JPH08268897A - Material for concentrating hematopoietic stem cell and concentration of hematopoietic stem cell - Google Patents

Abstract

PURPOSE: To obtain a material for concentrating hematopoietic stem cell capable of inexpensively, simply and safely using in medical site and provide a method for concentrating the cell. CONSTITUTION: This concentrating material comprises a concentrating material whose at least surface is a hydroxyapatite. The concentrating method comprises (1) pouring hematopoietic stem cell-containing solution into a column in which granules, fiber or porous body of the material for concentrating the hematopoietic stem cell is packed, retaining the solution therein and (2) pouring a washing solution into the column, making the solution flow and recovering the washing solution. Thereby, the hematopoietic stem cell can simply and effectively be concentrated and saving of freeze storage space and reduction in adverse effects due to dimethyl sulfoxide and circulating load can be expected. Further, the method is not required to use a reagent such as expensive monoclonal antibody and instrument and is extremely safe even when used as a clinic use, because sterilization is also readily carried out.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a material for specifically enriching hematopoietic stem cells from a cell population containing hematopoietic stem cells such as bone marrow or peripheral blood, and a method for enriching hematopoietic stem cells using the material. More specifically, the present invention relates to a hematopoietic stem cell concentrating material and a concentrating method in various hematopoietic stem cell transplants such as allogeneic bone marrow transplantation and autologous peripheral blood stem cell transplantation.

[0002]

Bone marrow transplantation therapy is widely used for hematopoietic disorders such as leukemia and other hematopoietic tumors and side effects of chemotherapy for 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 administer fatal high-dose radiation and / or high-dose chemotherapy to a patient, which leads to treatment of leukemia and solid cancer.

Furthermore, in recent years, it has been revealed that peripheral blood as well as bone marrow contains hematopoietic stem cells and / or hematopoietic progenitor cells (hereinafter abbreviated as hematopoietic stem cells) necessary for these treatments. . Usually, the content of these cells in the peripheral blood is considerably low, and it is difficult to collect and use them in place of bone marrow transplantation. However, anti-cancer agents and / or G-CSF (granulocyte colony-stimulating factor) are used. It has been clarified that the content of cytokines is increased by administering such cytokines, and compared with bone marrow collection, general anesthesia is not required and it is safe, and thus clinical application is actively carried out.

Here, the transplantation method is divided into allograft transplantation and autologous transplantation depending on who obtains the cells. The former uses cells of a healthy stranger (relative or unrelated), and the latter uses cells of the patient himself. Autologous transplantation is often used in the transplantation method using peripheral blood. Allogeneic transplantation is mainly on the day of transplantation (high dose radiation and / or
In contrast to autologous transplantation, cells are often collected immediately after high-dose chemotherapy or immediately after transplantation, whereas in autologous transplantation, patients' own cells are collected and mononuclear before high-dose radiation and / or high-dose chemotherapy. It is usually practiced to separate the cells into spheres and store them in a frozen state, then perform high-dose radiation and / or high-dose chemotherapy, thaw the cells stored in a frozen state, and transplant them.

Freezing storage is carried out in liquid nitrogen or in an electric refrigerator. At the time of freezing, the cell concentration is 1 to 5 × 10 ⁷ / ml in order to prevent cell aggregation during transplantation.
Since it has to be diluted to a certain degree, and the number of cases undergoing surgery has increased rapidly in recent years, the problem of increased storage space is beginning to be pointed out (for example, Masamichi Otsubo, et al .: Journal of the Japanese Society of Blood Transfusion,
40, No. 2, 336). Further, in the case of frozen storage, dimethyl sulfoxide (hereinafter referred to as DMSO) must be added in an amount of usually 10 to 20% in order to prevent cold damage, but DMSO is known to have toxicity (Yasuhiro Okamoto). , Et al: Clinical Blood, Vol. 33, No. 3.
No. 317), it is desirable to add as little DMSO as possible.

On the other hand, since the cells that are truly necessary in this transplantation method are hematopoietic stem cells that are contained in mononuclear cells in an amount of about 0.5 to 10%, monoclonal antibodies against surface antigens of these cells and magnetic beads are used. By using a combination of devices such as the above, concentrating and then cryopreserving, the volume of cells is reduced and the cryopreservation space is saved, and the amount of DMSO to be infused is reduced. Although it can be reduced, this method has drawbacks in that expensive reagents and instruments must be used, the operation is very complicated, and sterilization is difficult.

Even in the above-mentioned allogeneic transplantation which does not require cryopreservation, it is desirable that the infusion volume be as small as possible from the viewpoint of reducing the circulatory load of the infused patient. Further, if cryopreservation will be carried out in the same way as in the case of autotransplantation in the future, the same problems as in autotransplantation will occur.

[0008]

As described above, in autologous transplantation and allogeneic transplantation of hematopoietic stem cells,
Although the storage space for collected cells, side effects due to DMSO, and circulatory load are becoming problems, the method for concentrating hematopoietic stem cells using a monoclonal antibody is costly and complicated, and is not very suitable for daily clinical use. It was a proper method. In addition, it has a fatal defect in terms of safety for clinical use in which sterilization is difficult. Therefore, an object of the present invention is to provide a hematopoietic stem cell concentrating material and a concentrating method that can be used inexpensively, conveniently, and safely in a medical field.

[0009]

Means for Solving the Problems The present inventor has found that hydroxyapatite has a property of specifically concentrating hematopoietic stem cells as a result of intensive studies to achieve the above object, and completed the present invention. Came to do.

That is, the present invention is a hematopoietic stem cell concentrating material having at least the surface made of hydroxyapatite, and the present invention is a column packed with granules, fibers or porous bodies having at least the surface made of hydroxyapatite. ) A method for concentrating hematopoietic stem cells, which comprises injecting a hematopoietic stem cell-containing solution and retaining it inside the column, and 2) injecting a washing solution into the column and then pouring out and collecting.

The method for concentrating hematopoietic stem cells of the present invention does not use expensive reagents such as monoclonal antibodies and instruments and is very simple. Further, since it can be sterilized, it is extremely safe even when used for clinical use. Hydroxyapatite (hereinafter referred to as HAp) used in the present invention is a kind of calcium phosphate-based compound and has a chemical formula of Ca ₁₀ (PO ₄ ) ₆ (O
H) ₂ . An inorganic main component of vertebrate hard tissues (bones and teeth), biomaterials such as artificial bones and artificial roots,
It is used as a packing material for chromatography in separating enzymes.

The hematopoietic stem cell concentrating material of which at least the surface is HAp according to the present invention is preferably molded into a granular shape, a fibrous shape or a porous shape and then filled in a container, but a granular shape is particularly preferable. The average particle size of the granules is preferably 25 μm to 2500 μm, and more preferably 40 μm to 1000 μm. Average particle size is 25
If it exceeds 00 μm, the adsorption amount decreases, and if it is less than 25 μm, the pressure loss becomes too large, which is not preferable. When a fiber aggregate is used, its average fiber diameter is preferably 3 μm to 300 μm, more preferably 10 μm to 100 μm. Furthermore, Journal of the Textile Society Vol. 49 No. 11 29,
A non-woven fabric such as rayon coated by the method on page 30 can also be used.

Next, the chemical composition of hydroxyapatite will be described. Apatite is a Greek word meaning "to deceive people," and often uses various elements such as Ca and PO.
₄ , part of OH is replaced. In fact, vertebrate HAp
Ca is mainly Mg, Fe and □ (vacancy, vacancy)
In addition, PO ₄ is mainly CO ₂ , OH is mainly CO ₂ , F and C.
Although they were substituted with l, they are collectively referred to as HAp because there is almost no difference in structure and physical properties (substitutions with □ are sometimes referred to as calcium-deficient apatite). Therefore, the term HAp used in this specification has the same meaning.

In the method for concentrating hematopoietic stem cells according to the present invention, the above HAp is filled in a container and used. Examples of the container include commercially available disposable syringes, blood bags, and the like, but in order to use the concentrated hematopoietic stem cells in clinical applications, they must be sterilizable. The cell population used in the present invention is derived from blood, that is, peripheral blood (whole blood), a mononuclear cell fraction obtained by separating peripheral blood (whole blood) with a centrifuge, umbilical cord blood, etc., which has been attracting attention in recent years. Is mentioned. Examples of the bone marrow origin include bone marrow and a mononuclear cell fraction obtained by centrifugation of bone marrow.

The method of concentrating hematopoietic cells according to the present invention comprises: 1) injecting the above-mentioned hematopoietic stem cell-containing solution into a column filled with HAp and holding it inside the column; and 2) injecting a washing solution into the column. Pour out and collect with a plastic tube placed at the bottom outlet of the column. The washing liquid here is HB
Examples thereof include SS (Hanks's solution), D-PBS (Dulbecco's phosphate buffer), physiological saline, etc. However, in order to use concentrated hematopoietic stem cells for clinical use, they must be sterilized. Also, the recovery tube must be sterilized. Further, in order to use the concentrated hematopoietic stem cells for clinical use, it is necessary to perform the entire operation using an aseptic technique in a sterile environment such as a clean bench.

The cell population in which hematopoietic stem cells are enriched by the enrichment method of the present invention is filled in a cryopreservable container (conical tube, cryobag, etc.), and cryopreserved by adding an antifreezing agent and the like, It is infused into a patient, or is infused into a patient as it is, or is subjected to some kind of cell treatment (removal of cancer cells, further cell separation, hematopoietic stem cell expansion, etc.), and then stored or infused.

[0017]

The present invention is described in detail below with reference to examples, but the present invention is not limited to these. Preparation of Hematopoietic Stem Cell Concentrating Material HAp slurry synthesized by a known wet method using a phosphoric acid aqueous solution and a calcium hydroxide suspension is suction-filtered, then dried,
It was crushed to prepare HAp granules. After baking the granules at 1200 ° C for 4 hours, classify them with a stainless steel sieve to about 10
After the particle size was adjusted to 0 μm, 4 g of the present granules were filled in a 5 ml disposable syringe and subjected to high-pressure steam sterilization to obtain a sterilized column.

Specimens for Experiment Using a Spectra component blood sampling device manufactured by COBE from a patient with Ewing sarcoma after chemotherapy and G-CSF administration,
Peripheral blood containing peripheral blood stem cells collected by a conventional method is fractionated, and calcium-magnesium-free HBSS solution (hereinafter,
It was suspended in 0.2 ml as a sample for experiment. The total white blood cell count and hematopoietic stem cell (CD34-positive cell) content of this sample were as follows. Total white blood cell count 5.1 × 10 ⁶ Hematopoietic stem cell content rate 0.55%

Concentration procedure and results After priming the column with about 2 ml of HBSS, the experimental sample was gently run through the column. After incubating at 37 ° C for 15 minutes, about 3 ml of HBSS was washed out, and the washing liquid was collected in a 5 ml plastic tube for collection placed under the outlet at the bottom of the column. By this operation, the total white blood cell count,
The hematopoietic stem cell (CD34-positive cell) content was as follows. Total white blood cell count 7.2 × 10 ⁵ Hematopoietic stem cell content rate 4.7%

When the hematopoietic stem cell concentration ratio was calculated as the hematopoietic stem cell concentration ratio = hematopoietic stem cell content ratio after treatment / hematopoietic stem cell content ratio before treatment, it was about 8.5 times. When the behavior of cells other than hematopoietic stem cells was examined, the change in the number of cells before and after the treatment and the recovery rate (= 100 × the number of cells after treatment / the number of cells before treatment) were as shown in Table 1.

[0021]

[Table 1] From the results in Table 1, it can be seen that various T lymphocytes are nearly 50%, B cells are approximately 75%, and monocytes are almost removed, but hematopoietic stem cells can be recovered with almost no loss.

[0022]

Industrial Applicability As described above, the method for concentrating hematopoietic stem cells according to the present invention can simply and effectively concentrate hematopoietic stem cells, save frozen storage space, reduce side effects and circulatory load due to DMSO. Can be expected. Also,
Since this method does not use expensive reagents such as monoclonal antibodies and instruments and is easy to sterilize, it can be used extremely safely even when used for clinical applications.

Claims (2)

[Claims] 1. A hematopoietic stem cell concentrating material, at least the surface of which is made of hydroxyapatite. 2. A column filled with granules, fibers or a porous body having at least a surface made of hydroxyapatite, 1) injecting a hematopoietic stem cell-containing solution to retain it inside the column, and 2) injecting a washing solution into the column. A method for concentrating hematopoietic stem cells, which is characterized in that it is poured out and collected.