JPH11266852A - Cell separator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separator capable of recovering necessary cells from a mixture of the necessary cells with unnecessary cells in a high ratio by a simple operations in a short time, the necessary cells by capturing the necessary cells from a cell mixture solution in a cell capturing means such as a filter once and then recovering the captured cells. SOLUTION: This cell separator is used for a method for separating cells comprising introducing a cell-containing liquid into a cell capturing means for substantially capturing only the cells requiring the recovery, leading out the liquid containing the cells which are the object for the removal from the cell capturing means, then introducing a liquid into the cell capturing means and recovering the cells captured in the cell capturing means and requiring the recovery. The cell separator is equipped with a flat container having at least two liquid introduction port/lead-out port and a cell capturing material is filled so as to separate the introduction port for introducing the cell-containing liquid containing the cells requiring the recovery and the cells which are the object for the removal from the lead-out port for leading out the cells which are the object to be removed. The length (a) of a straight line connecting a point A where the cell-containing liquid initially comes into contact with the cell capturing material to a point B where the liquid introduced in order to recover the cells initially comes into contact with the cell capturing material is <=80% of the length (b) of a straight line connecting the point A and C which is the farthest in the cell capturing material from the point A.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an instrument for separating and recovering necessary cells from a mixed solution of various cells. The obtained 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 Japanese Patent Laid-Open No. 54-1 discloses a technique for collecting leukocytes by capturing leukocytes from a body fluid such as blood containing leukocytes (granulocytes, monocytes, lymphocytes) and erythrocytes with a filter.
No. 19012 discloses this. In hematopoietic stem cell transplantation, umbilical cord blood stem cells have attracted attention as a source of hematopoietic stem cell transplantation without donor invasion, and clinical application has been attempted in Europe and the United States. Umbilical cord blood stem cells
Since it is rare to be transplanted to a patient immediately after being collected from a donor like other hematopoietic stem cell transplants, ie, bone marrow transplantation or peripheral blood stem cell transplantation, it is necessary to preserve it from the time of collection to the time of use. Yes, especially for unrelated transplants. By the way, it is said that nucleated cells should be separated (removed red blood cells) in order to prevent side effects of destructed red blood cells after thawing and to reduce the volume during frozen storage during cryopreservation. Separation and storage are common (Nankodo, “Peripheral Blood Stem Cell Transplantation”, p. 173). Japanese Patent Publication No. 8-69 discloses the separation of umbilical cord blood by Ficoll-hypaque (centrifugation using a specific gravity solution) method (hereinafter abbreviated as Ficoll method) and details of a protocol thereof. However, the Ficoll method has a problem that it is an extremely complicated and time-consuming operation at the laboratory level. WO 96/17514 discloses a bag system, a method and a cell solution obtained by agglutinating and separating erythrocytes in cord blood using hydroxyethyl starch to obtain a nucleated cell concentrate. Although this method is slightly improved in comparison with the conventional Ficoll method in terms of complicated operations, it also requires a long centrifugal separation and therefore requires a long operation. By the way, hematopoietic stem cell separation methods instead of the Ficoll method and the hemagglutination removal have come to be seen occasionally. Japanese Patent Application Laid-Open No. 8-104463 discloses a method in which red blood cells are captured by a filter through which hematopoietic stem cells pass, and then a liquid flow is induced in a direction opposite to the first liquid flow direction to recover the cells. However, there is no description of containers suitable for recovery. JP-A-6-19
No. 7962 discloses a leukocyte removal filter for preventing side effects of blood transfusion, in which both the blood inlet and the blood outlet are located at the upper end of the container. However, the technical problem of the publication is how to easily perform a complicated operation of storing the filter and the blood bag in a centrifugal cup when the filter and the blood bag are placed in a centrifugal cup. The purpose of this study was to focus on the shape of the filter's container, with the aim of preventing it, and to capture the cells once in the filter, and how to increase the recovery rate when collecting the cells is a technical issue. It is a completely different technical idea.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a cell separator for recovering required cells from a mixture of required cells and unnecessary cells at a high rate by a simple and short-time operation. It is an object of the present invention to provide a cell separator capable of collecting required cells at a high rate in a cell separation method for once capturing required cells by a cell capturing means such as a filter and collecting the captured cells.

[0004]

Means for Solving the Problems In order to solve such problems, the present inventors, from the viewpoint of high recovery of captured cells,
As a result of repeated studies, it was surprisingly found that the distance between the cell-containing liquid inlet and the inlet of the liquid to be introduced for recovering cells affects the cell recovery rate, and the distance is such that the cell-containing liquid is A very high cell recovery rate is obtained when the length of the straight line connecting the point that first contacts the cell trapping material and the farthest point in the cell trapping material from the contact point is 80% or less. After confirmation, the present invention has been completed.
In other words, the present invention provides a cell-containing solution containing cells to be recovered and cells to be removed, which substantially captures the cells to be recovered, and the cells to be removed are introduced into a cell capturing means that substantially passes through, and the cells to be removed are removed. A cell separator for use in a cell separation method in which a liquid containing the liquid is introduced from the cell capturing means, and a liquid is introduced into the cell capturing means to collect the cells required for recovery which are captured by the cell capturing means. A flat container having at least two liquid inlets / outlets, filled with a cell trapping material so as to separate the inlet for introducing the cell-containing liquid and the outlet for extracting the cells to be removed, and The length of a straight line a connecting the point A at which the cell-containing liquid first contacts the cell trapping material and the point B at which the liquid introduced to collect the cells first contacts the cell trapping material is A and Farthest in cell trapping material from A A cell separator, characterized in that the straight line connecting the C is less than 80% of the length b.

Hereinafter, the present invention will be described in detail. The cells requiring recovery referred to in the present invention refer to cells that are separated and recovered and used for some purpose, and the cells to be removed are unnecessary for this purpose or are cells that need to be recovered because they are any pathogenic cells. This refers to cells that need to be actively removed because contamination causes a problem. Cell-containing liquids containing these include peripheral blood, bone marrow, umbilical cord blood (including not only those collected from umbilical cord blood vessels, but also those collected from placental blood vessels), lymph, and those subjected to some treatment such as centrifugation. Or re-suspended in some liquid after extraction from various organs or tissues. In the present invention, the cell capturing means that captures at least the cells to be recovered and substantially passes the cells to be removed is, for example, a liquid flow filled with a material that captures the cells to be recovered and passes the cells to be removed substantially. Refers to a container having an inlet and a liquid outlet. Any material can be used as long as it can capture the cells required for recovery and substantially pass through the cells to be removed, as long as it can selectively capture the cells required for recovery. Preferred examples in terms of low sterility and low cytotoxicity include polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, synthetic polymers such as polyurethane, agarose, cellulose, cellulose acetate, chitin, Natural polymers such as chitosan and alginate; hydroxyapatite; glass; inorganic materials such as alumina and titania; and metals such as stainless steel, titanium and aluminum. These trapping materials can be used as they are, but may be surface-modified as necessary, such as for selectively passing or trapping cells. For example, a method of coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group proposed in WO 87/05812 to improve platelet permeability can be mentioned. When cells are selectively captured, ligands having an affinity for specific cells, such as amino acids, peptides, saccharides, glycoproteins (including bioligands such as antibodies and adhesion molecules), can be used, for example, as disclosed in
For example, there is a method of fixing by the haloacetamide method proposed in Japanese Patent No. 261833. Examples of the shape of the trapping material include granules, fiber masses, woven fabrics, nonwoven fabrics, sponge-like structures, flat plates, and the like. However, granules, fiber masses, woven fabrics, nonwoven fabrics, sponge materials, and the like have a large surface area per volume. -Like structure is preferable, and further from the point of handleability, fiber mass,
A porous structure such as a woven fabric, a nonwoven fabric, and a sponge-like structure is preferable, and among them, a nonwoven fabric and a sponge-like structure are more preferable in terms of the flowability of cell liquid and the productivity. In the case of a non-woven fabric, when a so-called bioligand that specifically binds to a specific cell, such as an anti-CD34 monoclonal antibody, is not fixed on the surface, the fiber diameter is usually 1.0 μm or more and 30 μm or less, preferably 1.0 μm or more. It is 20 μm or less, still more preferably 1.5 μm or more and 10 μm or less. If it is less than 1.0 μm, cells that need to be collected may be firmly captured and may be difficult to collect, which is not preferable. If it exceeds 30 μm, the cells that need to be collected are more likely to pass through without being captured by the fibers. In either case, the recovery rate may be reduced, which is not preferable.
In the case of a sponge-like structure, the pore size is usually 2.0 μm.
Not less than 25 μm and preferably not less than 3.0 μm 2
0 μm or less, and still more preferably 4.0 μm or more and 15 μm or less. If it is less than 2.0 μm, the flowability is remarkably inferior, and it may be difficult to pass the liquid itself.
If it exceeds μm, the capture rate of the cells requiring recovery is reduced, and the recovery rate is reduced, which is not preferable. Examples of the material of the container that captures the cells that need to be collected and that is filled with a material through which the cells to be removed substantially pass are polyethylene, polypropylene, and polystyrene. Synthetic resins 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. However, the present invention is not limited to this.

In the present invention, "substantially capture cells required for recovery" means to capture at least 60% of cells required for recovery in a cell-containing solution, and "substantial passage of cells to be removed". This means that the cells to be removed in the cell-containing liquid pass through 60% or more. In the present invention, any liquid can be used as a liquid for recovering the cells required for recovery that have been captured by being introduced into the cell capturing means, as long as it is a physiological solution. PBS and HB
Examples include a buffer such as SS and a medium such as RPMI1640. These physiological solutions add cell protection, nourishment,
Dextran, hydroxyethyl starch, dimethyl sulfoxide, albumin, globulin, gelatin, glucose, saccharose, trehalose, etc. may be used for protection against frost damage during cryopreservation and for improving viscosity (may be effective for improving recovery rate). It may be added. The cell separator according to the present invention has at least two liquid inlets / outlets. The liquid inlet / outlet introduces a cell-containing liquid containing cells to be collected and cells to be removed into the cell separator. This is used to remove the cells to be removed from the cell separator, or to introduce a liquid to collect the cells captured by the cell separator, and may be used in common. The structure of the inlet / outlet may be a flat container having only a hole or a nozzle type. Those having only holes are advantageous in that they have a simple structure and are easy to manufacture, but are disadvantageous in that they are difficult to connect tubes and are easy to come off, so that a nozzle type is preferable. The term “flat container” as used in the present invention means a container in which the shorter one of the length and width is at least twice, preferably at least four times the thickness. The point A at which the cell-containing liquid first contacts the cell trapping material in the present invention refers to a point at which the cell-containing liquid comes into contact with the cell trapping material first when the cell-containing liquid is introduced into the cell separator through the liquid inlet. Further, the point B at which the liquid to be introduced for collecting the cells referred to in the present invention first comes into contact with the cell trapping material is defined as a point at which the collected liquid is first introduced into the cell trapping material when the collected liquid is introduced into the cell separator through the liquid inlet. Say the point of contact. The present invention is characterized in that the length a of the straight line connecting A and B is 80% or less of the length b of the straight line connecting A and A to the farthest point C in the cell trapping material. However, preferably a is no more than 50% of b, and even more preferably a is no more than 20% of b.

[0007] The cell separator according to the present invention is suitably used when the cells requiring recovery are nucleated cells. Nucleated cells are cells that have a nucleus in the cell, such as leukocytes,
Granulocytes, neutrophils, basophils, eosinophils, myelocytes, erythroblasts,
Lymphocytes, T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, suppressor T lymphocytes, B lymphocytes,
Examples include NK cells, NKT cells, monocytes, macrophages, osteoclasts, osteoblasts, osteocytes, hematopoietic stem cells, fibroblasts, chondroblasts and the like. Further, the hematopoietic stem cell-containing mononuclear cell fraction referred to in the present invention refers to a mononuclear cell population containing hematopoietic stem cells and / or hematopoietic progenitor cells (hereinafter, simply abbreviated as hematopoietic stem cells). Mononuclear cells are a general term for cells having one nucleus in the cell, and specifically include lymphocytes (T cells, B cells, NK cells), monocytes, hematopoietic stem cells, myeloid cells, blast cells, and the like. can give. The hematopoietic stem cell content in this mononuclear cell population is usually 0.01% to 99%, and the content differs depending on the type of the raw material cell population and the presence or absence of cell treatment. For example, it is usually around 0.01% in normal human peripheral blood, 0.05 to 1.0% in cord blood, and 0.5 to 2% in bone marrow. In addition, in the peripheral blood to which G-CSF (granulocyte colony stimulating factor) has been administered, individual differences are remarkable, being 0.1% to several%. When cells are separated by a monoclonal antibody, particularly by flow cytometry, the content may reach as high as 99%. In any case, the term hematopoietic stem cell-containing mononuclear cell fraction does not define the content of hematopoietic stem cells. The combination of the cells requiring collection and the cells to be removed according to the present invention is, for example, the cells requiring collection are nucleated cells, and the cells to be removed have no nucleus, that is,
Examples include, but are not limited to, erythrocytes and platelets, and cases where the cells requiring collection and the cells to be removed have different surface markers. Examples of combinations of cells requiring recovery and nucleated cells and cells to be removed having no nucleus and uses thereof are shown below, but are not limited thereto. 1. 1. Cells requiring recovery: leukocytes, cells to be removed: red blood cells, use: interferon production 2. Cells requiring recovery: lymphocytes, cells to be removed: red blood cells and platelets, use: adoptive immunotherapy Cells to be collected: Hematopoietic stem cell-containing mononuclear cell fraction, cells to be removed: erythrocytes and platelets, use: hematopoietic stem cell transplantation Examples of combinations of nucleated cells and uses thereof are shown below, but are not limited thereto. 1. 1. Cells requiring recovery: CD34-positive nucleated cells, cells to be removed: CD34-negative nucleated cells, use: CD34-positive cell transplantation Cells required for collection: CD8 + T lymphocytes, cells to be removed: CD8-negative T lymphocytes, use: adoptive immunotherapy In addition, cells required to be recovered are nucleated cells, cells to be removed have no nucleus, and cells required to be recovered Examples of combinations of nucleated cells having different surface markers and their uses are shown below, but are not limited thereto. 1. Cells required for collection: CD34-positive nucleated cells, cells to be removed: red blood cells, platelets, CD34-negative nucleated cells, use: C
1. D34 positive cell transplantation Cells requiring collection: CD8 + T lymphocytes, target cells to be removed: erythrocytes, platelets, CD8-negative T lymphocytes, use: adoptive immunotherapy. The inventors presume as follows. When the cell-containing liquid was introduced into the cell separator, the number of cells captured by the cell-capturing material was near the inlet into which the cell-containing liquid was introduced, that is, the point at which the cell-containing liquid first contacted the cell-capturing material. It is thought that there are many surroundings, and the farther from the point, the smaller. On the other hand, introducing liquid
The linear velocity of the collected liquid, which is important when collecting the captured cells, is near the liquid inlet, that is, the point where the collected liquid first contacts the cell capturing material, and the distance from that point is the fastest. It is thought that it gets slower as it gets farther. Therefore, by performing the introduction of the recovery liquid in the vicinity of the introduction port into which the cell-containing liquid has been introduced,
We believe that high recovery of cells may be achieved.

[0008]

EXAMPLES 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 This example is an example of cell separation in the case where the cell-containing liquid is cord blood, the cells requiring collection are hematopoietic stem cell-containing mononuclear cell fractions, and the cells to be removed are red blood cells and platelets. Cell separator The outer dimensions of the container (length × width × thickness) 41 × 41 × 18 mm, connecting the point A where the cell-containing solution first contacts the cell trapping material and the point C farthest in the cell trapping material from A Straight line length b is 4
In a 2 mm flat container made of polycarbonate, a straight line connecting a point A at which the cell-containing liquid first contacts the cell trapping material and a point B at which the liquid to be introduced to collect the cells first contacts the cell trapping material. Each length a is 14 mm (about 3 of b)
3%), 17mm (40%), 32mm (76%)
The liquid inlet / outlet was created so as to be as follows (FIGS. 1 to 3). 1. The average fiber diameter is 2.
Twelve 3 μm polyester non-woven fabrics were placed on the side of the cell exit port to be removed, and a polyester non-woven fabric 25 having an average fiber diameter of 12 μm was used.
The sheets were filled to produce three types of cell separators. The packing density was 0.24 g / cm ³ , the effective filtration area was 9 cm ² , and the effective filtration length was 12.4 mm. Further, for the purpose of imparting platelet permeability to this filter, coating with a hydrophilic polymer was performed. 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. Preparation of Recovered Liquid A commercially available dextran 40 physiological saline solution (trade name: Dextran 40 Injection-Midori, manufactured by Midori Cross) was prepared to contain 4% human serum albumin, and used as a recovered liquid. Cell separation operation and circuit system 15 vol% CP collected from placenta and umbilical cord after delivery
150 mL of umbilical cord blood containing D was divided into three equal parts to obtain a cell-containing liquid. As shown in FIG. 4, a blood bag 2 is connected to a three-way stopcock 4 to which a cell collection bag 5 is connected in the middle, a mesh chamber 3, and a rinsing saline bottle.
This was connected to the cell-containing liquid inlet side of the cell separator 1 prepared in the above.
The outlet of the cell separator 1 to be removed was connected to the drain bag 7 with a tube having a three-way cock 6 for connecting a syringe for recovery. The cell-containing liquid in the blood bag 2 was passed through the cell separator at a head of about 60 cm, and the red blood cell and platelet-containing liquid flowing out of the cell separator 1 was drained into the drain bag 7. Next, a physiological saline bottle was pierced into the spike 8, the clamp 9 was opened, and the inside of the cell separator was rinsed with about 20 mL of physiological saline, and a small amount of remaining red blood cells and platelets were washed away. Next, a 30 mL disposable syringe containing 25 mL of the recovery liquid is connected to the three-way cock 6, the three-way cock 6 is turned in a direction in which only the syringe and the cell separator communicate, and the three-way cock 4 is used for cell collection and cell collection. After the bag 5 is turned in the direction in which only the bag 5 communicates, the syringe is pushed to remove the cells captured in the cell separator 5.
Collected. Analysis The number of nucleated cells, the number of mononuclear cells, the number of red blood cells, and the number of platelets are measured by an automatic hemocytometer. The CD34 positive rate in nucleated cells is determined by FIT.
The measurement was carried out using a C-labeled anti-CD34 antibody and a flow cytometry method developed on SSC-FITC (Miyazaki et al .: Daily Practice and Blood, Vol. 5, No. 2, 21-24, 1995). The method of calculating the recovery rate and the removal rate is as follows. Recovery (%) = 100 × (number of cells after collection / number of cells in cell-containing solution) Removal rate (%) = 100−100 × (number of cells after collection / number of cells in cell-containing solution) Results Summary of results Are shown in Table 1. In each case, nucleated cells, mononuclear cells, and CD34-positive cells were recovered at a high rate in the recovered cell liquid, indicating that erythrocytes and platelets were removed at a high rate. In addition, the cells recovered with this recovery solution could be cryopreserved according to the protocol indicated in the instruction manual for the cryopreservative “CP-1” manufactured by Far East Pharmaceutical Co., Ltd. That is, after dimethyl sulfoxide was added to a final concentration of 5%, the mixture was frozen and stored in a deep freezer at −80 ° C., and thirty days after the freeze storage, rapidly thawed in a warm bath at 37 ° C. When the viability was measured by the blue exclusion method, the viability was maintained at a high value of 90.4%.

[0009]

Comparative Example 1 In this comparative example, as in Example 1, the cell-containing solution was cord blood, the cells required to be collected were hematopoietic stem cell-containing mononuclear cell fractions, the cells to be removed were red blood cells and platelets, and the cell-containing solution was the first. A point A at which the cell-capturing material comes into contact with the cell-capturing material, a length b of a straight line connecting the farthest point C in the cell-capturing material from A; B where the liquid introduced to recover the cells first contacts the cell trapping material
42 mm (100 of b)
%) Is compared with Example 1 by using the cell separator of (%). Cell separator The length b of a straight line connecting the point A where the cell-containing liquid first contacts the cell trapping material and the point C farthest in the cell trapping material from A
And a point A where the cell-containing liquid first contacts the cell trapping material;
The length a of the straight line connecting the point B where the liquid to be introduced for recovering the cells first contacts the cell trapping material is equal to 4
The same cell separator as in Example 1 was used except for 2 mm (100% of b). Cell separation operation and circuit system The same operation method and circuit system as in Example 1 were used. Analysis The same analysis as in Example 1 was performed. Results Table 2 summarizes the results. The recovery rate of nucleated cells, mononuclear cells, and CD34-positive cells in the recovered cell solution was lower than that in Example 1.

[0010]

Example 2 This example is an example of cell separation in the case where the cell-containing solution is cord blood, the cells requiring collection are hematopoietic stem cells (CD34-positive cells), and the cells to be removed are red blood cells and platelets. Cell separator The outer dimensions of the container (length × width × thickness) 41 × 41 × 18 mm, connecting the point A where the cell-containing solution first contacts the cell trapping material and the point C farthest in the cell trapping material from A Straight line length b is 4
A straight line connecting a point A where the cell-containing liquid first contacts the cell trapping material and a point B where the liquid introduced for collecting the cells first contacts the cell trapping material in a 2 mm flat container made of polycarbonate. Length a is 14 mm (about 33
%). Twelve polyester non-woven fabrics having an average fiber diameter of 12 μm were placed on the cell-containing liquid introduction port side of this container, and a mouse anti-human CD34 monoclonal antibody having an average fiber diameter of 2.3 μm (Coulter Co., clone name Immu133,
Abbreviated as CD34 antibody) immobilized polystyrene nonwoven fabric 25
Sheets were filled. The packing density of this filter is 0.2g / c
m ³ . The immobilization of the mouse anti-human CD34 monoclonal antibody on polystyrene is described in JP-A-2-2618.
This was carried out by a known haloacetamide method proposed in JP-A-33-33. That is, for the purpose of activating the polystyrene nonwoven fabric, the above-mentioned polystyrene nonwoven fabric (cut in advance to the dimensions described above) was added to a reaction solution obtained by adding 3.6 g of hydroxymethyliodoacetamide and 25 g of trifluoromethanesulfonic acid to 165 mL of sulfolane at room temperature. For 5 hours to react. After washing the activated non-woven fabric with D-PBS, the activated non-woven fabric is impregnated with 10 mL of a CD34 antibody solution adjusted to 20 μg / mL with D-PBS for 2 hours for the purpose of immobilizing the antibody on the activated non-woven fabric, DP
After washing with BS, vacuum drying was performed to obtain an antibody-immobilized nonwoven fabric. Preparation of Recovery Liquid Prepared in the same manner as in Example 1. Cell separation operation and circuit system Example 1 except that a cell separator was used.
The same operation as described above was performed using the same circuit. Analysis The same analysis as in Example 1 was performed. Results Table 3 summarizes the results. CD in the recovered cell fluid
It can be seen that 34 positive cells were recovered at a high rate, and erythrocytes, platelets, and CD34 negative cells were removed at a high rate.

[0011]

As described above, according to the present invention, useful cells such as hematopoietic stem cells can be recovered at a high rate with a simple and short operation, and thus the cell treatment process in the field of hematopoietic stem cell transplantation or adoptive immunotherapy is performed. It is extremely important to contribute to labor saving in

[Brief description of the drawings]

FIG. 1 is a cell separator of a / b = 33% used in Example 1 (left: front view, right: side view).

FIG. 2 is a cell separator of a / b = 40% used in Example 1 (left: front view, right: side view).

FIG. 3 is a cell separator of a / b = 76% used in Example 1 (left: front view, right: side view).

FIG. 4 is a schematic diagram of the cell separation system used in Examples 1 and 2.

FIG. 5 is used in Comparative Example 1. a / b = 100% cell separator (left: front view, right: side view).

[Explanation of symbols]

 A A point at which the cell-containing solution first contacts the cell trapping material B A point at which the cell collection solution first contacts the cell trapping material C A point farthest from the point A in the cell trapping material 1 Cell separator 2 Blood bag 3 Mesh chamber 4 Three-way stopcock 5 Cell collection bag 6 Three-way stopcock 7 Drain bag 8 Spike 9 Clamp

Claims (3)

[Claims] Claims: 1. A cell-containing solution containing cells to be recovered and cells to be removed is substantially captured by the cells to be recovered, and the cells to be removed are introduced into a cell capturing means that substantially passes through. A cell separator for use in a cell separation method in which a liquid containing the liquid is introduced from the cell capturing means, and a liquid is introduced into the cell capturing means to collect the cells required for recovery which are captured by the cell capturing means. In a flat container having at least two liquid inlets / outlets, a cell capturing material is provided so as to separate an inlet for introducing a cell-containing liquid containing cells to be recovered and cells to be removed and an outlet for extracting cells to be removed. The length of a straight line connecting the point A where the cell-containing liquid is first contacted with the cell trapping material and the point B where the liquid introduced for collecting the cells is first contacted with the cell trapping material. A
Is 80% or less of the length b of the straight line connecting the points A and A to the farthest point C in the cell trapping material. 2. The cell requiring recovery is a nucleated cell.
The cell separator according to claim 1. 3. The cell separator according to claim 2, wherein the nucleated cells are hematopoietic stem cell-containing mononuclear cell fractions.