittance was calculated from the CD34 positive cell concentration in the cell suspension before passing through the column. The results are shown in Fig. 9. CD34 with smaller transmittance
It means that the positive cell adsorption performance is excellent.

After completion of the above adsorption test, 4 ml / m
40 ml of BSA / PBS was passed through the module at a flow rate of in to remove cells that were not stably adsorbed. Then 8
While advancing BSA / PBS at a flow rate of ml / min, a vibrator (made by GL Sciences, for packing a column for gas chromatography) was applied to the side surface of the module to vibrate for 4 minutes to collect the adsorbed cells. The concentration of cells in the recovered solution can be measured by the same method as above,
Cell viability and CD34 positive cell purity were measured by trypan blue incorporation. The results are shown in Table 3.

[0039]

[Table 3] In the table, the functional group content is the amount of carboxyl group introduced, the concentration of cells in the recovered solution is the concentration of CD34-positive cells in the recovered solution, the cell viability is the viability of the recovered cells, and the cell purity is the CD34-positive cells in the recovered cells. Indicates purity.

Example 2 The same PET nonwoven fabric as that used in Example 1 was prepared. AA 0.86g, en0.3
6 g and 2 g of methylenebisacrylamide (hereinafter abbreviated as MBAA) were dissolved in 500 ml of methanol, and the PET nonwoven fabric was dipped in this solution to apply each reagent.
After it was sufficiently dried (30 ° C., 6 hours), an electron beam (hereinafter abbreviated as EB) was irradiated at a dose of 5 Mrad on one side to graft a carboxyl group onto the surface of the PET nonwoven fabric.

The surface-modified PET nonwoven fabric obtained by the above operation was washed in the same manner as in Example 1 to obtain AA-introduced PET nonwoven fabric PET-AA-2. PE in the same manner as in Example 1
Quantification of the carboxyl group content of T-AA-2, calculation of the graft ratio, and eluate test were carried out, and the results are shown in Table 1. The anti-CD34 antibody was immobilized in the same manner as in Example 1 to obtain an antibody-immobilized nonwoven fabric PET-AA-ab-2. The antibody immobilization amount was calculated in the same manner as in Example 1, and is shown in Table 2. Furthermore, PET-
The leukocyte permeability of AA-ab-2 was measured, and the results are shown in FIG. Subsequently, an evaluation module was prepared in the same manner as in Example 1 using PET-AA-ab-2, and CD34 was used.
The adsorption performance of positive cells and the collection of adsorbed cells were performed.
The results are shown in FIG. 9 and Table 3.

Comparative Example 1 A module for evaluation was prepared in the same manner as in Example 1 using the carboxyl group-introduced nonwoven fabric PET-AA-1 obtained in Example 1 to evaluate the adsorption performance of CD34-positive cells. The results are shown in Fig. 9. In addition, the adsorbed cells were collected by the same method as in Example 1, and the cell concentration, viability, and purity of CD34-positive cells in the collected solution were measured. The results are shown in Table 3.

Comparative Example 2 Using the carboxyl group-introduced nonwoven fabric PET-AA-2 obtained in Example 2, a module for evaluation was prepared in the same manner as in Example 1 and the adsorption performance of CD34-positive cells was evaluated. The results are shown in Fig. 9. In addition, the adsorbed cells were collected by the same method as in Example 1, and the cell concentration, viability, and purity of CD34-positive cells in the collected solution were measured. The results are shown in Table 3.

Comparative Example 3 As in Example 1, CD34-positive cells were adsorbed by the evaluation module prepared using the antibody-immobilized nonwoven fabric PET-AA-ab-1, and the same method as in Example 1 was performed. After adsorption, the column was washed. Then, the evaluation column was disassembled, the filled non-woven fabric was taken out, and the chymopapain solution (10 units / ml-P
It was immersed in 20 ml of BS solution) for 30 minutes. The cells recovered by centrifugation are separated, and BSA / PB is added several times.
After washing by centrifugation using S, 32 ml of BSA
/ Resuspended in PBS. From this recovered cell suspension, cell concentration, viability, and purity of CD34-positive cells were measured. The results are shown in Table 3.

From the results of Example 1 and Comparative Example 1 shown in FIG. 9, it can be seen that the non-woven fabric on which the antibody was not immobilized showed almost no adsorption of CD34-positive cells. On the other hand, focusing on the results of Example 2 and Comparative Example 2 in FIG. 9, the adsorption of CD34-positive cells on the antibody-immobilized nonwoven fabric is also observed, but the same tendency is exhibited by the nonwoven fabric on which the antibody is not immobilized. ing. This is the hydrophilic chain content (weight increase rate)
It is considered that the cells are non-specifically adsorbed because the excluded volume effect of the hydrophilic chain is not sufficiently exerted because of low. In addition, from the results of Example 1 and other examples shown in Table 3, it can be seen that the efficiency of the target adsorption / recovery of CD34-positive cells is improved by introducing a carboxyl group and a hydrophilic chain. In Comparative Example 3, although the specifications of the column used were the same as those in Example 1, the viability of the obtained cells was lowered because the enzyme was used to collect the adsorbed cells.

[0046]

INDUSTRIAL APPLICABILITY According to the method for separating stem cells described in the present invention, the target stem cells can be separated efficiently and at high speed, the purity of the obtained stem cells is high, and the function of the separated stem cells can be deteriorated. It is not very popular. Furthermore, the adsorbed stem cells can be easily recovered and the column can be regenerated. Due to such characteristics, it can be effectively used for stem cell autologous transplantation in hematopoietic malignant tumors and various solid cancers.

[Brief description of drawings]

FIG. 1 is a schematic view of an example of a stem cell separation column that can be used in the present invention.

FIG. 2 is a schematic view of another example of a stem cell separation column that can be used in the present invention.

FIG. 3 is a schematic view of another example of a stem cell separation column that can be used in the present invention.

FIG. 4 is a schematic view of another example of a stem cell separation column that can be used in the present invention.

FIG. 5 is a schematic view of an example of a method of packing a stem cell separating material that can be used in the present invention in a column.

FIG. 6 is a schematic diagram of another example of a method for packing a stem cell separating material that can be used in the present invention into a column.

FIG. 7 is a schematic diagram of a leukocyte adhesion behavior evaluation module used in Examples.

FIG. 8 is a diagram showing white blood cell adsorption behavior in Examples.

FIG. 9 is a diagram showing the transmittance of CD34-positive cells in Examples and the like.

[Explanation of symbols]

 1. Main body 1 '. Main body 2. Space for filling the separating material (nonwoven fabric) 3. Treatment liquid inflow side lid 3 '. Treatment liquid outflow side lid 4. Treatment liquid inflow side filter 4 '. Processing solution outflow side filter 5. Processing liquid inlet 6. Processing liquid outlet 7. Convex portion for holding non-woven fabric 8. Non-woven fabric 9. spacer

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 30/48 T (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu, Shiga Prefecture Toyobo Co., Ltd. Research Institute, Inc.

Claims (7)

[Claims] 1. A method for separating stem cells, which comprises using a stem cell separating material in which an antibody against an antigen on the surface of a stem cell membrane is immobilized on a carrier prepared by introducing a carboxyl group and a hydrophilic chain into a non-woven fabric made of fibers. 2. The carboxyl group content is 0.01 to 3.0.
The method for separating stem cells according to claim 1, wherein a nonwoven fabric having 0 meq / g-fiber and a hydrophilic chain content of 0.1 to 30% by weight based on the fiber is used. 3. A fiber diameter of 5 to 150 μm and a basis weight of 3
The method for separating stem cells according to claim 1, wherein a nonwoven fabric of 0 to 400 g / m ² is used. 4. The method for separating stem cells according to claim 1, wherein the antibody to the antigen on the surface of the stem cell membrane is an anti-CD34 antibody. 5. A stem cell separation column packed with a fiber packing density of 0.03 to 0.80 g / cm ³ after immobilizing an anti-CD34 antibody on a nonwoven fabric into which a carboxyl group and a hydrophilic chain have been introduced. The method for separating stem cells according to claim 4, which is used. 6. A method for adsorbing CD34-positive cells from bone marrow fluid or peripheral blood using a stem cell separation column, removing unnecessary cells by washing, and adsorbing the adsorbed CD34-positive cells by vibration. Claim 4 characterized by the above-mentioned.
The described method for separating stem cells. 7. Adsorbing CD34-positive cells from bone marrow fluid or peripheral blood using a stem cell separation column, removing unnecessary cells by washing, and adsorbing the adsorbed CD34-positive cells by stopped flow. The method for separating a stem cell according to claim 4, wherein