JPH09322757A - Cell separating purification material, cell separating purifier and cell separating purification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separating purification material, cell separating purifier and a method for cell separating purification to carry out positive and negative selection with one device. SOLUTION: This cell separating purification material comprises a water- insoluble carrier having a first and a second ligands on the same surface with a relatively large force to cells that express both a first and second markers as compared with a capturing force to cells that express the first marker but does not express the second marker. The cell separating purifier is a vessel that is provided with a liquid inflow opening and a liquid outflow opening, filled with a cell separating purification material and sterilized. The method for cell separating purification exfoliates and recovers only cells that express the first marker but does not express the second marker from the cell separating purification material.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell separation and purification material, a cell separation and purification device, and a cell separation and purification method for separating and purifying specific cells from a cell population. More specifically, in cell separation performed using two markers used in immunology, hematology, cell biology, etc., namely, expression of both 2 markers, expression of only 1 marker, and expression of a cell population not expressing 2 markers It relates to a method for separating cells expressing only a marker.

[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 for recovering a fatal hematopoietic disorder caused by transplanted bone marrow, so that it is possible to perform fatal high-dose radiation and / or high-dose chemotherapy (hereinafter referred to as “high-dose chemotherapy”) for patients, and leukemia. And solid cancer treatment. Further, 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 peripheral blood is quite low, making it difficult to collect and use in place of bone marrow transplantation,
It has been clarified that the content of cytokines is increased by administering anti-cancer agents and / or cytokines such as G-CSF (granulocyte colony stimulating factor), and compared with bone marrow collection, general anesthesia is unnecessary and safe. , Is being clinically applied. 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. Hereinafter, in the present specification, transplantation using peripheral blood and cord blood is also represented by the term bone marrow transplantation.

[0003] Here, transplantation methods are classified into allogeneic transplantation and autotransplantation depending on who obtains the cells. The former is one in which a healthy other person (related or unrelated) serves as the cell donor (donor), and the latter uses the patient's own cells. In any of the transplantation methods, the donor is heavily invaded in order to obtain the required amount of cells for transplantation. That is, when bone marrow is used, general anesthesia is required, and when peripheral blood is used, general anesthesia is not required, but an anticancer agent and / or G-CSF is required.
It is necessary to administer such cytokines for several days and collect by apheresis for several hours. On the other hand, in the case of umbilical cord blood, it can be said that there is no donor invasion at all, but the amount of cells that can be obtained is limited, so that transplantable patients are limited to children. In order to solve these problems, that is, in order to enable transplantation in bone marrow by collecting only local anesthesia, and also in transplanting cord blood in adults, attempts to expand hematopoietic stem cells by in vitro culture have recently been attempted. , Very active (Tokuheiyo Hyo 4-506153, Tokuhyo Hyo 6-5)
08987, Tokushuhei 7-504570 and many others). In the culture, since hematopoietic stem cells are present in the mononuclear cell fraction, initially, mononuclear cells separated by a centrifugation method using a density gravity centrifugation method (for example, Ficoll manufactured by Pharmacia) are used as raw material cells. Was used for. A high amplification factor was obtained in this culture method for mouse cells, but not so good for human cells. Therefore, recently, cells purified to CD34-positive cells have been used as starting cells. As a result, not only the improvement of the amplification factor is seen and not only the CD34 is separated as an index, but also
Furthermore, various markers (CD15, CD38, HLA-D
It is more preferable to purify the cells with R or the like, and thus, the culture using these purified cells as the starting cells is actively carried out (Tatsutoshi Nakahata: “In vitro of hematopoietic stem cells”).
Ro amplification ”, 1995, Nankodo“ Peripheral blood stem cell transplant ”
Pp. 176-184). Ibid and JP-A-5-7635.
4, among them, CD34-positive and CD38-negative cells are the most undifferentiated stem cells, and it is most preferable to use them.
Heretofore, in order to obtain these cells, for example, the following extremely complicated operation involving many steps has been performed. That is,
Separate the mononuclear cell fraction from the bone marrow by the density gradient centrifugation method described above,
Then, a known CD34-positive cell separation method, for example, "Microselector CD" manufactured by Applied Immun Science Co., Ltd.
34 "to separate CD34-positive cells (positive selection). The obtained CD34-positive cells are used as anti-human CD3
8 Mouse monoclonal antibody is used for labeling, and CD38-positive cells are removed by the indirect magnetic bead method (for example, Dynabeads manufactured by Dynal) (negative selection). All of the experimental instruments and reagents used for these operations are not sterilizable, and although they are performed aseptically in a clean bench, they are non-closed systems, so there is a risk of bacterial contamination. By the way, by using a so-called cell sorter, CD34-positive C
Although D38-negative cells can be obtained, the cell sorter is a very expensive device and it is still a non-closed system operation. From the above, there has been a great demand for the development of a method capable of safely separating and purifying cells with a small number of steps without using a simple and expensive device.

[0004]

DISCLOSURE OF THE INVENTION The present invention is a method for separating and purifying a specific cell from a cell population, which is simple and does not use an expensive device, and is safe and has a small number of steps, that is, positive selection and negative selection. An object of the present invention is to provide a cell separation / purification material, a cell separation / purification device, and a cell separation / purification method that can be performed with various types of instruments.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present invention has been completed. That is, the present invention relates to a ligand having a selective affinity for the first marker (hereinafter referred to as “first ligand”) and a ligand having a selective affinity for the second marker (hereinafter referred to as “first ligand”). Second ligand ”) on the same surface and has a first capturing power for cells expressing both the first marker and the second marker.
Expressing the first marker, but expressing the first marker, but expressing the second marker, characterized in that it has a relatively higher trapping power for cells that do not express the second marker. The present invention relates to a cell separation and purification material for separating and purifying cells that have not been treated. Further, the present invention relates to a cell separation and purification device, which is obtained by filling the cell separation and purification material in a container having a liquid inlet and a liquid outlet and then sterilizing the container. Furthermore, a cell liquid containing cells expressing at least the first marker is brought into contact with a cell separation / purification material comprising a water-insoluble carrier having a first ligand and a second ligand on the same surface, Cells expressing the second marker and cells expressing the second marker were captured by the separation / purification material, and then cells expressing the second marker were captured from the captured cells. The present invention relates to a method for separating and purifying cells, characterized in that only cells expressing the first marker but not expressing the second marker are separated from the separating and purifying material and recovered. The term "marker" as used in the present invention refers to a protein that is present on the surface of a cell and serves as an index for identifying the cell, a saccharide, a glycoprotein, or a peptide, and specifically, an antigen or an adhesion molecule such as selectin or integrin. , Interleukin 6 receptor (hereinafter referred to as “IL6R”)
,), G-CSF receptor, c-kit and other receptors.

As the first ligand of the present invention, the above-mentioned marker, that is, a cell surface antigen, an adhesion molecule, a monoclonal antibody against a receptor or a peptide fragment having a specific amino acid sequence constituting the variable region of this antibody is used. can give. In the case of adhesion molecules and receptors, those that are the original binding partners under physiological conditions, specifically, ICAM for integrin, GlyCAM-1 for L-selectin, and so-called ligand-receptor related ones. To be The cell antigen is not particularly limited and may be appropriately selected depending on the purpose.
For example, in hematopoietic stem cells the CD34 antigen is preferred and in T lymphocytes the CD3 antigen is preferred. Similarly, the adhesion molecule and the receptor are appropriately selected according to the purpose.
For example, in hematopoietic stem cells, c-kit is preferable.
The second ligand of the present invention is, like the first ligand, a monoclonal antibody against a cell surface antigen, an adhesion molecule, a receptor, or a peptide fragment having a specific amino acid sequence constituting the variable region of this antibody. To be Further, in the case of adhesion molecules and receptors, those that are original binding partners under physiological conditions, specifically, ICAM for integrin, Gly CAM-1 for L-selectin, and so-called ligand-receptor related ones. can give. For example, in hematopoietic stem cells CD2, CD3, CD4, CD5, CD6, CD
7, CD8, CD9, CD10, CD14, CD15,
CD19, CD20, CD33, CD38, HLA-D
An antibody against each antigen such as R and an antibody against IL6R can be mentioned. Among them, more undifferentiated hematopoietic stem cells are CD34-positive CD33-negative, CD34-positive CD38-negative, CD34-positive IL6R-positive and CD34-positive HLA-.
Since it is said to be DR negative, CD33, CD3
More preferred are antibodies against 8, HLA-DR, IL6R.

As the material of the water-insoluble carrier used in the present invention, any material can be used as long as it is water-insoluble.
The preferred ones in terms of low moldability and low cytotoxicity are synthetic polymer compounds such as polystyrene, polyethylene, polypropylene, nylon, acrylic resin, fluororesin, and polycarbonate, and natural polymers such as cellulose, chitin, dextran, and agarose. Molecular compound,
Examples thereof include ceramics such as alumina, hydroxyapatite, titania and silica, and inorganic compounds such as glass. Examples of the shape of the base material include flat plates, beads, fibers, non-woven fabrics, and porous bodies, but beads, fibers, non-woven fabrics, and porous bodies are preferable because they have a large surface area per volume. The cell separation and purification material according to the present invention comprises the first ligand and the second ligand immobilized on the same surface of a water-insoluble carrier. Fixation is a well-known panning method,
That is, it is achieved by contacting the ligand solution with the carrier for several hours, but when it is desired to strengthen the binding between the ligand and the carrier and / or to reduce the amount of the ligand used, it is disclosed in, for example, JP-A-2-261833. The known fixing method using a chemical bond may be used.

The cell separation / purification device according to the present invention is obtained by filling the above-mentioned cell separation / purification material into a container having a liquid inlet and a liquid outlet (which may serve as both) and then sterilized. The material of the container is not particularly limited, but if molding, mechanical strength, and stability in sterilization are listed, synthetic polymers such as polystyrene, polyethylene, polypropylene, nylon, acrylic resin, fluororesin, and polycarbonate are listed. Examples thereof include compounds and metals such as stainless steel, titanium, aluminum and alloys thereof. Examples of the sterilization method include high-pressure steam sterilization, radiation sterilization, ethylene oxide gas sterilization, etc., and a sterilization method suitable for a member to be used is appropriately selected. According to the present invention,
One type of cell separation and purification device can collect cells in which the first marker is positive and the second marker is negative. This is because the cell separation and purification material of the present invention has the ability to capture cells in which both the first marker and the second marker are expressed, but the first marker is expressed but the second marker is not expressed. This is because it is relatively higher than the trapping power for cells, but as a method for achieving this, the value of the ratio of the density of the ligands on the surface of the second ligand density / the first ligand density is larger than 1. Using the difference in affinity of antigen-antibody interaction, the method of immobilizing the first ligand on the substrate is weakly bound by physical adsorption by the panning method, and the method of immobilizing the second ligand is strongly bound by covalent bonding. Introducing a spacer into the first ligand (or introducing a spacer into both ligands, but introducing the spacer length of the spacer into the first ligand into the second ligand) Longer than the length in) can be achieved with such weakening the trapping force. As the spacer to be introduced, polyethylene glycol or the like which has been conventionally suitably used as a spacer can be used.

Further, in the cell separation / purification method according to the present invention, at least the first marker is expressed in a cell separation / purification material comprising a water-insoluble carrier having a first ligand and a second ligand on the same surface. A cell liquid containing cells that are in contact with each other to capture the cells expressing the first marker and the cells expressing the second marker in the separation and purification material, and then from the captured cells The cells expressing the second marker are left trapped, and only the cells expressing the first marker but not the second marker are separated from the separation and purification material and recovered. However, in order to achieve this, the above-mentioned trapping power for cells expressing the first marker, which also expresses the second marker together, causes the second marker to express together. Not the first mar In addition to the method of using a cell separation / purification device having a relatively higher capture power for cells expressing -1), the binding between the first ligand and the cells captured by the ligand is cleaved, but the second ligand is separated. A method in which a reagent that does not cleave the bond between the ligand and cells captured by the ligand is added to the recovery solution. 2) The binding site of the cell that binds to the first ligand is previously coated with a substance that partially inhibits the binding. There are ways to keep it. Examples of the reagent used in the method 1) include enzymes such as chymopapain, and examples of the substance used in the method 2) include CD in the case of hematopoietic stem cells.
Monoclonal antibody against 34 antigen, anti-selectin-
Monoclonal antibodies against adhesion factors such as P antibody are mentioned.

The cells separated and purified according to the present invention are used depending on the purpose. That is, to give an example of hematopoietic stem cells, necessary operations such as transplantation to a patient, gene transfer, etc., are performed as they are, or after being thawed after cryopreservation and then thawed and then infused into the patient, or after being in vitro cultured and amplified. After that, it is transplanted to the patient and contributes to the recovery of the hematopoietic function of the patient. Alternatively, it is used for the production of differentiated blood cells such as the production of platelets disclosed in JP-T 5-502385. In the example of the field of immunity, in T lymphocyte subset isolation, the whole T lymphocytes (CD4 negative CD8 negative, CD4 negative CD8 positive, CD4 positive CD8 negative, CD4 positive CD8 positive 4
Helper T cells (CD4 positive CD8 negative) and suppressor T cells (CD
4 negative CD8 positive) and so on.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Preparation of Cell Separation / Purification Device A commercially available surface-activated flask (“Microselector” manufactured by Applied Immun Science Co., Ltd.) was used in accordance with the company's technical data “Method for immobilizing ligand” and a monoclonal antibody against CD34 antigen and CD38. A monoclonal antibody against the antigen was immobilized. That is, a commercially available CD34 monoclonal antibody and CD38 monoclonal antibody were mixed at a ratio of 1: 4.
5 m of a Dulbecco's phosphate buffer solution (hereinafter, D-PBS) solution of the antibody prepared by mixing at a ratio of 50 μg / ml.
1 was put and left at room temperature for 4 hours. After that, the unbound antibody was washed three times with D-PBS, and the non-binding portion of the flask was blocked with human serum albumin, and then gamma sterilization was performed to obtain a cell separation and purification device. Preparation of Hematopoietic Stem Cell-Containing Liquid Bone marrow collected from a healthy donor was subjected to mononuclear cell separation by a known Ficoll specific gravity centrifugation method to obtain a hematopoietic stem cell-containing liquid. The composition of the obtained cell liquid (measured with a flow cytometer) is as shown in Table 1, and D-PBS (Ca,
It is suspended in 3 ml (without Mg). Cell separation Cell separation was performed according to the instruction manual of the micro selector. That is, D-PB of the hematopoietic stem cell-containing liquid prepared in
D-PBS3 containing 0.5% of human gamma globulin in S3 ml
After replacing with ml, the cell solution was placed in the flask prepared by and incubated at room temperature for 1 hour. D-PBS4
After gently washing the flask with ml to collect non-adherent cells, D-PBS 4m containing 0.02% EDTA.2Na
1 was introduced into the flask, the flask was shaken as much as possible, and the adherent cells were detached and collected. The composition of the adherent cells recovered is shown in Table 2, and the recovery rate calculated from the values in Table 2 and Table 1 is shown in Table 3.
It was as follows.

[0012]

Example 2 Preparation of Cell Separation and Purification Device 10 polyester nonwoven fabrics having an average fiber diameter of 3.9 μm and a size of 1.8 × 1.8 cm were subjected to a monoclonal antibody solution (commercially available CD34 monoclonal antibody and CD38 monoclonal antibody 1: 4). 15mg /
10 ml (prepared in D-PBS) so that the amount of the solution became 10 ml, the sample was immersed at room temperature for 5 hours for coating. This non-woven fabric was filled in a housing having an internal area of 2.15 × 2.15 cm, and then gamma ray sterilization was performed to obtain a cell separation and purification device. Preparation of Hematopoietic Stem Cell-Containing Liquid The same cells as in Example 1 were used. However, D-PB
It is suspended in 10 ml of S (without Ca and Mg). A 10 ml disposable syringe containing the cell suspension for cell separation was connected to the inlet of the hematopoietic stem cell separation and purification device, and 10 ml of the cell suspension was introduced by slowly pushing the syringe. The non-adherent cell liquid was collected by a conical tube placed on the outlet side. After incubation for 15 minutes at room temperature, 10
As a rinse liquid, ml of physiological saline was passed through the inlet, and this rinse liquid was also collected in the above-mentioned conical tube. Next, 20 ml of a chymopapain solution adjusted to 20 mg / ml with D-PBS was introduced from the inlet side, and the cell solution flowing out from the outlet side was received by a conical tube for collecting adherent cells different from the above-mentioned conical tube. After 15 minutes of incubation at room temperature, 20 ml of D-PBS was passed 4 times, and this rinse was also received in the above-mentioned conical tube for collecting adherent cells. The composition of the collected adherent cells is shown in Table 4, and the recovery rate calculated from the values in Table 4 and Table 1 is shown in Table 5.

[0013]

INDUSTRIAL APPLICABILITY According to the present invention, the separation and purification of specific cells from a cell population can be carried out easily and safely, without using an expensive apparatus, and in a small number of steps, that is, one type of positive selection and negative selection. It is possible to provide a cell separation / purification material, a cell separation / purification device, and a cell separation / purification method that can be performed by the above-mentioned instrument.

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Claims (3)

[Claims] 1. A ligand having a selective affinity for a first marker (hereinafter referred to as “first ligand”) and a ligand having a selective affinity for a second marker (hereinafter referred to as “first ligand”). Second ligand ”) on the same surface and comprises a first marker and a second marker.
Of the first marker is expressed relative to cells co-expressing the second marker but is relatively higher than that to cells not expressing the second marker. A cell separation and purification material for separating and purifying cells that express the marker 1 but not the second marker. 2. A cell separation and purification material according to claim 1, which comprises a water-insoluble carrier having a first ligand and a second ligand on the same surface, after being filled in a container having a liquid inlet and a liquid outlet. A cell separation and purification device characterized by being sterilized. 3. A cell separation and purification material comprising a water-insoluble carrier having a first ligand and a second ligand on the same surface is contacted with a cell fluid containing cells expressing at least the first marker. , The cells expressing the first marker and the cells expressing the second marker are captured by the separation and purification material, and then the cells expressing the second marker are selected from the captured cells. A method for separating and purifying cells, which is characterized in that only cells expressing the first marker but not expressing the second marker are left as they are captured and separated from the separating and purifying material and collected.