JP2000166541A - Human undifferentiated hematopoietic stem cell and its separation and separation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain human undifferentiated hematopoietic stem cells which are positive for CD 45 antigen and negative for CD 34 antigen and differentiated antigens and are useful for transplantation therapy, etc., in simple operations in a short time by recovering the human undifferentiated hematopoietic stem cells by the use of a filter capable of catching nucleated cells and allowing the passage of erythrocytes. SOLUTION: The human undifferentiated hematopoietic stem cells which are positive for CD 45 antigen and negative for CD 34 antigen and differentiated antigens are recovered using a filter capable of catching nucleated cells such as hematopoietic stem cells and allowing the passage of erythrocytes. The method for separating the human undifferentiated hematopoietic stem cells preferably comprises introducing a nucleated cell-containing liquid containing nucleated cells to the filter capable of catching nucleated cells such as hematopoietic stem cells and allowing the passage of erythrocytes, and then introducing a liquid to recover the nucleated cells. The nucleated cells are positive for CD 45 antigen and negative for CD 34 antigen and differentiated antigens. A device for separating and recovering the human undifferentiated hematopoietic stem cells preferably uses the filter obtained by charging a vessel having a liquid flow inlet and a liquid flow exit with a nucleated cell-catching material comprising a porous structure material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a human undifferentiated hematopoietic stem cell, a method for separating the cells, and a separation device. The obtained human undifferentiated hematopoietic stem cells can be used in the treatment of various diseases using cells, such as hematopoietic stem cell transplantation therapy, and in basic science fields such as immunology and cell biology.

[0002]

2. Description of the Related Art Hitherto, as a surface marker of human hematopoietic stem cells, CD34 antigen has been generally known.
Among the antigen-positive cell population, CD38 antigen-negative and differentiated antigen-negative cells were more undifferentiated (Japanese Patent Laid-Open No.
6354). However, in recent years, there have been many reports that CD34-negative cells are more undifferentiated in mice (for example,
57th Annual Meeting of the Japanese Society of Hematology, abstract numbers 490, 1995
Years), and more recently, even in humans, reports have emerged that more undifferentiated hematopoietic stem cells are CD34-negative (eg, Goodell, eta).
l: Nature Medicine, vol. 3, N
o. 12, 1997, Zanjani, et al: Ex
p. Hematol. vol. 26, 1998). In these reports, CD34 antigen negative cells (more specifically C
D45 antigen-positive and CD34 antigen negative and differentiation antigen-negative, below, CD45 ⁺ CD34 ^- Lin ^- The separation of abbreviated), and using a special cell sorting device and a special fluorescent dyes and / or a number of monoclonal antibodies, It requires a great deal of cost and time, and the operation is very complicated, far from the practical level.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for obtaining human undifferentiated hematopoietic stem cells by a simple operation at a low cost and in a short time.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve such problems. Here, the present inventors have determined that it is difficult to achieve the present object by employing a separation technique using a surface antigen, which is a common means in the art and is also employed in the above-mentioned prior art. , Tried a solution by completely new technical means. In other words, focusing on the properties of the target human undifferentiated hematopoietic stem cells, particularly on the capturing behavior of the cells in the material, the cells have a capturing behavior common to nucleated cells in general, but the desorption behavior is higher than that of ordinary nucleated cells. After conducting various studies on the assumption that it is much higher (easy to detach), it was found that the target human undifferentiated hematopoietic stem cells could be separated at a very high rate using a filter that captures nucleated cells. The inventor has found a surprising effect, which has led to the present invention.

That is, the present invention relates to a human undifferentiated hematopoietic stem cell which is substantially CD45 antigen-positive, CD34 antigen-negative and differentiated antigen-negative and has been captured from a filter substantially capturing nucleated cells and substantially passing through erythrocytes. In addition, the present invention introduces a nucleated cell-containing liquid containing CD45 antigen-positive, CD34-negative and differentiated antigen-negative nucleated cells into a filter that substantially captures nucleated cells and substantially passes through erythrocytes. Then, a method for separating human undifferentiated hematopoietic stem cells, wherein a liquid is introduced into the filter to collect CD45 antigen-positive and CD34 antigen-negative and differentiated antigen-negative nucleated cells captured by the filter, The present invention relates to CD45
An apparatus for separating and collecting antigen-positive and CD34 antigen-negative and lineage-negative human undifferentiated hematopoietic stem cells, wherein a container having a liquid inlet and a liquid outlet is filled with a nucleated cell-capturing material comprising a porous structure. 3 is an apparatus for separating human undifferentiated hematopoietic stem cells.

Hereinafter, the present invention will be described in detail. The nucleated cell referred to in the present invention refers to a cell having a nucleus in the cell, and specifically, leukocytes, granulocytes, neutrophils, eosinophils, basophils, myeloid cells, erythroblasts, Examples include lymphocytes, T lymphocytes, B lymphocytes, monocytes, hematopoietic stem cells, hematopoietic progenitor cells, and the like.

[0007] The term "filter that substantially captures nucleated cells and substantially passes red blood cells" as used in the present invention means, for example, a material that substantially captures nucleated cells and substantially transmits red blood cells. Filled in a container having a liquid inlet and a liquid outlet. Any material can be used as the material that substantially traps nucleated cells and substantially passes red blood cells, as long as it is a commonly used cell trapping material, but has low moldability, sterility, and low cytotoxicity. Illustrative of preferred are polyester, polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polycarbonate, synthetic polymers such as polyurethane, cellulose, cellulose acetate, chitin, natural polymers such as chitosan alginate, hydroxyapatite, glass, Examples include inorganic materials such as alumina and titania, and metals such as stainless steel, titanium, and aluminum.

These capture materials can be used as they are, but may be surface-modified as required for the purpose of enhancing the selective passage of cells. For example, in order to enhance the platelet permeability, a method of coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group proposed in WO 87/05812 can be mentioned.

Examples of the shape of the trapping material include granules, fiber masses, woven fabrics, nonwoven fabrics, sponge-like porous bodies, flat plates, etc. However, in terms of a large surface area per volume, granules, fiber masses, and woven fabrics are used. , Non-woven fabric, sponge-like porous body is preferable,
Further, a nonwoven fabric and a sponge-like porous body are more preferable in terms of manufacturability and flowability. In the case of a non-woven fabric, the fiber diameter is usually 1.0 μm or more and 30 μm or less, preferably 1.0 μm or less.
It is not less than μm and not more than 20 μm, even more preferably not less than 1.5 μm and not more than 10 μm. If it is less than 1.0 μm, the target cells, that is, human undifferentiated hematopoietic stem cells that are CD45 antigen-positive, CD34 antigen-negative and differentiated antigen-negative, may be firmly captured and may be difficult to collect. If it exceeds 30 μm, CD45 antigen positive and C
D34 antigen-negative and differentiated antigen-negative human undifferentiated hematopoietic stem cells are more likely to pass through without being captured by the nonwoven fabric.
In either case, the recovery rate may be reduced, which is not preferable. In the case of a sponge-like structure, the pore diameter is usually from 2.0 μm to 30 μm, preferably from 2.5 μm to 25 μm, and more preferably from 3.0 μm to 20 μm. 2.0 μm
If it is less than 25 μm, the flowability will be remarkably inferior, and it may be difficult to pass through the liquid itself. Not preferred.

[0010] As a material for the container which is filled with a material that substantially captures nucleated cells and substantially passes red blood cells, polyethylene, polyethylene, and the like are preferable in terms of moldability, sterility, and low cytotoxicity. Polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, synthetic polymers such as polyvinyl chloride, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel,
Examples include metals such as titanium and aluminum.

The term "substantially captures nucleated cells" as used in the present invention means that 60% or more of nucleated cells in a nucleated cell-containing liquid are captured, and "substantially passes erythrocytes.""Do" means that the erythrocytes in the nucleated cell-containing solution pass through 60% or more. The nucleated cell-containing solution referred to in the present invention includes, for example, bone marrow, cord blood (including those collected from placental blood vessels as well as cord blood vessels), and peripheral blood (hematopoietic factors such as granulocyte colony stimulating factor). And blood collected after administration) and those subjected to some treatment such as centrifugation. According to the experience of the present inventors, good results were obtained when cord blood was converted to a mononuclear cell fraction by specific gravity centrifugation. Also, any processing includes freezing and thawing.

In the present invention, any liquid may be used as a liquid for recovering CD45 antigen-positive and CD34-negative recovery required cells, which are target cells introduced into the cell capturing means and captured. Some examples include physiological saline, buffers such as D-PBS and HBSS, and media such as RPMI1640. EDTA, dextran, etc. may be added to these physiological solutions for the purpose of protecting cells, supplementing nutrients, preventing frost damage during cryopreservation, and improving viscosity (which may be effective for improving the recovery rate).
Hydroxyethyl starch, dimethyl sulfoxide, albumin, globulin, gelatin, glucose, saccharose, trehalose and the like may be added. The experience of the present inventors has shown that human serum albumin and EDTA
Good results have been obtained with added D-PBS.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 Cell Separator The outer dimensions of the container (length × width × thickness) were 41 × 41 × 18 mm, and the average fiber diameter was 2.3 μm on the inlet side of a polycarbonate container having a liquid outlet and a liquid inlet on a diagonal line. Eighteen polyester non-woven fabrics were filled on the outlet side with 16 polyester non-woven fabrics having an average fiber diameter of 12 μm to prepare a cell separator. In addition,
The packing density is 0.2 g / cm ³ and the effective filtration area is 12.25.
cm ² and an effective filtration length of 12.4 mm.

[0014] In order to impart platelet permeability to the cell separator, a coating of a hydrophilic polymer was applied. That is, after passing a 1% ethanol solution of a hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer (molar ratio of hydroxyethyl methacrylate to dimethylaminoethyl methacrylate = 97: 3) from the inlet side of the filter, nitrogen gas was passed. Let dry.

Cell Separation Procedure Umbilical cord blood was separated into mononuclear cell fractions by known Ficoll-Hypaque specific gravity centrifugation. The obtained mononuclear cell fraction was subjected to 0.1% human serum albumin-added EDTA-DPBS2.
The suspension was resuspended in 00 ml (hereinafter referred to as solution A) to obtain a raw cell solution. This raw cell solution was transferred to a 200 ml blood bag. This blood bag was connected to the inlet side of the cell separator 1 manufactured by using a tube having a three-way cock 4 and a mesh chamber 3 to which a cell collection bag 5 was connected in the middle. The outlet side of the cell separator 1 is a drain bag 7 with a tube having a three-way stopcock 6 for connecting a syringe for collection in the middle.
Connected. The nucleated cell-containing liquid in the source blood bag 2 is passed through the cell separator at a head of about 60 cm, and the liquid containing a small amount of red blood cells flowing out of the cell separator 1 is drained into the drain bag 7.
Was drained. Next, a 30 ml disposable syringe containing 30 ml of the solution A is connected to the three-way cock 6, and the three-way cock 6 is turned in a direction in which only the syringe and the cell separator communicate with each other.
The three-way stopcock 4 is connected to the cell separator 1 and the cell collection bag 5.
After turning in the direction in which only the cells communicated, the syringe was pushed and the cells captured in the cell separator were collected in the cell collection bag 5.

Analysis The number of nucleated cells was measured by an automatic hemocytometer, and undifferentiated hematopoietic stem cells were analyzed for CD2, 3, 1 by flow cytometry.
4, 16, 19, 20, 33, 34, 41, 56, Gl
The determination was performed by quantifying a group of ycohorin A negative and CD45 positive cells. The method for calculating the concentration rate and the recovery rate is as follows. Concentration rate (%) = 100 × the cell positive rate / the cell positive rate in the source cell solution Recovery rate (%) = 100 × (the number of the cells after collection / the number of cells in the source blood)

Results Positive rate of undifferentiated hematopoietic stem cells before cell separation was 0.74
%Met. Positive rate of the cells after separation is 50.2%
And the cells were highly concentrated by the separation operation.
The concentration ratio was 67.83%. In addition, the recovery rate was 42.8.
%Met. The time required for the separation operation was extremely short, about 10 minutes.

[0018]

As described above, according to the present invention, human undifferentiated hematopoietic stem cells can be separated inexpensively, simply and in a short time.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a cell separation circuit system used in Example 1.

[Explanation of symbols]

 Reference Signs List 1 cell separator 2 blood bag 3 mesh chamber 4 three-way cock 5 cell collection bag 6 three-way cock 7 drain bag

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Yoshihiko Nakamura 2-28-4 Tomigaya, Shibuya-ku, Tokyo F-term within Tokai University 4B029 AA07 AA27 BB11 CC01 FA02 FA11 4B065 AA93X AA94X BC42 BD18 CA44 4D019 AA03 BA01 BA02 BA04 BA06 BA12 BA13 BB02 BB03 BB07 BB10 BB12 BB13 BC13 BD01

Claims (3)

[Claims] Claims: 1. An undifferentiated human hematopoietic stem cell that is CD45 antigen-positive, CD34 antigen-negative and differentiated antigen-negative and is substantially free of nucleated cells and recovered from a filter substantially passing through erythrocytes. 2. A filter that substantially captures nucleated cells and that is positive for CD45 antigen and CD on a filter that substantially passes erythrocytes.
A nucleated cell-containing liquid containing nucleated cells that is negative for CD34 antigen and differentiated antigen is introduced.
4. A method for separating human undifferentiated hematopoietic stem cells, comprising collecting nucleated cells that are antigen-negative and differentiated antigen-negative. 3. An apparatus for separating and recovering human undifferentiated hematopoietic stem cells that are CD45 antigen-positive, CD34 antigen-negative, and lineage-negative, comprising a porous structure in a container having a liquid inlet and a liquid outlet. An apparatus for separating human undifferentiated hematopoietic stem cells, which is a filter filled with a cell capturing material.