JP2003116521A - Method and apparatus for separating and concentrating sp cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and an apparatus for concentrating SP cells simply and inexpensively in a short time. SOLUTION: This method for separating and concentrating SP cells comprises introducing a nucleated cell-containing solution containing SP cells into a filter which catches nucleated cells and passes erythrocyte and then introducing a liquid into the filter to recover the SP cells caught by the filter. This apparatus therefor is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and apparatus for enriching SP cells. The obtained SP cells can be used for basic science fields such as immunology and cell biology, and for treatment of various biological tissue lesions and / or defects.

[0002]

2. Description of the Related Art SP cells are described by Goodel et al. (J. Ex.
p. Med. vol. 183, 1996) undifferentiated cells, and when analyzed by flow cytometry, when a fluorescent dye called Hoechst33342 was incorporated into the cells and excited by UV, 405 nm and 60 nm were obtained.
Appeared at a different position on the cytogram from normal cells that fluoresce at 0 nm (cells other than undifferentiated cells) (dark part of fluorescence at the lower left side, ie, "Hoechst Blue weak positive and Hoechst Red weak positive") It is a cell population that does. Side Population, which is abbreviated as SP cell, is abbreviated as "Side Population" in the sense of being laterally displaced.

Usually, the above-mentioned cell sorting method (fluorescent cell sorting method) utilizing flow cytometry is used to separate SP cells, but it took a very long time. Therefore, in order to shorten the time, it is theoretically possible to remove differentiated antigen-positive cells by magnetic beads on which a monoclonal antibody is immobilized (negative selection) as concentration before applying to cell sorting, but this is also complicated. An expensive and time-consuming method, a simple and inexpensive method for concentrating SP cells in a short time has been desired.

By the way, in Japanese Patent Laid-Open No. 2000-166541, a filter that substantially captures nucleated cells and substantially passes erythrocytes is positive for CD45 antigen and CD34.
A liquid containing nucleated cells containing nucleated cells that are negative and different from the differentiation antigen is introduced, and then the liquid is introduced into the filter to capture the CD45 antigen-positive, CD34 antigen-negative, and differentiation antigen-negative liquids. Although a method for separating human undifferentiated hematopoietic stem cells, which is characterized by recovering nuclear cells, is disclosed, there is no description suggesting enrichment of SP cells in this publication.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and a device for concentrating SP cells in a simple and inexpensive manner in a short time.

[0006]

Means for Solving the Problems The inventors of the present invention have made extensive studies in order to solve such problems. We have
It is an approach that is usually used in such fields,
We believe that it would be extremely difficult to solve the problems of simple, inexpensive, and short-time operation by developing a separation technique using a surface antigen such as a new monoclonal antibody, and tried to solve it by a completely new technical means that does not use a monoclonal antibody. . That is, the present inventors have made a deep consideration from a developmental biology point of view, not from a cell biology / immunological point of view, and SP cells are similar to the aforementioned CD45-positive, CD34-negative, and differentiation antigen-negative hematopoietic stem cells. We made a bold hypothesis that it might have the above behavior and conducted an examination. Specifically, SP
Focusing on the adhesiveness of cells, SP cells have an adhesion behavior that is common to nucleated cells in general, but the desorption behavior is much higher than that of normal nucleated cells and they are easily desorbed, and are CD45-positive, CD34-negative, and differentiation antigens. As a result of various studies under the assumption that it may have a property similar to that of negative hematopoietic stem cells, as with CD45-positive, CD34-negative, and differentiation antigen-negative hematopoietic stem cells, a filter that captures nucleated cells SP The present invention has been completed by discovering a surprising effect that cells can be concentrated at an extremely high rate.

That is, the present invention captures nucleated cells,
The present invention relates to a method for separating SP cells by introducing a nucleated cell-containing liquid containing SP cells into a filter that passes red blood cells. Further, the present invention is characterized in that a nucleated cell-containing liquid containing SP cells is introduced into a filter that passes red blood cells, and then the liquid is introduced into the filter to recover the SP cells captured by the filter. This is a method for separating and concentrating SP cells. Furthermore, the present invention is a device for separating and concentrating SP cells, wherein a container having a liquid inlet and a liquid outlet is filled with a nucleated cell trapping material that traps nucleated cells and passes red blood cells. And a means for connecting a means for injecting a solution containing nucleated cells containing SP cells upstream of the filter, a means for injecting a liquid for recovering the SP cells from the filter, A device including a means for collecting SP cells. Furthermore, the present invention also relates to an SP cell-containing liquid recovered from a filter that captures nucleated cells and passes red blood cells. In the present invention, as described above, an unexpected result that SP cells can be separated and concentrated by a simple means of using a filter that captures nucleated cells and passes red blood cells can be obtained.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The nucleated cell referred to in the present invention refers to a cell having a nucleus in the cell, specifically, white blood cells, granulocytes, neutrophils, eosinophils,
Basophils, myeloid cells, erythroblasts, lymphocytes, T lymphocytes,
Examples thereof include B lymphocytes, monocytes, hematopoietic stem cells, hematopoietic progenitor cells and the like.

In the present invention, "capturing nucleated cells and passing red blood cells" means substantially trapping nucleated cells and substantially passing red blood cells. It refers to capturing 60% or more of nucleated cells in a liquid containing nucleated cells and passing 60% or more of red blood cells in a liquid containing nucleated cells. For example, a filter that captures nucleated cells and allows red blood cells to pass through is provided with, for example, a nucleated cell capturing material composed of a porous structure that substantially captures nucleated cells and substantially allows red blood cells to pass through the liquid inlet. An example is a container filled with a liquid outlet.

As a material for capturing nucleated cells and passing erythrocytes, any commonly used cell-trapping material can be used, but it is preferable in terms of low moldability, sterility and cytotoxicity. For example, polyester, polyethylene, polypropylene, polystyrene,
Synthetic polymers such as acrylic resin, nylon, polycarbonate, polyurethane, natural polymers such as cellulose, cellulose acetate, chitin, chitosan and alginate, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel, titanium, aluminum, etc. The metal is. Above all, it is more preferable to use polyester, polyethylene, polypropylene, or polyurethane that is easily available for medical use and is easily processed into a capturing material having a preferable shape.

These capturing materials can be used as they are, but may be surface-modified if necessary for the purpose of enhancing selective passage of cells. For example, in order to enhance platelet permeability, there is a method proposed in WO87 / 05812 by coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group.
In addition, amino acids, peptides, sugars, glycoproteins, etc. (antibodies,
(Including bioligands such as adhesion molecules)
For example, the method of fixing by the haloacetamide method proposed in JP-A-2-261833 can be preferably used.

Examples of the shape of the trapping material include granules, fiber lumps, woven fabrics, non-woven fabrics, sponge-like porous bodies, flat plates, and the like. , Non-woven fabric, sponge-like porous body is preferred,
In addition, a nonwoven fabric and a sponge-like porous body are more preferable from the viewpoint 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 more and 20 μm or less, and more preferably 1.5 μm
It is 10 μm or less. If it is less than 1.0 μm, SP cells, which are the target cells, are firmly captured, and concentration may be difficult, which is not preferable. If it exceeds 30 μm, SP cells are more likely to pass through without being captured by the nonwoven fabric. In either case, the concentration rate may be reduced, which is not preferable.

In the case of a sponge-like structure, the pore size is usually 2.0 μm or more and 30 μm or less, preferably 2.
It is 5 μm or more and 25 μm or less, and more preferably 3.0 μm or more and 20 μm or less. If it is less than 2.0 μm, the flowability is remarkably poor, and it may be difficult to pass the liquid. If it exceeds 25 μm, the capture rate of SP cells may be decreased, which may lead to a decrease in concentration rate, which is not preferable.

Preferred examples of the material for the container filled with the material for capturing nucleated cells that captures nucleated cells and passes erythrocytes are preferable in terms of moldability, sterility and low cytotoxicity. Ropylene, polystyrene,
Examples thereof include synthetic polymers such as acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane and vinyl chloride, inorganic materials such as hydroxyapatite, glass, alumina and titania, and metals such as stainless steel, titanium and aluminum.

The nucleated cell-containing liquid referred to in the present invention is, for example, bone marrow, umbilical cord blood (including blood collected from not only umbilical cord blood vessels but also placental blood vessels), peripheral blood (hematopoietic factors such as granulocyte colony stimulating factor). (Including blood collected by administration of) and those that have been subjected to other treatments such as centrifugation, or cells extracted from various tissues such as kidneys and various organs and muscles by enzyme digestion, etc. Something is floating. Other processes such as centrifugation include freeze-thawing. According to the study conducted by the present inventors, good results were obtained from the mononuclear cell fraction obtained by subjecting bone marrow to specific gravity centrifugation. Preference is given to using the nuclear fraction.

In the present invention, any liquid can be used as a liquid for collecting the captured SP cells as long as it does not adversely affect the cells, but some examples are physiological saline and D-PBS. Buffer solution such as or HBSS,
Examples of the medium include RPMI1640. These liquids are used for cell protection, nutritional supplementation, frost damage prevention during cryopreservation,
EDTA, dextran, hydroxyethyl starch, dimethylsulfoxide, albumin, globulin, gelatin, glucose, saccharose, trehalose, citric acid compounds, polyvinyl as necessary for the purpose of improving viscosity (which may be effective in improving recovery rate). Pyrrolidone, glycerin, chitin derivatives and the like may be added.

The liquid referred to herein includes not only a liquid alone but also a mixture of air, argon, nitrogen and other gases that do not adversely affect cells. The liquid may be introduced in the same direction as the liquid passing through for the first filtration or in the reverse direction, but the reverse direction is more preferable because a higher recovery rate tends to be obtained. In addition, before introducing the liquid for recovery, rinsing may be performed for the purpose of washing away a small amount of contaminating cells remaining in the filter. As the rinsing solution, any liquid can be used as long as it does not adversely affect cells, but some examples include physiological saline, Dulbecco's phosphate buffer (D-PBS) and Hanks' solution (HBSS). Examples of the medium include buffer solutions and RPMI1640. The rinse liquid may be introduced in the same direction or in the opposite direction to the first liquid passing direction, but the same direction is more preferable because the trapped cells are less likely to leak.

The SP cell referred to in the present invention is the Go of the discoverer.
As defined by Odel et al., when a fluorescent dye called Hoechst33342 was taken up by cells and analyzed by flow cytometry and excited by UV, 405 nm and 600 n were detected.
Normal cells that fluoresce in m (cells other than undifferentiated cells)
Therefore, it refers to a cell population that appears at different positions on the cytogram (dark portion of the lower left lateral fluorescence, that is, weak positive for Hoechst Blue and weak positive for Hoechst Red).

The SP cells obtained by the present invention are as they are,
Or if necessary, further separation and purification, culture, activation,
After being subjected to various treatments such as differentiation induction, amplification, gene transfer, and cryopreservation, it is used in the fields of basic science such as immunology and cell biology, and various biological tissue lesions and / or defects.

[0021]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

First Embodiment 1. Cell concentrator Polyester nonwoven fabric with an average fiber diameter of 2.3 μm
0 g / m ² , bulkiness of about 0.3 mm) 18 sheets and average fiber diameter 1
2 μm polyester non-woven fabric (Basis weight: approx. 100 g / m ² ,
16 pieces (bulkiness of about 0.47 mm) were stacked and cut into 35 mm square with a push-cutting cutter to obtain a material for capturing nucleated cells. This nucleated cell capture material is placed on the outside of the container (length x width x thickness) 41 x 41 x 1
A filter (1) was prepared by filling a polycarbonate container having a liquid outlet and a liquid inlet on a diagonal line of 8 mm so that a polyester nonwoven fabric having an average fiber diameter of 12 μm would come to the outlet side. A three-way stopcock having a spike (2) for connecting a means for injecting a liquid containing nucleated cells at the inlet side of the filter and a branch to a collection bag (6) which is an SP cell recovery means on the way. The tube having (4) was connected. Further, a three-way stopcock (5) was provided on the outlet side of the filter (1), and a tube whose end was connected to the drain bag (3) was connected to the SP cell concentrator shown in FIG.

2. Bone marrow from rat legs (femur and tibia) using Dulbecco's phosphate buffer (hereinafter, D-PBS) containing 2% fetal calf serum (hereinafter, FCS) supplemented with 2 μg / ml of raw material cell suspension gentamicin And collect this with NYCOMED PHA
Product name LY made by RMAA (Oslo, Norway)
A mononuclear cell fraction was separated by a known specific gravity centrifugation method using MPHOPREP, and diluted with D-PBS containing 2% FCS to obtain 100 ml of a raw material cell suspension.

3. Cell concentration procedure 1. 1. In the spike (2) of the SP cell concentrator prepared in 2. A 200 ml blood bag (hereinafter, referred to as a raw material bag) containing the raw material cell suspension of was connected. The three-way stopcock (4) is in the direction in which only the raw material bag and the filter (1) are in communication, and the three-way stopcock (5) is in the direction in which only the filter (1) and the drain bag (3) are in communication. The liquid was filtered through the drop, and the liquid flowing out from the filter was drained into the drain bag (3). Next, 25 ml of D-PBS containing 2% FCS was placed in the three-way stopcock (5) 3
An injection means (7) for SP cell recovery liquid consisting of a 0 ml syringe (luer lock port) is connected, the three-way stopcock (5) is oriented so that only the syringe and the filter (1) communicate, and the three-way stopcock (4) is a filter. Only the (1) and the collection bag (6) communicated with each other. Next, the cells captured by the filter (1) were collected in the collection bag (6) by pushing the plunger of the syringe (7) by hand. The time required for these operations was about 3 minutes.

4. Analysis Before and after filtering cells,
Goodell, MAet al: “Isolat
ion and Functional Proper
ties of Murine Hematopoie
tic Stem Cells that are R
"Elicating In Vivo", J. Ex.
p. Med. , Vol. 183, 1797-1806
Analysis was performed according to the SP cell analysis method by flow cytometry described in 1996, and Hoechst Bl was used.
The abundance was calculated by setting the cell population of ue weak positive and Hoechst Red weak positive as SP cells. Figure 2 shows the flow cytometry cytogram of the cells before filtering.
The flow cytometry cytogram of the cells after the filter treatment is shown in FIG. In FIGS. 2 and 3, the portion surrounded by the line is the SP cell dot.

The concentration factor was calculated by the following formula. Here, the SP cell abundance rate before the filter treatment is
The abundance of SP cells in all the cells contained in the nucleated cell-containing solution before introduction into the SP cell separating and concentrating device and the SP cell abundance after filtering are the S in all cells contained in the recovery solution.
It is the abundance rate of P cells. Concentration factor = SP cell abundance after filter treatment / SP cell abundance before filter treatment

5. Results The results are summarized in Table 1. S by filtering
It can be seen that P cells are highly enriched.

[Table 1]

[0027]

Industrial Applicability As described above, according to the present invention, it is possible to provide a method for concentrating SP cells in an extremely simple and short-time operation. Therefore, the field of basic science such as immunology and cell biology, and various biological tissue lesions It is of great importance to contribute to the development of the field of treatment of defects and / or defects.

[Brief description of drawings]

FIG. 1 is a schematic diagram of an SP cell concentrator of Example 1.

FIG. 2 is a flow cytometry cytogram before filtering.

FIG. 3 is a flow cytometry cytogram after filtering.

[Explanation of symbols]

1 filter 2 spikes 3 drain bag 4 three-way stopcock 5 three-way stopcock 6 SP cell recovery bag 7 SP cell recovery liquid injection means

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/48 G01N 33/48 S C12N 5/00 EF term (reference) 2G045 BA13 BB04 BB10 BB14 BB31 BB41 CA02 CA11 CA25 FA37 HA06 2G052 AA30 AA31 AA33 AB18 AB20 AD29 AD52 CA03 CA08 DA09 EA03 EA04 ED06 ED17 FD01 GA11 GA29 GA33 JA07 JA11 JA14 JA15 JA16 4B065 AA93X AC20 BA21 BD15 BD18 CA46

Claims (8)

[Claims] 1. A nucleated cell-containing liquid containing SP cells is introduced into a filter that traps nucleated cells and allows red blood cells to pass through SP.
How to separate cells. 2. An SP cell trapped in the filter by capturing the nucleated cells, introducing a liquid containing SP cells containing the nucleated cells into a filter that passes red blood cells, and then introducing the liquid into the filter. A method for separating and concentrating SP cells, which comprises recovering 3. The method according to claim 1, wherein the nucleated cell-containing solution containing SP cells has been pretreated by specific gravity centrifugation. 4. The method according to claim 1, wherein a filter filled with one or more of polyester, polyethylene, polypropylene and polyurethane is used. 5. A container having a liquid inlet and a liquid outlet,
A device for separating SP cells, characterized in that a filter filled with a material for capturing nucleated cells that captures nucleated cells and passes erythrocytes is used. 6. A filter in which a container having a liquid inlet and a liquid outlet is filled with a material for capturing nucleated cells, and a liquid containing nucleated cells containing SP cells upstream of the liquid inlet of the filter is injected. An apparatus for separating and concentrating SP cells, comprising connecting means for connecting the means, means for injecting a liquid to recover SP cells from the filter, and means for recovering SP cells. 7. The device according to claim 5, wherein one or more kinds of polyester, polyethylene, polypropylene and polyurethane are used as the material for capturing nucleated cells. 8. An SP cell-containing liquid that has captured nucleated cells and has been recovered from a filter that passes red blood cells.