JP2003210161A - Concentrator for hepatic stem cell and method for treating liver disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly concentrating a hepatic steam cell according to simple operation, a tool therefor, and a method for treating liver diseases. <P>SOLUTION: The method for concentrating the hepatic stem cell comprises a step of feeding a cell suspension containing the hepatic stem cell from a liquid inflow port of a container containing a water-insoluble carrier having the surface which is a β2 microglobulin-affinitive substance, adsorbing a β2 microglobulin-positive cell on the carrier and taking out a fraction freed of the β2 microglobulin-positive cell from a liquid outflow port. The method for treating the liver diseases comprises returning the hepatic stem cell concentrated and preserved by the method using the cell suspension containing the hepatic stem cell collected from a tissue or an organ other than the liver of a patient suffering from chronic hepatitis by the method to the patient before leading to cirrhosis. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for enriching hepatic stem cells, an instrument used therefor, and a method for treating liver disease. The hepatic stem cells obtained by the present invention can be used in basic science fields such as immunology and cell biology, and in the treatment of various biological tissue lesions and / or defects.

[0002]

2. Description of the Related Art By using stem and / or progenitor cells of a tissue or an organ of a living body (hereinafter, simply referred to as a tissue) to form the tissue in vitro or in vivo, a lesion and / or a tissue So-called regenerative medicine (regenerative medicine) for treating defects has attracted a great deal of attention, and research and development are being actively conducted in countries around the world (for example, Non-Patent Documents 1 and 2). Among them, the liver is one of the most promising ones because it is originally known as an organ with strong regenerative power, and research is actively conducted. Very recently, the existence of stem and / or progenitor cells of the liver (hereinafter simply referred to as hepatic stem cells) in the bone marrow and a method of separating the same have been reported (Non-patent Document 3), which has been extremely attracting attention. .

This method uses the immunomagnetic bead method, and more specifically, the method is as follows. That is, the bone marrow cell fluid is incubated with beads to which a monoclonal antibody against β2 microglobulin is immobilized (“negative selection”), and then red blood cells are hemolyzed with a hemolytic agent, and further, beads to which a monoclonal antibody against Thy1 antigen is immobilized. And a β2 microglobulin-negative and Thy1 antigen-positive cell and a bead complex on which a monoclonal antibody against the Thy1 antigen is immobilized are obtained. According to this method, the cells cannot be removed from the beads,
In a strict sense, it cannot be said that hepatic stem cells have been isolated and cannot be used for human transplantation. Furthermore, the method itself called the immunomagnetic bead method is a method generally used in immunology, cell biology, clinical laboratory medicine, etc., but it is complicated and is a laboratory level method having a long time such as incubation time. , Is not generally accepted as a routine medical practice.

On the other hand, β2 microglobulin has a molecular weight of 11
It is a protein of 600 and is known as a causative protein of dialysis amyloidosis in the field of artificial dialysis (in dialysis patients, the value of β2 microglobulin existing in free state in plasma is high), and β2 Artificial dialysis using a high-flux dialysis membrane that actively removes microglobulin and removal with an adsorbent have been performed, but this is a completely different technical field from regenerative medicine. By the way, in general, hepatitis patients generally develop chronic hepatitis, and then gradually progress in their pathological condition, leading to cirrhosis and dying, or further progressing to liver cancer and dying. In cirrhosis, the liver is highly fibrotic. It is expected that the transplantation of liver cells before this highly fibrosis will suppress the progress of fibrosis. However, transplanting the liver cells of another person is very difficult considering the current situation of Japan's shortage of donors.

On the other hand, it is theoretically possible to cryopreserve the liver cells of the patient himself and to thaw and transplant them when necessary, but in many cases of chronic hepatitis, viruses are the cause and Contamination of the virus is unavoidable, and the virus will be reinjected at the time of transplantation. On the other hand, since it is considered that the hepatitis virus is almost absent in the bone marrow (containing hepatic stem cells as described above) of the patient himself, it is extremely excellent to save this bone marrow and use it for transplantation when necessary. Considered to be a cure for liver disease. By the way, cord blood is cryopreserved at the time of birth, and when the owner of the stored cord blood becomes a blood disease such as leukemia in the future, it is thawed and used for cord blood hematopoietic stem cell transplantation (for example, Patent Document 1 1), the so-called cord blood bank for autologous transplantation, has been started in Japan as well as in the US and Europe.

[0006]

[Non-Patent Document 1] Latest Medical Supplement "Regenerative Medicine" 2000
Year

[Non-patent document 2] Monthly tissue culture engineering, September 2001, "Development from cell engineering to tissue engineering / organ engineering"

[Non-Patent Document 3] Avita, I. et al: Bi
electrical and Biophysical
Research Communication 28
8,156-164,2001

[Patent Document 1] Japanese Unexamined Patent Publication No. 2002-165500

[0007]

The object of the present invention is to provide a method for rapidly enriching hepatic stem cells by a simple operation, an instrument used for the method, a method for storing the enriched hepatic stem cells, and a method for treating liver disease. To provide.

[0008]

Means for Solving the Problems The inventors of the present invention have made extensive studies to solve the above problems. As a result, the present invention has been completed as a result of repeated investigations focusing on the technology for removing β2 microglobulin in plasma in the field of artificial dialysis, which is a technical field completely different from tissue engineering. That is, the present invention is as follows. (1) A cell suspension containing hepatic stem cells from a liquid inlet of a container having a liquid inlet and a liquid outlet, at least a surface of which contains a water-insoluble carrier composed of a substance having an affinity for β2 microglobulin And adsorbing β2-microglobulin-positive cells in a cell suspension containing hepatic stem cells to the carrier, and taking out a fraction from which the β2-microglobulin-positive cells have been removed, from the liquid outlet. Method for enriching hepatic stem cells.

(2) The method for enriching hepatic stem cells according to (1), which comprises a step of selecting Thy1 antigen-positive cells after removing β2-microglobulin-positive cells. (3) The method for concentrating hepatic stem cells according to (1), wherein the cell suspension containing hepatic stem cells is collected from a tissue or organ other than the liver of a chronic hepatitis patient. (4) A hepatic stem cell concentrator, wherein a water-insoluble carrier having a substance having an affinity for β2 microglobulin on at least the surface is contained in a container having a liquid inlet and a liquid outlet. (5) Treatment of a liver disease characterized by collecting and preserving tissues or organs other than the liver of a chronic hepatitis patient and returning it to the patient before hepatitis progresses and leading to cirrhosis to prevent the progression to cirrhosis. Method.

(6) Using a cell suspension containing hepatic stem cells collected from a tissue or organ other than the liver of a chronic hepatitis patient, hepatic stem cells contained in the tissue or organ are concentrated by the method described in (1). Then, the stored hepatic stem cells are returned to the patient before hepatitis of the patient progresses and cirrhosis occurs,
A method for treating a liver disease, which comprises preventing the progression to cirrhosis. (7) A method for preserving hepatic stem cells, which comprises concentrating and preserving hepatic stem cells contained in the same tissue or organ by the method according to (1) using a cell suspension containing hepatic stem cells.

The present invention will be described in detail below. One of the present invention is a method for removing β2 microglobulin-positive cells in a cell suspension containing hepatic stem cells and an instrument used therefor. Cell suspensions containing hepatic stem cells include, for example, bone marrow, umbilical cord blood (including those collected from not only umbilical cord blood vessels but also placental blood vessels), peripheral blood (hematological factors such as granulocyte colony stimulating factor) And those that have been subjected to some treatment such as centrifugation, or those obtained by resuspending cells extracted from various tissues such as liver and various organs such as muscle by enzyme digestion method in some liquid. To be Above all, good results are obtained when bone marrow is used. Here, some processing includes freeze-thawing.

The substance having an affinity for β2 microglobulin as used in the present invention is generally called β2 microglobulin.
It is a substance that has an affinity for microglobulin. Examples of such a substance include a monoclonal antibody against β2 microglobulin, a substance containing a urea bond or a thiourea bond proposed in JP-A-9-234246, and a molecular weight proposed in Japanese Patent No. 2665526. Aspect ratio (c-axis / a-axis) of 0.5 or more, and specific surface area of 1 m ² proposed in JP-A-6-262065, such as polyamino acid, polysaccharide, synthetic polymer electrolyte body of 1000 or more. Hydroxyapatite having an amount of / g or more and β2 microglobulin adsorbent “Rixel” (registered trademark) (produced by Kanegafuchi Chemical Industry Co., Ltd.) are preferably used. However, in addition to these, as long as it has an affinity for β2 microglobulin, it is not limited at all.

The water-insoluble carrier referred to in the present invention is a carrier having a substance having an affinity for the aforementioned β2 microglobulin at least on its surface. Examples of such a carrier include those in which a water-insoluble carrier is formed by the substance itself, and those in which the substance is present on the surface by coating or chemical bonding on a substrate. As the water-insoluble carrier, any material can be used as long as it is a commonly used carrier, but preferable examples in terms of good moldability, low sterility and low cytotoxicity include polyester, polyethylene, polypropylene and polystyrene. , Synthetic resin such as acrylic resin, nylon, polycarbonate, polyurethane, cellulose, cellulose acetate, chitin, chitosan,
Examples thereof include natural polymers such as alginates, inorganic materials such as hydroxyapatite, glass, alumina and titania, and metals such as stainless steel, titanium and aluminum.

These carriers can be used as they are, but for the purpose of enhancing selective passage of cells,
The surface may be modified if necessary. For example, in order to enhance platelet passage, WO 87/05812
The method of coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group, which is proposed in Japanese Patent No. Examples of the shape of the carrier include granules, fiber lumps, woven fabrics, non-woven fabrics, sponge-like porous bodies, flat plates, and the like, but in terms of large surface area per volume, granules, fiber lumps, woven fabrics, non-woven fabrics, sponges. A porous body and the like are preferable.

According to the present invention, hepatic stem cells are introduced from a liquid inlet of a container having a liquid inlet and a liquid outlet, in which a water-insoluble carrier at least the surface of which is made of a substance having an affinity for β2 microglobulin is contained. Is supplied to adsorb β2-microglobulin-positive cells in the cell suspension containing hepatic stem cells to the carrier, and the fraction from which the β2-microglobulin-positive cells have been removed is taken out from the liquid outlet. Usually, β2-microglobulin-positive cells are
Since it is instantly adsorbed to a substance that has an affinity for β2 microglobulin, the cell suspension containing hepatic stem cells is introduced and passed through the liquid inlet at a drop, or by using a syringe (by hand or by using a syringe pump). ) It may be passed.

The temperature during the treatment is not generally limited (generally, the cell treatment is carried out at 0 ° C or higher and 40 ° C or lower).
It is generally known that cells are damaged at higher or lower levels than this), and there may be an optimum temperature depending on contaminating cells mixed in a cell suspension containing hepatic stem cells. That is, when monocytes or macrophages are present as contaminating cells, they become more adherent at higher temperatures,
Since it may adversely affect the adsorption of 2 microglobulin-positive cells to the water-insoluble carrier, it is preferably 37 ° C or lower, more preferably 20 ° C or lower.

On the other hand, when platelets are present as contaminating cells, the cells are activated at a low temperature and increase in adhesiveness, which may also adversely affect the adsorption of β2 microglobulin-positive cells on the water-insoluble carrier. The temperature is preferably 4 ° C or higher, more preferably 10 ° C or higher. It is preferable to remove a large amount of red blood cells existing in bone marrow or peripheral blood by a specific gravity centrifugation method, a hemolysis method, a filter method or the like because the adsorption efficiency is enhanced. In particular, it is not necessary to hold the cell suspension in the container and incubate it, but since the adsorption efficiency may be increased by incubation, it may be carried out as necessary.

The hepatocyte concentrator of the present invention comprises a container having a liquid inlet and a liquid outlet. Usually, the liquid inlet is located at the upper part of the container and the liquid outlet is located at the lower part of the container. However, the liquid is introduced from the liquid inlet, and the liquid is introduced into the water-insoluble carrier composed of a substance having an affinity for β2 microglobulin. The structure is not limited to this as long as the liquid is discharged from the liquid outlet after the liquid comes into contact with it. As the shape of the container, any shape such as a cylinder, a cube, a rectangular parallelepiped, a rhombus, a sphere, and a polygon can be used, but a cylindrical shape and a rectangular shape are preferable in consideration of the production of the container and the accommodation of the water-insoluble carrier. .

As a material for the container having a liquid inlet and a liquid outlet used in the present invention, preferred examples are those having low moldability, sterility and low cytotoxicity.
Polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, synthetic polymers such as vinyl chloride, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel, titanium,
Examples of the metal include aluminum. In the case of further increasing the concentration of hepatic stem cells, the aforementioned literature (Avital,
I. , Et al: Biochemical and
Biophysical Research Comm
unitation 288,156-164,200
It is preferable to further add the selection using the Thy1 antigen shown in 1).

The selection using the Thy1 antigen is a method by flow cytometry, that is, fluorescently labeled Th.
Method of incubating with y1 antibody and selecting with a cell sorter, β2 microglobulin negative cell suspension obtained by the above operation in a container having a liquid inflow port and a liquid outflow port containing a water-insoluble carrier having Thy1 antibody immobilized on the surface After passing the liquid, the β2-microglobulin-negative Thy1 antigen-positive cells bound to the water-insoluble carrier are recovered from the liquid inlet or the liquid outlet (usually a physiological solution such as a medium or a buffer solution. For this purpose, a thickener that does not adversely affect cells such as dextran, polyvinylpyrrolidone, and hydroxyethyl starch may be added).
That is, a method of releasing by physical (shearing force) or by a biochemical method (the presence of a drug such as an enzyme that destroys the bond between cells and an antibody in the recovery liquid) is recovered.

According to the method for treating liver disease according to the present invention, tissues or organs other than the liver of a chronic hepatitis patient are collected and preserved and returned to the patient before hepatitis progresses and cirrhosis develops. The reason why the liver is not preferable here is that most of the chronic hepatitis is caused by viruses, and it is unavoidable that the virus is mixed into the cells of the liver, and there is a possibility that the virus will be re-injected at the time of transplantation or patients with chronic hepatitis. This is because the cells in the liver may have reduced function. Tissues or organs other than the liver include, for example, bone marrow, umbilical cord blood (including not only umbilical cord blood vessels but also placenta blood vessels),
Peripheral blood (including blood collected by administering hematopoietic factors such as granulocyte colony stimulating factor) Various organs such as pancreas, stomach and intestine,
Examples include various tissues such as muscle, bone, fat and placenta. It is preferable to subject these to some treatment such as centrifugation, and to resuspend cells extracted by an enzyme digestion method in some liquid to obtain a cell suspension. Of these, bone marrow is particularly preferable.

As a method for collecting and preserving bone marrow, a usual method for collecting and preserving autologous bone marrow transplant for the purpose of treating leukemia and the like may be used (for example, in “New Technique of Hematopoietic Cell Transplantation” published by Nanzandou). p112 “D. Autologous bone marrow transplantation 2. Bone marrow collection and preservation”). That is, under anesthesia, bone marrow is collected with a bone marrow puncture needle (usually, bone marrow can be collected around 1000 ml at a time under general anesthesia, and can be collected multiple times if there are several months apart. Erythrocytes are removed by a known specific gravity centrifugation method or a sedimentation method using hydroxyethyl starch, or concentrated into hepatic stem cells by a hepatic stem cell concentration method described later to prepare a cell suspension. In this cell suspension, a frost damage protectant (commercial name CP-1 manufactured by Kyokuto Pharmaceutical Co., Ltd.
Is preferably used. This product contains 14.7% of dimethyl sulfoxide and 17.6% of hydroxyethyl starch as a cryoprotective agent in physiological saline) and commercially available human serum albumin or the plasma (self plasma) of the person. After addition, store frozen in a -80 ° C freezer or in liquid nitrogen.

At that time, the tissue or organ other than the liver is subjected to the method for concentrating hepatic stem cells according to the present invention, that is, a liquid flow containing a water-insoluble carrier at least the surface of which is made of a substance having an affinity for β2 microglobulin. A cell suspension containing hepatic stem cells is supplied from a liquid inlet of a container having an inlet and a liquid outlet, and β in the cell suspension containing hepatic stem cells is supplied.
By a method for concentrating hepatic stem cells, which comprises a step of adsorbing 2 microglobulin-positive cells to the carrier and taking out a fraction from which the β2-microglobulin-positive cells are removed from the liquid outlet.
It is desirable to concentrate in advance before freezing.

Infusion into a patient is performed, for example, as follows. That is, the cryopreserved cell solution is thawed at a bedside in a hot bath of 37 to 40 ° C., and the solution is infused into the spleen or the portal vein. At this time, there is also a finding that washing (removal of dimethylsulfoxide) after thawing is preferable as a cell viability (Ayumi of Medicine, vol. 201N).
o. 11, P825, 2002). Further, according to the results of recent animal experiments, it is considered that cells may be intravenously injected into the injured liver or may reach the liver ("homing"), so that intravenous injection may be performed.

Regarding the number of times of collection and the number of times of infusion, 1) a method of collecting a plurality of times and collectively infusing at the time of infusion,
2) A method of collecting multiple times and dividing the infusion into multiple infusions,
3) There is a method of single infusion with a single infusion as it is, 4) a method of single infusion with multiple infusions, each of which has advantages and disadvantages. Therefore, it is appropriately selected according to the patient's situation. That is, the method of 1) is expected to have a high effect because a large amount of cells are infused at the time of infusion, but there is an invasion to the patient by collecting a plurality of times. In addition, it takes time to infuse a large amount of cells at one time during infusion, which may cause thrombosis or pulmonary embolism. The method 2) does not have the problem of mass transfusion of the method 1) because the method is divided into a small amount and transfused, but the number of cells may be too small to exert the effect. Also, the point that the patient is invaded by multiple collection is similar to the method of 1). Since the method of 3) is a single sampling, it is considered that the patient is minimally invaded.
There is a possibility that the effect will not be exhibited due to the small number of cells. On the other hand, if the number of transfused cells is increased, it is possible that the patient will be invaded during collection / transfusion, and the method in 4) is also expected to cause less invasion to the patient because it is a single collection, Since it is a transfusion, it is expected that the invasion during the transfusion will be small, but the number of cells during the transfusion may be small and the effect may not be exerted. The total number of transfused cells should be at least 1 x 1
If it is 0 ⁷ , the effect can be expected.

The hepatic stem cells obtained by the present invention are as they are,
Alternatively, after subjecting to various treatments such as further separation and purification, culture, activation, differentiation induction, amplification, gene transfer, and cryopreservation as necessary, the fields of basic science such as immunology and cell biology and various biological tissue lesions and And / or used to treat defects. Treatment of various biological tissue lesions and / or defects of interest is not limited to liver disease. That is, recently, plasticity of stem cells has been attracting attention, and it may be used for inducing insulin-secreting cells from hepatic stem cells and used for treating diabetes (see, for example, CMC Publishing Co., Ltd., “The Cutting Edge of Regenerative Medicine Engineering”, p241. This is because. The use of the hepatic stem cells preserved by the preservation method according to the present invention is not limited to the treatment of liver diseases.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0028]

Example 1 (1) Hepatic Stem Cell Concentrator Container size 41 × 41 × 18 mm, and a polycarbonate square container having a liquid outlet and a liquid inlet on a diagonal line, and a fiber cut into a 35 mm square with a push cutter. 16 pieces of polyethylene terephthalate nonwoven fabric having a diameter of about 12 μm (area weight: about 100 g / m ² , thickness: about 0.47 mm) and human anti-β2 microglobulin antibody (50 ml of an antibody solution having a concentration of 2 μg / ml was used for fixation) Two
No. 2618133, a polypropylene non-woven fabric having a fiber diameter of about 2 μm fixed by using a known haloacetamide activation method (basis weight: about 60 g / m ² , thickness: about 0.
18 mm (3 mm) were housed to form a hepatic stem cell concentrator.

(2) Cell Concentration Procedure 3 ml of human bone marrow fluid was collected by a conventional method, and immediately 6 units / ml heparin-containing Dulbecco's phosphate buffer 3 was added.
MEM (Minimum Essential Meme) containing 15% fetal bovine serum immediately before cell separation.
It was diluted with 50 ml of a medium to obtain a raw material cell suspension. This was injected from the inlet side of the hepatic stem cell concentrator prepared in (1) using a 50 ml disposable syringe,
A 50 ml conical tube was placed on the outlet side and the passed cell suspension was collected. The time required for this operation was about 3 minutes.

(3) Analysis Avita, I. , Et al: Biochemic
al and Biophysical Research
ch Communication 288, 156-
Based on the finding that 25% of hepatic stem cells were concentrated in the β2-microglobulin negative fraction in 164, 2001, β2-microglobulin-negative cells were analyzed by flow cytometry (ie, β2 microglobulin was simulated. Globulin-negative cells were used as hepatic stem cells). The concentration factor was calculated by the following formula. Concentration factor = β2 microglobulin negative cell abundance ratio after concentration / β2 microglobulin negative cell abundance ratio before concentration (4) Results Table 1 shows a summary of the results. It can be seen that β2 microglobulin negative cells according to the present invention are highly enriched.

[0031]

[Table 1]

[0032]

EFFECTS OF THE INVENTION According to the present invention, hepatic stem cells can be rapidly concentrated by a simple operation, and therefore, it greatly contributes to the practical application of regenerative medicine.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/16 A61P 3/10 3/10 19/04 19/04 31/12 31/12 C12M 3/00 Z C12M 3/00 C12N 5/00 E

Claims (7)

[Claims] 1. Cells containing hepatic stem cells from a liquid inlet of a container having a liquid inlet and a liquid outlet, in which a water-insoluble carrier at least the surface of which is made of a substance having an affinity for β2 microglobulin is contained. A step of supplying a suspension, adsorbing β2 microglobulin positive cells in a cell suspension containing hepatic stem cells to the carrier, and taking out a fraction from which the β2 microglobulin positive cells have been removed, from the liquid outlet. A characteristic method for enriching hepatic stem cells. 2. The method for enriching hepatic stem cells according to claim 1, further comprising the step of selecting Thy1 antigen-positive cells after removing β2-microglobulin-positive cells. 3. The method for concentrating hepatic stem cells according to claim 1, wherein the cell suspension containing hepatic stem cells is collected from a tissue or organ other than the liver of a chronic hepatitis patient. 4. A hepatic stem cell concentrator, wherein a water-insoluble carrier having a substance having an affinity for β2 microglobulin on at least its surface is contained in a container having a liquid inlet and a liquid outlet. 5. A liver disease characterized in that tissues or organs other than the liver of a chronic hepatitis patient are collected and preserved, and returned to the patient before hepatitis progresses to cause cirrhosis to prevent the progression to cirrhosis. How to treat. 6. A cell suspension containing hepatic stem cells collected from a tissue or organ other than the liver of a patient with chronic hepatitis,
Concentrating hepatic stem cells contained in the same tissue or organ by the method according to claim 1, and returning the preserved hepatic stem cells to the patient before hepatitis progresses in the patient, leading to cirrhosis, and A method for treating a liver disease, which comprises preventing progress. 7. A method for preserving hepatic stem cells, which comprises concentrating and preserving hepatic stem cells contained in the same tissue or organ by the method according to claim 1 using a cell suspension containing hepatic stem cells.