JPH0923879A - Concentration of myelocyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To concentrate myelocytes for bone marrow transplantation, etc., in high efficiency by diluting myelocytes with a physiological saline solution, etc., introducing the obtained myelocyte suspension in a membrane-type plasma separator, separating the suspension into a cell-free liquid component and a cell component and recovering the separated cell component. SOLUTION: A blood bag filled with a myelocyte suspension obtained by diluting myelocytes with a physiological saline solution, etc., is connected to a membrane-type plasma separator 4 through a tube 3 provided with a clamp 2. The myelocyte suspension is introduced through the tube 3 into the membrane- type plasma separator 4 by the head by opening the clamp 2, the liquid component free from cell is permeated through the membrane and recovered to a waste liquid bag 5 and the cell component remained without passing through the membrane is recovered as it is in a recovery bag 6. The myelocyte for bone marrow transplantation, etc., can be concentrated in a short time with a simple operation without using a centrifugal separator.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention removes liquid components from bone marrow cells or peripheral blood mononuclear cells etc. (hereinafter simply referred to as "bone marrow cells") diluted with physiological saline, various buffers or media. And a method for enriching bone marrow cells.

[0002]

2. Description of the Related Art For hematopoietic disorders, which are the main side effects in chemotherapy of hematopoietic tumors such as leukemia and solid cancer,
Bone marrow transplant is widely practiced. Bone marrow transplantation is a method for recovering a fatal hematopoietic disorder caused by transplanted bone marrow, so that a patient can be given fatal high-dose radiation and / or high-dose chemotherapy, which leads to the cure of leukemia and solid cancer. Furthermore, in recent years, in peripheral blood or cord blood as well as bone marrow,
It has been revealed that hematopoietic stem cells and / or hematopoietic progenitor cells necessary for these treatments are contained. The transplants using these are officially referred to as peripheral blood stem cell transplant and cord blood stem cell transplant, respectively, but here, they are unified into the term bone marrow transplant. Bone marrow transplantation is divided into allogeneic transplantation (a healthy person is a donor of bone marrow) and autologous transplantation (using a patient's own bone marrow) depending on the relationship between the donor of bone marrow cells and the transplant patient. GVHD (graft-versus-host disease in allogeneic transplantation.
The biggest problem is a complication called "digestive system disorder, skin symptoms appearing" due to the immune reaction that the donor's lymphocytes attack the patient. In autologous transplantation, cancer cells are mixed because the patient's own bone marrow is used. Recurrence is a problem. With the recent progress of biotechnology, a cell separation instrument for separating cells by an antigen-antibody reaction using a monoclonal antibody has been developed, and cell isolation from the collected bone marrow using the instrument (purpose: GVHD in allogeneic transplantation). Removal of T lymphocytes that cause the above or selective collection of hematopoietic stem cells,
In autologous transplantation, removal of cancer cells or selective collection of hematopoietic stem cells) is being actively studied. In such cell separation, usually, bone marrow cells are diluted with a separation liquid (physiological saline, buffer solution, medium, etc.) for cell separation operation. Therefore, the recovered product after the separation contains a large amount of the separation liquid. Therefore, the separation liquid is removed from the collected material for the purpose of reducing the load on the circulatory organ at the time of transplantation and improving the handleability. Usually, a centrifuge is used for this operation, and in many cases, it is performed by aliquoting the centrifuge into a centrifuge tube and then sucking out the supernatant (separation liquid) with a pipette. . These operations must be performed aseptically (excluding centrifuge),
It is extremely complicated and must be very skilled to avoid contamination. In order to overcome these problems, a method is also used in which the blood bag is placed in a large-scale centrifuge, the blood bag is connected by an aseptic tube connector, the supernatant is drained, and the remaining cells are used. However, this still requires expensive large centrifuges and aseptic connectors for tubes,
Further, the centrifugation operation for about 30 minutes once is still necessary.

On the other hand, ordinary bone marrow transplantation in which cells are not separated by these cell separation instruments is, of course, widely practiced. In allogeneic transplantation, if the donor and patient have different blood groups, it is necessary to remove red blood cells from the donor's bone marrow.
Also, in autologous transplantation, bone marrow is kept frozen until the time of transplantation, but it is also necessary to remove red blood cells to prevent hemolysis during thawing. To remove red blood cells, a centrifugation method called a density gradient centrifugation method using a specific gravity liquid (for example, Ficoll manufactured by Pharmacia) is used. Specific gravity fluid remains in the red blood cell-removed bone marrow separated by this method, and since this specific gravity fluid is harmful, it is also necessary to wash and remove it with a centrifuge. Since the centrifugal separation operation is accompanied by such problems, a simple method for separating a liquid has been desired.

[0004]

As described above, in the bone marrow transplantation, removal of the separating liquid, that is, concentration of bone marrow cells is carried out by using a centrifuge, but the operation is complicated and contamination is not caused. However, cost, length of processing time, etc. have become problems. An object of the present invention is to provide a method for concentrating bone marrow cells, which can be separated easily in a short time and at low cost without using a centrifuge.

[0005]

Means for Solving the Problems As a result of intensive studies by the present inventors, when a bone marrow cell suspension in which bone marrow cells were diluted with physiological saline was introduced into a membrane plasma separator, the It has been found that the liquid component and the cell component that do not contain can be separated and the cell component can be collected in a blood bag. Further, the separation time was shorter than that of centrifugation, and the operation was extremely simple. That is, the present invention introduces a bone marrow cell suspension in which bone marrow cells are diluted with physiological saline or the like into a membrane plasma separator, separates a cell-free liquid component and a cell component, and recovers the cell component. It is a characteristic method for enriching bone marrow cells. The membrane plasma separator used in the present invention is not limited in terms of membrane material and membrane shape as long as it can efficiently separate a liquid component and a cell component. Cellulose acetate, polyvinyl alcohol, polymethylmethacrylate type Although a flat membrane or hollow fiber membrane made of resin, polyethylene, polypropylene, polysulfone, polyacrylonitrile, or the like can be used, it is preferable that priming / washing operation with a physiological solution is unnecessary before use. It is most desirable that the liquid component and the cell component can be completely separated, but the liquid may contain some cells. The liquid components removed by the membrane plasma separator include physiological saline and Dulbecco's phosphate buffer (hereinafter referred to as D-
Examples thereof include buffer solutions such as PBS) and culture media such as RPMI, but these are determined by the cell separation instrument used and are not limited. For example,
D-PBS containing EDTA, heparin, and human gamma globulin is used in the cell separation device "Selector T cell" of Applied Immun Sciences,
RPMI to which human serum albumin is added is used in the cell separation instrument “Isolex” manufactured by Baxter. Also, the cellular component separated from the liquid component according to the present invention,
Depending on the cell separation device used, for example, "selector T cell" is bone marrow from which T lymphocytes have been removed,
"Isolex" is bone marrow purified into hematopoietic stem cells (CD34-positive cells). That is, in the bone marrow cell suspension that is the target of bone marrow cell concentration according to the present invention, cells purified by these various cell separation instruments are physiological saline, D-
It is diluted with a liquid component such as a buffer solution such as PBS and a medium such as RPMI.

[0006]

Embodiment 1 One example of an embodiment of the present invention will be described with reference to the drawings. FIG. 1 shows the usage state. The cell suspension collected by "Selector T cell" or "Isolex" is contained in the blood bag (1). For example, in the case of “Selector T cell”, about 2 × 10 ⁹ bone marrow cells from which T lymphocytes have been removed by this cell separation device are 530 ml of a separation liquid (EDTA, heparin, human gamma globulin in D-PBS). ) Is a cell suspension diluted with. The clamp (2) is opened, and the cell suspension is introduced into the membrane plasma separator (4) by a drop through the tube (3). As the membrane type plasma separator, for example, BCC-I manufactured by Asahi Medical Co., Ltd. is used. This separator is hydrophilic polyethylene resin, an effective membrane area of 0.1 m ² membrane 23
It is enclosed in a polycarbonate container of mmφ × 143 mm. The liquid component is filtered by the membrane and is collected in the drainage bag (5), and the cell component is directly collected in the collection bag (6) without passing through the membrane. In the case of the above-mentioned "selector T cell", the separation time was about 10 minutes, which was far shorter than the time required for the centrifugation operation (about 30 minutes). The next operation of the collected cells differs depending on the use. For example, when used for allograft transplantation, a port of the collection bag (6) is pierced with a needle of a syringe, sucked and taken out, and then intravenously injected into a patient. At that time, usually, the cells are not left as they are, but they are slightly diluted with a small amount of physiological saline and intravenously injected. When it is used for autologous transplantation, it is similarly aspirated with a syringe and taken out, but it is very rare that the patient is intravenously injected immediately thereafter, and most of them are cryopreserved. That is, the taken out cells are put in a freezing bag or a freezing tube together with a cryoprotective agent and frozen and stored in liquid nitrogen until use.

[0007]

INDUSTRIAL APPLICABILITY The method for concentrating bone marrow cells according to the present invention can separate a liquid component and a cell component without using a centrifuge, the separation time is shorter than that of centrifugation, and the operation is extremely simple. .

[Brief description of drawings]

FIG. 1 is an explanatory diagram of an embodiment.

[Explanation of symbols]

 1 Blood Bag 2 Clamp 3 Tube 4 Membrane Plasma Separator 5 Drainage Bag 6 Collection Bag

Claims (1)

[Claims] 1. A method in which a bone marrow cell suspension obtained by diluting bone marrow cells with physiological saline or the like is introduced into a membrane-type plasma separator and separated into a cell-free liquid component and a cell component, and the cell component is recovered. And a method for concentrating bone marrow cells.