JP4412621B2 - Cell separation method - Google Patents

Description

【００１５】
【発明の効果】
以上示したように、本発明による細胞分離方法は簡便な操作且つ短時間で、必要細胞と不要細胞の混合物から必要細胞を高率に回収することができ、また得られた細胞浮遊液はその後の煩雑な細胞浮遊液調製操作を経ることなく凍結保存が可能なので、造血幹細胞移植分野や養子免疫療法分野の細胞処理工程における省力化に貢献するところ大である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the separation of how to isolate a particular cell from a cell population.
[0002]
[Prior art]
Bone marrow transplantation 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 recovery from fatal hematopoietic damage caused by transplanted bone marrow, so that it is possible to carry out high-dose radiation and / or high-dose chemotherapy (hereinafter abbreviated as high-dose chemotherapy) that is fatal to patients. It leads to the cure of solid cancer. In recent years, it has been clarified 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 them and use them instead of bone marrow transplantation, but anticancer agents and / or G-CSF (granulocyte colony stimulating factor) It has been clarified that the content rate is increased by administering cytokines such as the above, and since general anesthesia is unnecessary and safe compared with bone marrow collection, it has been actively applied clinically. In recent years, it has become clear that cord blood contains hematopoietic stem cells at a much higher concentration than peripheral blood, and clinical application has begun. Here, transplantation methods are classified into allogeneic transplantation and autologous transplantation depending on who is the donor (donor) of the cells. In the former, a healthy other person (related or unrelated) is a donor, and in the latter, the patient is a donor. In autotransplantation, and in allogeneic transplantation, most cases are cryopreserved until transplantation when cord blood is used. Prior to cryopreservation, red blood cells are usually removed. This is because, when stored in whole blood, side effects due to storage space and destroyed red blood cells during thawing become a problem. Conventionally, erythrocyte removal has been performed by a centrifuge, and a specific gravity centrifuge method using a specific gravity liquid (for example, Ficoll manufactured by Pharmacia) is employed to increase the separation efficiency. This method is a cumbersome operation requiring very skill, for example, the liquid surface should not be disturbed when the raw material cells are layered on the specific gravity solution. Many attempts have been made in JP 61-84577 A, JP 2-134564 A, etc. to solve the troublesome operation, but there is no change in using a specific gravity liquid, and it is a radical solution. Has not reached. By the way, there have been some proposals of a method for removing red blood cells without using centrifugation. In JP-A-8-104643, a cell population containing erythrocytes and hematopoietic stem cells and / or hematopoietic progenitor cells is passed through a filter that substantially passes erythrocytes and leukocytes are captured. There has been proposed a method for removing red blood cells, which induces a liquid flow in the direction and collects the captured white blood cells. However, there is no description that the recovery rate is improved when a recovery liquid having a specific viscosity is used.
By the way, Japanese Patent Application Laid-Open No. 55-130917 is characterized in that a liquid having a viscosity of 2 centipoise (CP) or more is used as a body fluid extrusion liquid when collecting body fluid remaining in a filter, an adsorbent packed column or the like from a container. A method for recovering body fluid is disclosed. However, according to the publication, it is not preferable to use a low viscosity such as physiological saline because the cells adhered to the filter are also recovered. Therefore, a high viscosity is used in order not to collect the adherent cells. That is, in this publication, it is a method for recovering the body fluid that has almost passed through the filter and remained slightly in the filter, which is a technical idea completely different from the present application.
[0003]
[Problems to be solved by the invention]
In view of the above-mentioned problems of the prior art, the present invention is a method for recovering necessary cells at a high rate from a mixture of necessary cells and unnecessary cells in a simple operation and in a short time. For example the intended white blood cells, platelets, and to provide a way to selectively high rate recovery of white blood cells from a mixture of red blood cells.
[0004]
[Means for Solving the Problems]
The present inventors have completed the present invention as a result of intensive studies to solve the above-mentioned problems. That is, the present invention captures leukocytes or CD34-positive cells from bone marrow, peripheral blood, umbilical cord blood or a cell population obtained by roughly separating these cells, which contains leukocytes or CD34-positive cells and erythrocytes and platelets. And then introduced into a filter filled with a nonwoven fabric in a container, and then introduced into the filter a liquid having a viscosity of 5 mPa · s or more and 31.8 mPa · s or less in a direction opposite to the flow direction of the cell population , The cell separation method is characterized in that the leukocytes or CD34-positive cells captured by a filter are detached from the filter and the leukocytes or CD34-positive cells are collected. Further, in the cell separation method, a liquid having a viscosity of 5 mPa · s or more and 31.8 mPa · s or less can be used as a preservative for the leukocytes or CD34 positive cells.
[0005]
[Action]
In the cell separation method of the present invention, by using a liquid having a specific viscosity of 5 mPa · s or more and 31.8 mPa · s or less as a collection liquid, leukocytes or CD34 positive cells captured by the capture means are highly recovered. In addition, it can be peeled and collected in a short time with a simple operation. Furthermore, a cell suspension obtained by using a cryopreservation solution as a recovery solution having a specific viscosity can be cryopreserved as it is.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the capturing means used in the present invention include a container filled with a capturing material. Any material can be used as the capturing material as long as it is insoluble in water. Examples of preferable materials in terms of low moldability, sterility, and cytotoxicity include polyethylene, polypropylene, polystyrene, acrylic resin, nylon. Synthetic polymers such as polyester, polycarbonate, polyacrylamide, polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, alginate, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel, titanium And the like. In addition, these capture materials can be used as they are, but if necessary, ligands having affinity for specific cells such as amino acids, peptides, glycoproteins (including bioligands such as antibodies and adhesion molecules) are immobilized. May be. Examples of the shape of the capturing material include granular, fiber mass, woven fabric, non-woven fabric, flat plate, sponge-like porous body, and the like, but a non-woven fabric is preferable in terms of a large surface area per volume.
Examples of the cell population containing at least leukocytes or CD34 positive cells and erythrocytes and platelets in the present invention include bone marrow, peripheral blood, umbilical cord blood, or those obtained by roughly separating them with a centrifugal separator or the like. The cells trapped in the cell are collected with a liquid having a specific viscosity, and this viscosity is 5 mPa · s or more and 31.8 mPa · s or less. When the viscosity is less than 5 mPa · s, the recovery rate is low, and when it exceeds 31.8 mPa · s, workability is inferior. As the component, those having little adverse effect on cells are preferable. Some examples include synthetic polymer solutions such as polyethylene glycol, polyvinyl pyrrolidone and polyvinyl alcohol, natural polymer solutions such as methyl cellulose, gelatin, hydroxyethyl starch, dextran, chitin derivatives, collagen, fibronectin, albumin and globulin, glucose and saccharose. , Organic solutions such as maltose, sorbitol, glycerin, dimethyl sulfoxide, and mixtures thereof. Examples of the solvent for dissolving them include physiological saline, buffer solutions such as D-PBS (Dulbeccoline buffer solution) and HBSS (Hanks solution), and culture media such as RPMI 1640.
[0007]
Moreover, it is more preferable that the liquid having a specific viscosity according to the present invention is used for freezing or liquid storage as it is. That is, taking cryopreservation of stem cells as an example, usually after washing the cell population from which red blood cells have been separated by the Ficoll method described above, a cryopreservation agent is added to prepare a cell suspension, which is then stored in liquid nitrogen. Alternatively, cryopreservation is performed in a freezer. In the present invention, a cryopreservation agent is used for a liquid having a specific viscosity, so that a cell suspension for cryopreservation can be obtained without adding a complicated operation after removing red blood cells. Can do.
Examples of the method of passing a liquid having a specific viscosity through the capturing means according to the present invention include use of a pump, injection by a syringe, crushing a bag storing the liquid to induce a liquid flow, and a method using a drop. In addition, in the case of a capturing means consisting of separate containers for the liquid inlet and the liquid outlet, it can be divided into whether the recovered liquid is passed in the same direction as the flow direction of the raw blood or in the reverse direction. Generally, the recovery rate tends to be higher in the reverse direction. Furthermore, not only the liquid may be simply passed, but vibration may be applied to the capturing means or a stopped flow may be used. In addition, 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 mechanical / superfluous. Cells are collected by applying sonic vibration.
[0008]
【Example】
EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
[Example 1]
Production of 1 cell separator Twenty- five polyester nonwoven fabrics having an average fiber diameter of 12 μm 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, and an average fiber diameter on the outlet side 12 sheets of 2.3 μm polyester nonwoven fabric were filled. The filter packing density was 0.2 g / cm ³ .
In addition, for the purpose of imparting platelet permeability to this filter, a hydrophilic polymer was coated. That is, a 1% ethanol solution of a hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer was passed through the liquid inlet of the filter and then dried with nitrogen gas.
2- cell separation operation
After passing 50 ml of peripheral whole blood through the cell separator prepared in 1 through a drop (flow rate of about 5 ml / min) from the liquid inlet, 30 ml of physiological saline was added for the purpose of washing out the remaining red blood cells and platelets. The liquid was passed. Thereafter, 30 ml of a 3.5% polyvinylpyrrolidone (average molecular weight 360,000) aqueous solution was passed through the liquid outlet at a rate of 100 ml / min using a pump, and the cells were recovered from the liquid inlet. The recovered liquid had a viscosity of 20.3 mPa · s. The leukocyte collection rate, red blood cell removal rate, and platelet removal rate in this cell separation operation were 75%, 99%, and 98%, respectively. The method for calculating the recovery rate and removal rate is as follows.
Recovery rate (%) = 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]
(1) Production of cell separator The same cell separator as in Example 1 was used.
(2) Cell separation operation The same operation as in Example 1 was carried out except that a 40% bovine serum albumin physiological saline solution was used instead of the 3.5% polyvinylpyrrolidone aqueous solution. The recovered liquid had a viscosity of 17.2 mPa · s. The leukocyte collection rate, red blood cell removal rate, and platelet removal rate in this cell separation operation were 98%, 95%, and 80%, respectively.
[0010]
[Example 3]
(1) Production of cell separator The same cell separator as in Example 1 was used.
(2) Cell separation operation Examples were used except that a commercially available cryopreservation agent (“CP-1” manufactured by Kyokuto Pharmaceutical Co., Ltd., about 18% hydroxyethyl starch, about 15% dimethyl sulfoxide) was used instead of the 3.5% polyvinylpyrrolidone aqueous solution. The same operation as 1 was performed. The viscosity of the recovered liquid was 31.8 mPa · s. The leukocyte collection rate, red blood cell removal rate, and platelet removal rate in this cell separation operation were 75%, 96%, and 88%, respectively. The cells recovered with this recovery solution could then be cryopreserved according to the protocol attached to the aforementioned cryopreservation agent.
[0011]
[Example 4]
(1) Production of cell separator The same cell separator as in Example 1 was used.
(2) Cell separation operation Instead of a 3.5% polyvinylpyrrolidone aqueous solution, a liquid obtained by adding bovine serum albumin to 25% to a commercially available hydroxyethyl starch physiological saline solution (Russel Morishita “6-HES”) is used. Except that, the same operation as in Example 1 was performed. The recovered liquid had a viscosity of 17.4 mPa · s. The leukocyte collection rate, red blood cell removal rate, and platelet removal rate in this cell separation operation were 74%, 96%, and 98%, respectively. The cells recovered with this recovery solution could then be cryopreserved by the same operation as the protocol attached to the cryopreservation agent of Example 3.
[0012]
[Example 5]
Preparation of 1 cell separator 12 polyester nonwoven fabrics with an average fiber diameter of 12 μm 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, and an average fiber diameter on the outlet side 25 sheets mouse anti-human CD34 monoclonal antibodies secured polystyrene nonwoven 2.3μm was filled. 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.
2- cell separation operation
In order to wash out red blood cells, platelets, and CD34 negative cells remaining in the filter after passing 50 ml of umbilical cord whole blood through the cell separator prepared in 1 with a drop (flow rate of about 5 ml / min) from the liquid inlet. 30 ml of saline solution was passed through. Thereafter, a commercially available cryopreservation agent (“CP-1” manufactured by Kyokuto Pharmaceutical Co., Ltd.) added with a 25% human serum albumin solution according to the instructions for use of the cryopreservation agent (12% hydroxyethyl starch, 10% dimethyl sulfoxide, 8% human serum albumin) was passed through the liquid outlet at 100 ml / min using a pump, and the cells were collected from the liquid inlet. The recovered liquid had a viscosity of 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 were 80%, 93%, 98%, and 98%, respectively. The cells recovered with this recovery solution could be cryopreserved according to the protocol attached to the aforementioned cryopreservation agent.
[0013]
[Comparative Example 1]
(1) Production of cell separator The same cell separator as in Example 1 was used.
(2) Cell separation operation The same operation as in Example 1 was carried out except that physiological saline was used in place of the 3.5% polyvinylpyrrolidone aqueous solution. The recovered liquid had a viscosity of 1.0 mPa · s. The leukocyte collection rate, erythrocyte removal rate, and platelet removal rate in this cell separation operation were 31%, 99%, and 95%, respectively, and the leukocyte collection rate was low.
[0014]
[Comparative Example 2]
(1) Production of cell separator The same cell separator as in Example 5 was used.
(2) Cell separation operation The same operation as in Example 5 was carried out except that physiological saline was used in place of the commercially available cryopreservation agent added with human serum albumin. The recovered liquid had a viscosity of 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.
Table 1

[0015]
【The invention's effect】
As described above, the cell separation method according to the present invention can recover necessary cells from a mixture of necessary cells and unnecessary cells at a high rate in a simple operation and in a short time. Therefore, it can be cryopreserved without going through the complicated cell suspension preparation procedure, which greatly contributes to labor saving in the cell treatment process in the field of hematopoietic stem cell transplantation and adoptive immunotherapy.

Claims (2)

Bone marrow, peripheral blood, umbilical cord blood containing leukocytes or CD34 positive cells and erythrocytes and platelets or a cell population obtained by roughly separating them is captured in the vessel, and the red blood cells and platelets substantially pass through the container. A liquid having a viscosity of 5 mPa · s or more and 31.8 mPa · s or less is introduced into the filter filled with the nonwoven fabric in the direction opposite to the flow direction of the cell population , and is captured by the filter. A method for separating cells, comprising separating the leukocytes or CD34 positive cells from the filter and collecting the leukocytes or CD34 positive cells.   The cell separation method according to claim 1, wherein a liquid having a viscosity of 5 mPa · s to 31.8 mPa · s can be used as a preservative for the leukocytes or CD34 positive cells.