JPH10201470A - Cell separation and cell floating solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recovering necessary cells such as leucocyte from a mixture (e.g. a mixture of leucocyte, platelet and erythrocyte) of necessary cells and unnecessary cells in high ratio by a simple operation in a short time and to obtain a cell floating solution obtained by the method. SOLUTION: This method for separating cells comprises supplying a cell group comprising at least recovery requiring cells and target removal cells to a means for catching at least the recovery requiring cells and substantially passing the target removal cells, then introducing a liquid having >=2mPa.s and <=1,000mPa.s viscosity, releasing the caught recovery requiring cells from the means and recovering the recovery requiring cells. This cell floating solution comprises a liquid and the recovery requiring cells which are obtained by supplying the cell group comprising at least the recovery requiring cells and the target removal cells to the means for catching at least the recovery requiring cells and substantially passing the target removal cells and then introducing a liquid having >=2mPa.s and <=1,000mPa.s viscosity, releasing and recovering the recovery requiring cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a separation method for separating specific cells from a cell population, 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 and white blood cells are passed through a filter for capturing, and then a liquid flow in the opposite direction to the flow-through method is induced 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 viscosity. By the way, Japanese Patent Application Laid-Open No. 55-130917 discloses a liquid having a viscosity of 2 cm./cm.
A method for collecting bodily fluids characterized by using a liquid of poise (CP) or more is disclosed. However, according to the publication, it is not preferable to use a low-viscosity solution such as a physiological saline solution, since cells adhering in the filter are also collected. Therefore, high viscosity is used in order not to collect adherent cells. That is, the publication discloses a method for recovering a bodily fluid that almost passes through a filter and slightly remains in the filter, and is a technical idea completely different from that of the present application.

[0003]

SUMMARY OF THE INVENTION In view of the above-mentioned problems in the prior art, the present invention provides a method for recovering required cells from a mixture of required cells and unnecessary cells at a high rate with a simple operation and in a short time.
For example, it is an object of the present invention to provide a method for selectively recovering leukocytes from a mixture of leukocytes, platelets, and erythrocytes at a high rate and a cell suspension obtained by the method.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, completed the present invention. That is, the present invention introduces a cell population containing at least the cells required to be recovered and the cells to be removed into at least the means for capturing the cells required to be recovered, and the cells to be removed are substantially introduced into the passing means. s or more and 1000 mPa
A cell separation method comprising introducing a liquid having a viscosity of not more than s, detaching the cells required for collection trapped by the means from the means, and collecting the cells required for collection. Further, there is provided a cell separation method in which the liquid having a viscosity of 2 mPa · s or more and 1000 mPa · s or less is used as a preservative in a subsequent storage step of cells requiring recovery. Further, 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 introduced into a means through which the cells substantially pass. s
A cell suspension obtained by introducing a liquid having the following viscosity and peeling and recovering the cells, the cells being required to be recovered, and the liquid.

[0005]

In the cell separation method of the present invention, 2 mPa ·
By using a liquid having a specific viscosity of s or more and 1000 mPa · s or less as a recovery liquid, the required cells captured by the capturing means can be separated and recovered at a high recovery rate with a simple operation and in a short time. Can be. Further, the cell suspension obtained by using a cryopreservation solution as a recovery solution having a specific viscosity can be cryopreserved as it is.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the capturing means used in the present invention include a container 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, and nylon. , 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 that have affinity for specific cells, such as amino acids, peptides, and glycoproteins (including bioligands such as antibodies and adhesion molecules). You may. Examples of the shape of the trapping material include granular, fibrous mass, woven fabric, non-woven fabric, flat plate, sponge-like porous material, and the like, but granular, fibrous mass, woven fabric, and non-woven fabric have a large surface area per volume. preferable. 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. 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. Some examples of combinations of cells requiring collection and cells to be removed are also shown. When the cells requiring collection 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 the liquid having the specific viscosity according to the present invention. Leukocytes are collected. If the cells that need to be collected are lymphocytes and the cells to be removed are red blood cells, platelets, granulocytes, and monocytes, white blood cells (granulocytes + monocytes + lymphocytes)
Is captured by the capturing means, red blood cells and platelets pass through, and only lymphocytes are collected from the white blood cells captured by the capturing means by the liquid having the specific viscosity according to the present invention. Further, when the cells requiring collection are CD34-positive cells and the cells to be removed are red blood cells, platelets, and CD34-negative cells,
CD34-positive cells are captured by the capturing means, and red blood cells, platelets, and CD34-negative cells pass through and are captured by the capturing means by the liquid having a specific viscosity according to the present invention.
Positive cells are collected. In the present invention, the cells captured by the capturing means are collected with a liquid having a specific viscosity.
0 mPa · s or less, preferably 5 mPa · s or more and 500
mPa · s or less, more preferably 10 mPa · s or more 2
00 mPa · s or less. If the viscosity is less than 2 mPa · s, the recovery rate is low, and if it exceeds 1000 mPa · s, even if a pump is used, it becomes extremely difficult to pass the liquid and the workability is poor. In addition, there is a possibility that the pressure may rise and the connection between the filter and the tube may be disconnected, which is dangerous.
As the components, those having little adverse effect on cells are preferable.
Some examples are synthetic polymer solutions such as polyethylene glycol, polyvinylpyrrolidone, and polyvinyl alcohol; natural polymer solutions such as methyl cellulose, gelatin, hydroxyethyl starch, dextran, chitin derivatives, collagen, fibronectin, albumin, and globulin; glucose, saccharose. , Maltose, sorbitol, glycerin, dimethyl sulfoxide, and other organic solutions and mixtures thereof. Examples of the solvent for dissolving these include physiological saline, buffer solutions such as D-PBS (Dulbecoline salt buffer) and HBSS (Hanks solution), and culture media such as RPMI1640.

[0007] Further, it is more preferable that the liquid having a specific viscosity according to the present invention is used for frozen storage or liquid storage as it is. 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 in the present invention, by using a cryopreservative for a liquid having a specific viscosity, it is possible to prepare a cell suspension for cryopreservation without adding complicated operations after removing red blood cells. Can be. Examples of the method for passing a liquid having a specific viscosity through the capturing means according to the present invention include use of a pump, injection with a syringe, crushing a bag storing the liquid to cause a liquid flow, and a method using a head. Further, 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 flowing direction of the source blood or flowing 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.

[0008]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Preparation of Cell Separator Twelve polyester nonwoven 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 a liquid outlet and a liquid inlet on a diagonal line. Polyester non-woven fabric 25 having an average fiber diameter of 2.3 μm
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. After flowing 50 ml of peripheral whole blood from the liquid inlet into the cell separator prepared by the cell separation operation by a head (flow rate of about 5 ml / min), physiological saline was used to wash out red blood cells and platelets remaining in the filter. 30 ml was passed. Then, 30 ml of a 3.5% aqueous solution of polyvinylpyrrolidone (average molecular weight: 360,000)
Was passed through the liquid outlet at a rate of 100 ml / min using a pump, and cells were collected from the liquid inlet. The viscosity of the recovered liquid was 20.3 mPa · s. The leukocyte recovery rate, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 75%, 99%, and 98%, respectively. 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)

[0009]

Example 2 Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell separation operation 40% instead of 3.5% polyvinylpyrrolidone aqueous solution
The same operation as in Example 1 was performed except that a bovine serum albumin physiological saline solution was used. The viscosity of the recovered liquid is 1
It was 7.2 mPa · s. The leukocyte recovery rate, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 98, respectively.
%, 95% and 80%.

[0010]

Example 3 Production of Cell Separator The same cell separator as in Example 1 was used. Cell separation operation A commercial cryopreservative instead of a 3.5% aqueous solution of polyvinylpyrrolidone ("CP-1" manufactured by Far East Pharmaceutical Co., about 18% of hydroxyethyl starch, about 15% of dimethyl sulfoxide)
The same operation as in Example 1 was performed except for using. In addition,
The viscosity of the recovered liquid was 31.8 mPa · s. The leukocyte recovery rate, red blood cell removal rate, and platelet removal rate in this cell separation operation were 75%, 96%, and 88%, respectively. In addition,
The cells recovered with this recovery solution could then be cryopreserved according to the protocol attached to the cryopreservative described above.

[0011]

Example 4 Production of Cell Separator The same cell separator as in Example 1 was used. Cell separation operation The procedure was carried out except that a liquid obtained by adding bovine serum albumin to a commercially available aqueous solution of hydroxyethyl starch ("6-HES" manufactured by Luceru Morishita) to a concentration of 25% was used instead of the aqueous solution of 3.5% polyvinylpyrrolidone. The same operation as in Example 1 was performed. The viscosity of the recovered liquid is 17.4 mPa ·
s. The leukocyte recovery rate, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 74%, 96%, and 98%, respectively.
%Met. The cells recovered with this recovery solution could be cryopreserved by the same operation as the protocol attached to the cryopreservative of Example 3.

[0012]

Example 5 Preparation of Cell Separator Twelve polyester nonwoven fabrics having an average fiber diameter of 12 μm were placed on the outlet side of a polycarbonate container having a container size of 41 × 41 × 18 mm and a liquid outlet and a liquid inlet on a diagonal line, and an outlet side. Was filled with 25 pieces of mouse non-human CD34 monoclonal antibody-immobilized polyester nonwoven fabric having an average fiber diameter of 2.3 μm. The packing density of this filter was 0.2 g / cm ³ . The mouse anti-human CD34 monoclonal antibody was immobilized on polystyrene by a known haloacetamide method proposed in JP-A-2-261833. After passing 50 ml of umbilical cord whole blood from the liquid inlet into the cell separator prepared by the cell separation operation at a drop (flow rate of about 5 ml / min), rinsing red blood cells, platelets, and CD34 negative cells remaining in the filter. Then, 30 ml of physiological saline was passed. Thereafter, a 25% human serum albumin solution was added to a commercially available cryopreservation agent (“CP-1” manufactured by Far East Pharmaceutical Co., Ltd.) according to the instructions for use of the cryopreservation agent (12% hydroxyethyl starch, 10% dimethyl sulfoxide, 8% human serum albumin) from the liquid outlet using a pump to 100 ml /
And the cells were collected from the liquid inlet. The viscosity of the recovered liquid was 19.0 mPa · s. The CD34-positive cell recovery rate, CD34-positive cell purity, erythrocyte removal rate, and platelet removal rate in this cell separation operation are 80%, respectively.
93%, 98% and 98%. In addition, the cells collected with this collection solution could be cryopreserved by the protocol attached to the above-mentioned cryopreservative.

[0013]

Comparative Example 1 Preparation of Cell Separator The same cell separator as in Example 1 was used. Cell separation operation The same operation as in Example 1 was performed except that physiological saline was used instead of the 3.5% polyvinylpyrrolidone aqueous solution. The viscosity of the recovered liquid was 1.0 mPa · s. The leukocyte recovery rate, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 31%, 99%, and 95%, respectively, and the leukocyte recovery rate was low.

[0014]

Comparative Example 2 Preparation of Cell Separator The same cell separator as in Example 5 was used. Cell separation operation The same operation as in Example 5 was performed except that physiological saline was used instead of a commercially available cryopreservative to which human serum albumin was added. The viscosity of the recovered liquid is 1.0 mPa ·
s. The CD34-positive cell recovery rate, CD34-positive cell purity, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 10%, 73%, 99%, and 99%, respectively. Table 1 summarizes the results.

[0015]

As described above, the method for separating cells according to the present invention is capable of recovering the required cells from the mixture of the required cells and the unnecessary cells at a high rate with a simple operation and in a short time. Since the cell suspension can be cryopreserved without going through a complicated cell suspension preparation operation thereafter, it 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 (3)

[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 liquid having a viscosity of 2 mPa · s or more and 1000 mPa · s or less is introduced into the means, and the cells required for collection trapped by the means are separated from the means, and the cells required for collection are collected. Characteristic cell separation method. 2. The cell separation method according to claim 1, wherein a liquid having a viscosity of 2 mPa · s or more and 1000 mPa · s or less can be used as a preservative for the cells requiring recovery. 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 liquid having a viscosity of 2 mPa · s or more and 1000 mPa · s or less is introduced into the means, and a cell suspension comprising the cells requiring recovery and the liquid obtained by peeling and recovering.