JPH11335289A - Removal of blood platelet and cell composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for removing blood platelets from a nucleated cell-containing liquid at a high rate by a simple and short time process without using an expensive reagent. SOLUTION: This method for removing blood platelets comprises a process for transducing a nucleated cell-containing liquid containing at least the platelets and nucleated cells but substantially not containing erythrocyte into a cell- catching means to release the platelet from the cell-catching means, and a subsequent process for transducing the recovered liquid into the cell-catching means to recover the caught nucleated cells. The cell-catching means can catch the nucleated cells bus substantially allows the passage of the platelets.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a hematopoietic stem cell and / or hematopoietic progenitor cell (hereinafter, referred to as "hematopoietic stem cell") containing substantially no erythrocytes obtained by, for example, specific gravity centrifugation or hemagglutination sedimentation.
The present invention relates to a method for removing platelets from a liquid containing nucleated cells such as "hematopoietic stem cells") and lymphocytes, and a cell composition obtained thereby.

[0002]

2. Description of the Related Art Cord blood stem cells have attracted attention as a source of hematopoietic stem cell transplantation without donor invasion, and clinical applications thereof have been attempted mainly in Europe and the United States. Umbilical cord blood stem cells are rarely transplanted to patients immediately after being collected from a donor like other hematopoietic stem cell transplants, i.e., bone marrow transplants and peripheral blood stem cell transplants. (Especially for unrelated transplants). Japanese Patent Publication No. 8-69 discloses that after cord blood is cryopreserved,
It is disclosed that it can be thawed and used for transplantation. By the way, umbilical cord blood should be separated into mononuclear cells (removal of red blood cells) in order to prevent side effects of destructed erythrocytes after thawing and to reduce the volume during cryopreservation during cryopreservation. It is separated and preserved (Nankodo,
"Peripheral blood stem cell transplantation", p. 173). At present, the most frequently used method for removing erythrocytes from cord blood is a method of using a hydroxyethyl starch (hereinafter, abbreviated as "HES") to cause coinage formation in erythrocytes to remove aggregated sediment (for example, WO96 / 17514). Gazette). By the way, platelets are originally cells that carry out a hemostatic action, which is an important physiological function for a living body. However, when platelet utilization is not intended, its aggregation is often a problem. Aggregation of platelets is a phenomenon often observed after some kind of cell treatment or due to a change in temperature, but especially in hematopoietic stem cell transplantation, the generation of aggregates involving hematopoietic stem cells is a valuable loss of hematopoietic stem cells for transplantation. Must be avoided because it leads to In addition, since platelets release various physiologically active substances, it is feared that the target cells may be adversely affected during the cell treatment operation. By the way, in the above-mentioned HES method, red blood cells can be removed at a high rate, but platelets cannot be removed at a high rate. At the laboratory level, thrombin is added to aggregate and sediment platelets to remove platelets from the cell population.However, in an open system, washing and concentration by centrifugation must be repeated. , And the use of an expensive reagent called thrombin increases the cost. For these reasons, they are not at all available in routine clinical settings. As described above, in clinical practice, there is a long-awaited need for a practical level of platelet removal method for preventing precious cell loss for transplantation due to platelet aggregation.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for removing platelets from a nucleated cell-containing solution at a high rate by a simple and short-time operation without using an expensive reagent. .

[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 comprises a nucleated cell-containing liquid containing at least platelets and nucleated cells and substantially free of erythrocytes, the nucleated cells are captured, and the platelets are introduced into a cell capturing means that substantially passes through. From the cell-capturing means, and then recovering the captured nucleated cells by introducing a collection solution into the cell-capturing means. A nucleated cell-containing liquid containing cells and substantially free of red blood cells is captured by nucleated cells, and platelets are introduced into a cell capturing means that substantially passes through,
A cell composition obtained by a cell separation operation including a step of causing platelets to be led out from the cell capturing means, and then introducing a recovery liquid into the cell capturing means to recover the captured nucleated cells.

Hereinafter, the present invention will be described in detail. The nucleated cell as referred to in the present invention refers to a cell having a nucleus in the cell, and specifically, leukocytes, granulocytes, neutrophils, eosinophils, basophils, myelocytes, erythroblasts, lymphocytes Sphere, T lymphocyte, helper T lymphocyte, suppressor T lymphocyte, cytotoxic T
Lymphocytes, B lymphocytes, NK cells, NKT cells, monocytes,
Examples include macrophages, dendritic cells, hematopoietic stem cells, osteoclasts, osteoblasts, osteocytes, fibroblasts, chondroblasts and the like. Further, the nucleated cell-containing solution substantially free of red blood cells as referred to in the present invention is, for example, peripheral blood, bone marrow fluid, erythrocyte-removed liquid such as umbilical cord blood from which red blood cells have been removed by some artificial operation. There are artificial operations such as centrifugation (including continuous or intermittent centrifugal blood cell collection used for peripheral blood stem cell collection) and Ficoll-Hypaqu.
Specific gravity centrifugation using e, etc., coagulation sedimentation removal using HES or dextran, etc., and erythrocyte hemolysis treatment using ammonium chloride or the like. As used herein, substantially not containing red blood cells means that 60% or more of the red blood cells are removed from the blood before the artificial operation. The cell capturing means for capturing nucleated cells and substantially passing platelets as referred to in the present invention includes, for example, a container filled with a material for capturing nucleated cells and substantially passing platelets. The material capable of capturing nucleated cells and substantially passing platelets is, for example, a material coated with a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group proposed in WO 87/05812. can give. Here, any material can be used as the material of the carrier, and examples of preferable materials in terms of moldability, sterility and low cytotoxicity include polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, and poly. Synthetic polymers such as acrylamide and polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metals such as stainless steel, titanium, and aluminum can give. Examples of the shape of the carrier include a flat plate, a hollow fiber, a granule, a fiber mass, a woven fabric, a nonwoven fabric, a sponge-like porous body, and the like, but in view of a large surface area per volume, a granular shape, a fiber mass, a woven fabric, Nonwoven fabrics and spongy porous bodies are preferred. A container filled with a material that captures nucleated cells and platelets substantially pass through is preferably molded, sterilized, or low in cytotoxicity.
Examples include synthetic polymers such as polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metals such as stainless steel, titanium, and aluminum. As the recovering solution to be introduced into the cell capturing means for recovering nucleated cells according to the present invention, any physiological solution can be used, but some examples include physiological saline and Dulbecco's. Buffers such as phosphate buffer (D-PBS) and Hanks' solution (HBSS), RPMI164
0 and the like. If necessary, dextran, HES, albumin, globulin, glucose, saccharose, trehalose, etc. may be added to these solutions for the purpose of improving viscosity (may be effective for improving the recovery rate), protecting cells, supplementing nutrition, etc. Is also good.

In the present invention, a liquid containing nucleated cells containing at least platelets and nucleated cells and substantially free of red blood cells is introduced into a cell capturing means through which nucleated cells are captured and platelets substantially pass through. As a method, a bag or bottle containing a nucleated cell-containing liquid is connected to the cell capturing means via a tube, and a head, a roller pump, or a nucleated cell is introduced by crushing the bag to induce a liquid flow. What is necessary is just to connect a syringe containing the cell-containing solution, and to introduce the solution by manually pushing or using a syringe pump. When the nucleated cell-containing solution is introduced into the cell capturing means, the nucleated cells are captured in the means and platelets flow out of the means, but may remain slightly in the means. For the purpose of cleaning and removing, it is desirable to rinse the inside of the means. As the rinsing liquid, any physiological solution can be used, but some examples include physiological saline,
Buffers such as Dulbecco's salt buffer (D-PBS) and Hanks' solution (HBSS), and culture media such as RPMI1640. If necessary, dextran, HES, albumin, globulin, glucose, saccharose, trehalose, etc. may be added to these solutions for the purpose of cell protection, nutritional supplementation and the like. The direction in which the rinsing solution is introduced is preferably the same as the direction in which the nucleated cell-containing solution is introduced. In the opposite direction, nucleated cells may leak due to this rinsing operation. In the present invention, a method for collecting nucleated cells captured by the cell capturing means is performed by introducing the above-mentioned recovered solution into the cell capturing means. Then, a bag or a bottle containing the collected liquid is connected and the liquid is sent by a head, a roller pump, or a bag crusher, or a syringe containing the collected liquid is connected and the liquid is sent by a manual push or a syringe pump. At this time, the direction of feeding the collected liquid is as described above.
There are two directions, the same direction as the direction in which the nucleated cell-containing liquid is introduced, and the opposite direction, but the latter is generally preferable because the latter is higher. In the method for removing platelets according to the present invention, platelets derived from the cell capturing means are usually discarded, but may be used for platelet-related studies, platelet transfusion, and preparation of various release factors from platelets.

[0007] The cell composition according to the present invention refers to a centrifugal separation from a liquid originally containing erythrocytes such as peripheral blood, bone marrow fluid, umbilical cord blood, specific gravity centrifugation using Ficoll-Hypaque or the like, and coagulation sedimentation using HES or dextran. Removal, erythrocyte hemolysis using ammonium chloride, etc.
A nucleated cell-containing liquid substantially free of erythrocytes, from which erythrocytes have been removed by an artificial operation, is captured by nucleated cells, platelets are introduced into a cell capturing means that substantially passes, and platelets are collected by the cell capturing means. , And then obtained by a cell separation operation including a step of recovering the captured nucleated cells by introducing a recovery solution into the cell capturing means. Usually, the cell composition from which erythrocytes are artificially removed is obtained, for example, by centrifugation (for use in transplantation of allogeneic peripheral blood stem cells, granulocyte colony stimulating factor is administered to a healthy subject and then separated and collected by a centrifugal blood cell collection device). Stronsek, DF
Other: Transfusion, vol. 37, 411-
417, 1997) contains about 4 × 10 ¹⁰ nucleated cells and about 5 × 10 ¹¹ platelets, so that the ratio of nucleated cells to platelets is the number of nucleated cells / the number of platelets = 0.08. By the present cell separation operation, the concentration can be at least 0.1, usually 0.2 or more.

[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 Separation Filter Consisting of an upper container and a lower container, the inner dimension after assembly was 30
mm, width 30mm, height 12mm (effective filtration area 9c
m ² , inner volume of 11 cm ³ ), from the inlet side of a polycarbonate container having a liquid outlet and a liquid inlet on the longest diagonal line, the first layer has an average fiber diameter of 12 μm and a basis weight of 100 g / m 2.
The average fiber diameter of 2.3μm to 25 sheets ² of nonwoven polyester fabric for the second layer, non-woven polyester fabric 1 having a basis weight of 60 g / m ²
The two sheets were filled so as to be sandwiched between the upper container and the lower container, and separated into a container entrance space and an exit space. The porosity of the nonwoven fabric filled in the cell separation filter is 0.8.
1, packing density, first layer surface is 0.29 g / cm ^3, the second layer surface was 0.22 g / cm ^3. The hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer (molar ratio = 97: 3) 1
A 15 mL% ethanol solution was passed through the liquid inlet of the cell separation filter, excess polymer solution was purged with nitrogen gas, and then dried at 60 ° C. for 8 hours or more using a vacuum dryer. Cell Separation Procedure Red blood cells were removed from 50 mL of umbilical cord whole blood (nucleated cell count: 5.0 × 10 ⁸ , platelet count: 1.0 × 10 ¹⁰ ) by a known hemagglutination-sedimentation method using HES. After the resulting cell solution was passed through the liquid inlet of the cell separation filter created by the head to capture the nucleated cells, and the platelets were allowed to flow out, the platelets slightly remaining in the filter were also discharged from the liquid inlet. Rinse was performed by passing 30 mL of a commercially available saline solution through a head. Next, a liquid 50 obtained by adding human serum albumin to 3% to a 10% dextran physiological saline solution (“Dextran 40 Note” manufactured by Midori Cross Corporation).
A syringe was connected to the tube connected to the liquid outlet through the mL, and the liquid was passed by hand, and nucleated cells were collected from the liquid inlet. The time required for this cell treatment operation was about 6 minutes. Table 1 summarizes the results. The number of platelets and the number of nucleated cells were measured using an automatic blood cell counter (Hematology Analyzer manufactured by Coulter, Inc.), and cells that were positive for the CD34 antigen, a surface marker of hematopoietic stem cells (hereinafter, referred to as
CD34 ⁺ abbreviated as cells) Measurement of the content of nucleated cells of positive cells with FITC-labeled mouse anti-human CD34 antibody, Freund cytometry method to expand the SSC-FITC fluorescence intensity (Miyazaki, et al .: and clinical practice Blood, Volume 5 2
No. 21, pp. 24-24, 1995). The recovery rate, the removal rate, and the number of CD34 ⁺ cells were calculated by the following equations. Recovery rate (%) = 100 × (number of cells in recovered solution / number of original cells) Removal rate (%) = 100−100 × (number of cells in recovered solution / number of original cells) CD34 ⁺ cell number = CD34 ⁺ cell content (%) X number of nucleated cells / 100

[0009]

Example 2 Preparation of Cell Separation Filter The same cell separation filter as in Example 1 was used. Cell separation operation Continuous centrifugal blood cell collection device (AS10 manufactured by Fresenius)
Except for using 50 mL of the peripheral blood stem cell collection liquid (nucleated cell count: 6.1 × 10 ⁸ , platelet count: 5.0 × 10 ¹⁰ ) collected substantially in 4) which is substantially free of red blood cells, as an experimental sample The same operation and analysis as in Example 1 were performed. Table 2 summarizes the results.

[0010]

As described above, the method for removing platelets according to the present invention can remove platelets from a nucleated cell-containing solution at a high rate with a simple and short operation without using an expensive reagent. Therefore, it is extremely important to contribute to the field of cell medicine such as the field of hematopoietic stem cell transplantation.

Claims (3)

[Claims] 1. A nucleated cell-containing solution containing at least platelets and nucleated cells, and substantially free of erythrocytes, is captured by nucleated cells, and the platelets are introduced into a cell-trapping means that substantially passes through. From the cell capturing means, and then introducing a collecting solution into the cell capturing means to recover the captured nucleated cells. 2. A nucleated cell-capturing solution containing at least platelets and nucleated cells and substantially free of erythrocytes is captured by the nucleated cells, and the platelets are introduced into a cell capturing means that substantially passes through. From the cell-capturing means, and then introducing a collection solution into the cell-capturing means to recover the captured nucleated cells. 3. The ratio of the number of nucleated cells to the number of platelets is 0.
The cell composition according to claim 2, wherein the number is 1 or less.