JPH10313855A - Separation of cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recovering necessary cells from a mixture of the necessary cells with unnecessary cells in high yield in a short period according to simple operations, e.g. a method for selectively recovering leucocytes from the mixture of the leucocytes, blood platelets and erythrocytes in high yield. SOLUTION: This method for separating cells comprises introducing a cell population containing at least the necessary cells for recovery and the cells as an removal object into a means for capturing at least the necessary cells for the recovery and substantially passing the cells as the removal object therethrough, then introducing a physiological solution containing a dextran thereinto and recovering the necessary cells for the recovery captured by the means. The composition for recovering the cells is used for introducing the cell polulation containing at least the necessary cells for the recovery and the cells as the removal object into the means for capturing at least the necessary cells for the recovery and substantially passing the cells as the removal object therethrough and recovering the captured necessary cells for the recovery and is a physiological solution containing at least the dextran.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a composition for separating specific cells from a cell population, a separation method, and a cell suspension obtained by the separation method.

[0002]

2. Description of the Related Art Bone marrow transplantation therapy is widely used for hematopoietic disorders, which are side effects of chemotherapy for hematopoietic tumors such as leukemia and solid cancer. Bone marrow transplantation therapy is a method of recovering a fatal hematopoietic disorder caused by transplanted bone marrow, so that patients can receive high-dose radiation and / or high-dose chemotherapy (hereinafter, abbreviated as high-dose chemotherapy). It leads to cure of solid cancer. In recent years, it has been revealed that peripheral blood, as well as bone marrow, contains hematopoietic stem cells necessary for these treatments. Usually, the content of these cells in the peripheral blood is quite low and it is difficult to collect and use them in place of bone marrow transplantation, but it is difficult to obtain anticancer drugs and / or G-CSF (granulocyte colony stimulating factor). It has been clarified that the administration of such cytokines increases the content thereof. Compared with bone marrow collection, general anesthesia is unnecessary and safe. In recent years, it has become clear that cord blood contains hematopoietic stem cells at a much higher concentration than peripheral blood,
Clinical application has begun. Here, the transplantation method is divided into allogeneic transplantation and autotransplantation depending on who the cell donor (donor) is. In the former, healthy others (relatives or unrelated) become donors, and in the latter, patients themselves become donors. Cryopreservation is performed until autotransplantation, and in all cases of autotransplantation, and in the case of allogeneic transplantation, cord blood is almost always used until transplantation. Before cryopreservation, red blood cells are usually removed. This is because when stored with whole blood, side effects due to storage space and destructed red blood cells upon thawing become a problem.
Conventionally, erythrocyte removal has been performed by a centrifugal separator. In order to further increase the separation efficiency, a specific gravity centrifugation method using a specific gravity liquid (for example, Ficoll manufactured by Pharmacia) has been adopted. This method is a complicated operation that requires a great deal of skill, such that the liquid surface must not be disturbed when layering the raw material cells on the specific gravity solution. JP-A-61-84577, JP-A-2-1
Many attempts have been made to solve the complexity of the operation in, for example, Japanese Patent No. 34564, but there has been no change in using a specific gravity liquid, and no drastic solution has been reached. By the way, proposals for a method of removing red blood cells without using centrifugation have been seen occasionally. JP-A-8-104463 discloses a cell population containing erythrocytes and hematopoietic stem cells and / or hematopoietic progenitor cells.
A method for removing red blood cells, wherein substantially red blood cells pass through, and white blood cells pass through a filter for capturing, and then a liquid flow in a direction opposite to the flow direction is caused to collect the captured white blood cells. Has been proposed. However, there is no description that the recovery rate is improved by using a recovery liquid having a specific composition.

Dextran is a polysaccharide containing glucose as a monomer and mainly composed of α-1,6 bonds, and has been used as a leukocyte separating agent for a long time. However, in the separation of leukocytes by dextran, red blood cells in a test tube are agglutinated and sedimented (centrifuged as necessary), and leukocytes in the supernatant are collected with a pipette (edited by Shiro Miwa: Clinical Laboratory Techniques, Vol. 3, "Hematology"). 425
Page) as a hemagglutination promoter,
This is a technical idea completely different from the present application, that is, recovery after capture.
It should be noted that hydroxyethyl starch and the like also have a similar hemagglutination-promoting effect, and are not inherent in dextran.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a method for recovering required cells from a mixture of required cells and unnecessary cells at a high rate in a simple operation and in a short time, for example, a method for recovering leukocytes from a mixture of leukocytes, platelets, and erythrocytes. It is an object of the present invention to provide a method for selectively recovering at a high rate. More specifically, it is an object of the present invention to provide a cell separation method for once capturing cells and recovering the captured cells, in which the cells can be recovered at a high rate.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the problems of the prior art, and as a result, when cells are recovered with a physiological solution containing dextran, an extremely high recovery rate can be obtained. The present inventors have found the surprising property of dextran and completed the present invention. That is, the present invention captures at least the collection-required cells, the cell population containing at least the collection-required cells and the cells to be removed,
A cell characterized in that the cells to be removed are introduced into a means through which the cells substantially pass, and then a physiological solution containing dextran is introduced to collect the cells required for collection captured by the means from the means. It is a separation method. Further, the present invention captures a cell population containing at least the cells to be recovered and the cells to be removed, at least the cells to be recovered, and introduces the cells to be removed substantially into the passing means. A composition for use in recovering cells, which is a physiological solution containing at least dextran. Further, the present invention further comprises introducing a cell population containing at least the cells to be recovered and the cells to be removed into a means through which at least the cells to be recovered are captured and the cells to be removed substantially pass through, and then the means comprising dextran A method for obtaining a cell suspension comprising a physiological solution containing the cells requiring collection and dextran, wherein the cells requiring recovery are collected by introducing a physiological solution. Furthermore, the present invention introduces a cell population containing at least the cells to be recovered and the cells to be removed into a means for capturing at least the cells to be recovered and substantially passing through the cells to be removed, and then adding dextran to the means. This is a cell suspension comprising a physiological solution containing dextran and cells requiring collection obtained by introducing and collecting a physiological solution containing dextran.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below in more detail. Examples of the cell population containing at least the cells required to be recovered and the cells to be removed according to the present invention include bone marrow, peripheral blood, umbilical cord blood, or those obtained by roughly separating them using a centrifuge. As means for capturing at least the cells required to be recovered according to the present invention and for substantially passing the cells to be removed,
The container may be filled with a capturing material, or a molded container having a cell capturing surface on the inner surface of the container. As the capturing material, any material can be used as long as it is water-insoluble, but examples of preferable materials in terms of moldability, sterility and low cytotoxicity include polyethylene, polypropylene, polystyrene, acrylic resin, nylon, and polyester. , Polycarbonate, polyacrylamide, synthetic polymers such as polyurethane, agarose, cellulose,
Examples include natural polymers such as cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metals such as stainless steel and titanium. These capture materials can be used as they are, but if necessary, immobilize ligands such as amino acids, peptides, and glycoproteins (including bioligands such as antibodies and adhesion molecules) that have affinity for specific cells. May be. Examples of the shape of the trapping material include granular, fibrous mass, woven fabric, nonwoven fabric, sponge-like porous body, and flat plate, but in terms of a large surface area per volume, granular, fibrous mass, woven fabric, nonwoven fabric, A sponge-like porous body is preferred. In addition, examples of a molded container having a cell capturing surface on the inner surface of the container include a flask, a dish, a conical tube, and a syringe.

The term "dextran" as used in the present invention refers to a polymer of glucose whose bonds are mostly α-1,6 bonds, including its partial hydrolysates and derivatives such as sulfates. Any molecular weight can be used,
In consideration of solubility and availability, the preferred average molecular weight is 1,000 to 10,000,000, and more preferably 500 to 10,000,000.
It is from 0 to 5,000,000, still more preferably from 10,000 to 1,000,000. In the present invention, dextran is dissolved and used as a physiological solution. As the solvent, preferred ones in consideration of osmotic pressure and cytotoxicity include physiological saline, Dulbecco's salt buffer, Hanks' solution. Buffers such as R
Examples include, but are not limited to, media such as PMI1640. The solution may contain proteins such as albumin and globulin, sugars such as glucose, mannitol, trehalose, and hydroxyethyl starch, and anti-oxidants such as heparin, ACD, and CPD for the purpose of nutritional supplementation and cryopreservation of cells. A cryopreservative such as a coagulant, dimethyl sulfoxide, and glycerin may be added. The dextran concentration in the dextran-containing liquid according to the present invention is 0.5 w / v% or more and a saturation concentration or less, preferably 1 w / v% or more and a half or less of the saturation concentration. At 1 w / v%, the cell recovery rate may decrease, and when an amount exceeding one half of the saturation concentration is added,
When the viscosity increases and the liquid passes through the filter, the pressure increases, and the connection between the filter and the tube may be disconnected, which is dangerous.

[0008] As a method of passing a physiological solution containing dextran according to the present invention through the capturing means, use of a pump,
There are a method of injecting with a syringe, a method of causing a liquid flow by crushing a bag storing a liquid, and a method of using a head, but the use of a syringe is most preferable in terms of simplicity. In addition, in the case of the capturing means in which the liquid inlet and the liquid outlet are formed of separate containers, it is divided into passing the collected liquid in the same direction as the method of passing the source blood or passing in the opposite direction. Generally, the reverse direction tends to have a higher recovery rate. Further, instead of simply passing the liquid, vibration may be applied to the capturing means, or a stopped flow may be performed. When the liquid inlet and the liquid outlet are the same, for example, in the case of a flask, a liquid having a specific viscosity according to the present invention is introduced into the flask with a pipette or the like, and then the flask body is shaken, or mechanically The cells are recovered by applying sonic vibration.

Several examples of combinations of cells requiring recovery and cells to be removed in the present invention will be described. If the cells to be collected are white blood cells and the cells to be removed are red blood cells and platelets, the white blood cells are captured by the capturing means, the red blood cells and platelets pass through, and are captured by the capturing means by a physiological solution containing dextran according to the present invention. White blood cells are collected. If the cells to be collected are lymphocytes and the cells to be removed are erythrocytes, platelets, granulocytes, and monocytes, leukocytes (granulocytes + monocytes + lymphocytes) are captured by the capturing means, and erythrocytes and platelets pass. Then, only lymphocytes among the leukocytes captured by the capturing means are collected by the physiological solution containing dextran according to the present invention. When the cells to be collected are CD34-positive cells and the cells to be removed are erythrocytes, platelets, and CD34-negative cells, the CD34-positive cells are captured by the capturing means, and the erythrocytes, platelets, and CD34-negative cells pass through. The CD34-positive cells captured by the capturing means by the physiological solution containing dextran are collected.

The physiological solution containing dextran according to the present invention can be used as it is for liquid storage or cryopreservation. That is, taking the cryopreservation of stem cells as an example, usually, after washing the cell population from which erythrocytes have been separated by the Ficoll method described above, a cryopreservative is added to prepare a cell suspension, which is then placed in liquid nitrogen. Alternatively, cryopreservation is performed in a freezer, but since dextran itself can be a cryopreservative, it can be used as a cell suspension for cryopreservation without adding complicated operations after removing red blood cells. When it is desired to coexist not only dextran alone but also substances used for cryopreservation such as dimethyl sulfoxide, hydroxyethyl starch, and albumin, add these substances after cell collection or add them in advance to a physiological solution containing dextran. You should leave it. It is unclear at this time what mechanism can achieve high cell recovery with dextran. The present inventors assume that dextran may have the property of reducing the adhesion of cells to a substrate, but elucidation thereof is a subject to be solved in the future.

[0011]

EXAMPLES Examples of the present invention will be described below together with comparative examples, but the present invention is not limited to these examples.

EXAMPLE 1 Cell separation according to this example aims at collecting mononuclear cells containing CD34-positive cells and removing red blood cells and platelets. Preparation of Cell Separator Twelve polyester non-woven fabrics having an average fiber diameter of 12 μm were placed on the inlet side of a polycarbonate container having a container size of 41 × 41 × 18 mm and having a liquid outlet and a liquid inlet on a diagonal line, and an average fiber diameter of 2 was placed on the outlet side. .3 μm polyester non-woven fabric 25
Sheets were filled. The packing density of this filter is 0.2
g / cm ³ . Further, for the purpose of imparting platelet permeability to this filter, coating with a hydrophilic polymer was performed. That is, a 1% ethanol solution of a hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer was passed through a liquid inlet of the filter, and then dried by passing through a nitrogen gas. Cell Separation Operation A blood bag containing 50 ml of fresh human umbilical cord blood (anticoagulant CPD) was connected on the way to the inlet side of the cell separator prepared with a tube having a branch to a saline bag and a cell collection bag. . A blood bag for drain was connected to the outlet side of the cell separator with a tube having a three-way stopcock in the middle. 50 ml of fresh umbilical cord blood was passed through the head, and the erythrocyte-containing liquid flowing out of the filter was collected in a drain bag.
Thereafter, 30 ml of physiological saline was passed through for the purpose of washing away the red blood cells and platelets remaining in the filter. Thereafter, human serum albumin (hereinafter referred to as HSA) was added to a commercially available dextran physiological saline solution (Dextran 40 Note-Midori manufactured by Midori Cross, a 10 w / v% physiological saline solution of dextran having an average molecular weight of about 40,000) at 4 w / v%. A syringe containing 30 ml of a dextran-containing physiological solution (viscosity of about 10 mPa · s) prepared by adding a dextran was connected to a three-way cock of the outlet tube of the cell separator, and the syringe was pushed to release the dextran-containing physiological solution. The solution was injected into the cell separator, and the captured cells were collected in a cell collection bag connected to the inlet side. Analysis The leukocyte count, leukocyte subfraction, erythrocyte count, and platelet count were measured by an automatic hemocytometer, and the CD34 positive rate in leukocytes was determined by FIT
The measurement was carried out using a C-labeled CD34 antibody and a flow cytometry method developed on SSC-FITC (Miyazaki et al., Daily Practice and Blood, Vol. 5, No. 2, 21-23, 1995). The method of calculating the recovery rate and the removal rate is as follows. Recovery (%) = 100 × (number of cells after separation / number of cells before separation) Removal rate (%) = 100−100 × (number of cells after separation / number of cells before separation) Results Table 1 summarizes the results. Mononuclear cells and CD34-positive cells can be recovered at a high rate, and red blood cells and platelets have been removed at a high rate.

[0012]

Example 2 The purpose of the present embodiment is to collect mononuclear cells containing CD34-positive cells and to remove red blood cells and platelets. Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell Separation Procedure Dextran 40 (manufactured by Tokyo Chemical Industry, average molecular weight: about 40,000) and HSA as dextran-containing solutions were each 23 w / v.
%, 4 w / v%, and the same operation as in Example 1 was performed, except that the addition was performed. The viscosity of the recovered liquid was about 30 mPa · s. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. As in Example 1, mononuclear cells,
CD34-positive cells can be recovered at a high rate, and red blood cells and platelets have been removed at a high rate.

[0013]

[Embodiment 3] The purpose of this embodiment is to collect mononuclear cells containing CD34-positive cells and to remove red blood cells and platelets. Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell separation operation The procedure was performed except that dextran 70 (average molecular weight: about 70,000 manufactured by Tokyo Chemical Industry) and HSA were added to physiological saline as a dextran-containing solution at 19 w / v% and 4 w / v%, respectively. The same operation as in Example 1 was performed. The viscosity of the recovered liquid was about 30 mPa ·
s. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. As in Example 1, mononuclear cells,
CD34-positive cells can be recovered at a high rate, and red blood cells and platelets have been removed at a high rate.

[0014]

Embodiment 4 The purpose of this embodiment is to collect mononuclear cells containing CD34-positive cells and to remove red blood cells and platelets. Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell Separation Procedure Dextran for leukocyte separation (average molecular weight: about 200,000 manufactured by Nacalai Tesque) and HSA as dextran-containing solutions were added to a commercially available aqueous solution of hydroxyethyl starch saline (manufactured by Morishita Roussel, product name: 6-HES) at 10 w /
The same operation as in Example 1 was performed, except that the one added so as to be v% and 4 w / v% was used. The viscosity of the recovered liquid was about 20 mPa · s. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. As in Example 1, mononuclear cells,
CD34-positive cells can be recovered at a high rate, and red blood cells and platelets have been removed at a high rate. The cells recovered in this recovery solution were added with 5% w / v of dimethyl sulfoxide,
According to the I protocol (manufactured by Far East Pharmaceutical Co., Ltd.), cryopreservation in a deep freezer at -80 ° C was possible. That is, 3
After freezing and preservation on day 0, the mixture was rapidly thawed in a warm bath at 37 ° C., and the viability was measured by a conventional trypan blue exclusion method. As a result, the high value was maintained at 90.4%.

[0015]

Embodiment 5 The purpose of this embodiment is to collect CD34-positive cells and to remove erythrocytes, platelets, and CD34-negative nucleated cells (hereinafter, CD34-negative cells). Preparation of Cell Separator Twelve polyester non-woven fabrics having an average fiber diameter of 12 μm were placed on the inlet side of a polycarbonate container having a container size of 41 × 41 × 18 mm and having a liquid outlet and a liquid inlet on a diagonal line, and an average fiber diameter of 2 was placed on the outlet side. .3 μm mouse anti-human CD34 monoclonal antibody (Coulter, clone name Imm
u133, abbreviated as CD34 antibody hereinafter) 25 fixed polystyrene nonwoven fabrics were filled. The packing density of this filter was 0.2 g / cm ³ . The immobilization of the CD34 antibody was carried out by the known haloacetamide method proposed in JP-A-2-261833. That is, for the purpose of activating the polystyrene nonwoven fabric, the above-mentioned polystyrene nonwoven fabric (cut in advance to the above dimensions) was added to a reaction solution obtained by adding 3.6 g of hydroxymethyliodoacetamide and 25 g of trifluoromethanesulfonic acid to 165 ml of sulfolane.
At room temperature for 5 hours to react. Next, for the purpose of immobilizing the antibody on the activated nonwoven fabric, the activated nonwoven fabric was impregnated with 10 ml of a CD34 antibody solution adjusted to a concentration of 20 μg / ml with Dulbecco's salt buffer (hereinafter D-PBS) for 2 hours, After washing with D-PBS, vacuum drying was performed to obtain an antibody-immobilized nonwoven fabric. Cell Separation Operation A blood bag containing 50 ml of fresh human umbilical cord blood (anticoagulant CPD) was connected on the way to the inlet side of the cell separator prepared with a tube having a branch to a saline bag and a cell collection bag. . A blood bag for drain was connected to the outlet side of the cell separator with a tube having a three-way stopcock in the middle. 50 ml of fresh umbilical cord blood was passed through the head, and the erythrocyte-containing liquid (including CD34-negative cells) flowing out of the filter was collected in a drain bag. Thereafter, 30 ml of physiological saline was passed through for the purpose of washing away the red blood cells, platelets, and CD34-negative cells remaining in the filter. Thereafter, human serum albumin (hereinafter referred to as HSA) was added to a commercially available dextran physiological saline solution (Dextran 40 Note-Midori manufactured by Midori Cross, a 10 w / v% physiological saline solution of dextran having an average molecular weight of about 40,000) at 4 w / v%. Dextran-containing physiological solution (viscosity of about 10 m
Pa.s) A syringe containing 30 ml was connected to the three-way cock of the outlet tube of the cell separator, and the syringe was pushed to inject a physiological solution containing dextran into the cell separator.
The captured cells were collected in a cell collection bag connected to the inlet side. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. CD34 positive cells can be recovered at a high rate, and red blood cells, platelets, and CD34 negative cells have been removed at a high rate.

[0016]

Comparative Example 1 Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell Separation Procedure In place of the dextran-containing solution, physiological saline was used to which hydroxyethyl starch (manufactured by Ajinomoto Co., average molecular weight: about 200,000) and HSA were added at 10 w / v% and 4 w / v%, respectively. Except for the above, the same operation as in Example 1 was performed. The viscosity of the recovered liquid was about 10 mPa · s, which was the same as that in Example 1. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. The erythrocyte removal rate and platelet removal rate were as high as in the Examples, but the recovery rates of mononuclear cells and CD34-positive cells were low.

[0017]

Comparative Example 2 Production of Cell Separator The same cell separator as in Example 1 was used. Cell Separation Procedure Instead of the dextran-containing solution, a solution obtained by adding hydroxyethyl starch (manufactured by Ajinomoto Co., average molecular weight: about 200,000) and HSA to physiological saline to 20 w / v% and 4 w / v%, respectively, was used. The same operation as in Example 1 was performed except for the operation. The viscosity of the recovered liquid was about 30 mPa · s, which was the same as that in Example 2. Analysis was performed in the same manner as in Example 1. Results Table 1 summarizes the results. The erythrocyte removal rate and platelet removal rate were as high as in the Examples, but the recovery rates of mononuclear cells and CD34-positive cells were low.

[0018]

[0019]

As described above, the cell separation method according to the present invention can easily recover the required cells from the mixture of the required cells and the unnecessary cells in a simple operation and in a short time. The suspension can be cryopreserved without going through complicated cell suspension preparation operations thereafter, which greatly contributes to labor saving in the cell treatment process in the field of hematopoietic stem cell transplantation and the field of adoptive immunotherapy.

Claims (4)

[Claims] Claims: 1. A cell population containing at least cells to be recovered and cells to be removed is introduced into a means for capturing at least the cells to be recovered, and the cells to be removed substantially pass through;
Next, a physiological solution containing dextran is introduced, and the cells required for recovery captured by the means are recovered from the means. 2. The method according to claim 1, wherein a cell population containing at least the cells required to be recovered and the cells to be removed is captured by at least the cells required to be recovered, and the cells to be removed are substantially introduced into the passing means. A composition for use in recovering cells, which is a physiological solution containing at least dextran. 3. A cell population containing at least the cells to be recovered and the cells to be removed is introduced into a means for capturing at least the cells to be recovered, and the cells to be removed substantially pass through;
Next, a physiological solution containing dextran is introduced into the means, and the cells requiring recovery are recovered, thereby obtaining a cell suspension comprising a physiological solution containing the cells requiring recovery and dextran. 4. A cell population containing at least cells to be recovered and cells to be removed is introduced into a means for capturing at least the cells to be recovered and substantially passing through the cells to be removed,
Next, a cell suspension comprising a physiological solution containing dextran and cells required for recovery obtained by introducing and collecting a physiological solution containing dextran into the means.