JP2001136956A - Cell segregation recovery filter and cell segregation recovery material and cell segregation recovery system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-segregating and recovering filter for recovering only needed cells from a cell mixture in a simple operation in a high yield, to provide a cell-segregating and recovering material, and to provide a cell- segregating and recovering system. SOLUTION: This cell-segregating and recovering filter which is used for segregating and recovering recovery-needed cells from a cell group containing the recovery-needed cells and removal-targeted cells and in which a laminated filtration material is charged in a container having a liquid-charging port and a liquid-discharging port, characterized in that >=25% of the total side surface area of the terminal surfaces of the filter medium is sealed in a fluid-tight state. The cell-segregating and recovering material, characterized in that >=25% of the total side surface area of the terminal surface of the laminated filter medium is sealed in a fluid-tight state. The cell-segregating and recovering system using the cell-segregating and recovering filter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell separation / recovery filter, a cell separation / recovery material, and a cell separation method for use in a cell separation method in which specific cells are collected from a variety of cell liquids by being passed through a filter medium and once captured. Regarding the collection system.
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 Cord blood stem cells have attracted attention as a source of hematopoietic stem cell transplantation without donor invasion, and clinical applications thereof have been attempted mainly in Europe and the United States. Umbilical cord blood stem cells are rarely harvested from donors and transplanted to patients immediately, as in other hematopoietic stem cell transplants, i.e., bone marrow transplants or peripheral blood stem cell transplants. (Especially for unrelated transplants). It is said that umbilical cord blood should be separated from nucleated cells and erythrocytes should be removed in order to prevent side effects due to red blood cells that lyse after thawing during cryopreservation and to reduce the volume during cryopreservation. In most cases, isolation and preservation is performed (Nankodo, “Peripheral blood stem cell transplantation”, p. 173). Japanese Patent Publication No. 8-69 discloses details of a protocol for separating umbilical cord blood by a Ficoll-hypaque method (a centrifugal separation method using a specific gravity solution, which is hereinafter abbreviated as a Ficoll method). However, the Ficoll method has a problem that it is an extremely complicated and time-consuming operation at the laboratory level. WO96 / 17514 also discloses that red blood cells in cord blood are coagulated, sedimented and separated using hydroxyethyl starch.
A bag system and a method for obtaining a nucleated cell concentrate and a cell solution obtained by the method are disclosed. This method is slightly improved in comparison with the conventional Ficoll method in terms of reducing complicated operations, but it also requires a long centrifugal separation and therefore requires a long operation. On the other hand, a hematopoietic stem cell separation method instead of the Ficoll method and the hemagglutination method, that is, a so-called filter method in which cells are once captured by a filter medium and then collected, has also been found. Japanese Patent Application Laid-Open No. 8-104463 discloses a method in which hematopoietic stem cells are captured by a filter that allows red blood cells to pass therethrough, and then a liquid flow is induced in a direction opposite to the first flow direction to collect hematopoietic stem cells. . However, there is no mention of a study on a filter medium structure for improving the recovery rate of captured cells. Also, JP-A-10-137557
Japanese Patent Application Laid-Open No. 11-178920 discloses a method in which a filter medium is compressed to capture cells that need to be recovered, the compressed state is released, and a liquid is introduced into the filter medium to recover the captured cells. However, the operation was troublesome, and the structure of the filter was complicated. Japanese Patent Application Laid-Open No. 7-267871 discloses a white blood cell filter material in which a welded portion is formed in a blood filter material and a plurality of blood circuits are formed in the blood filter material. However, the publication discloses that, when removing leukocytes from blood for transfusion at a high rate (to prevent the side effect of blood transfusion caused by leukocytes), the end face of the filter medium is prevented in order to prevent blood leakage from the end face of the filter medium, which causes a reduction in the removal rate. This is a completely different technical idea from the present application in which the captured cells are recovered at a high rate.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to recover from a mixture of cells that need to be recovered by simple operation (hereinafter abbreviated as cells requiring recovery) and unnecessary cells (hereinafter abbreviated as cells to be removed). An object of the present invention is to provide a cell separation / collection filter, a cell separation / collection material, and a cell separation / collection system for collecting necessary cells at a high rate.

[0004]

Means for Solving the Problems The present inventors have intensively studied to solve such a problem. As a result, the present inventors have found a surprising effect of improving the collection rate of cells required for collection by sealing the end faces of the stacked filter media in the cell separation / collection filter in a liquid-tight manner, and completed the present invention. That is, the present invention provides a method for separating and collecting cells required for recovery from a cell population containing cells required for recovery and cells to be removed, in which a stacked filter medium is filled and packed in a container having a liquid inlet and a liquid outlet. In the filter, a cell separation / recovery filter is characterized in that at least 25% of the entire area of the end face of the filter medium is sealed in a liquid-tight manner, and the present invention also relates to the total area of the end face of the stacked filter medium. 2
A cell separation and recovery material characterized in that 5% or more is sealed in a liquid-tight manner. Further, the present invention includes a cell separation / collection filter in which at least a stacked filter medium is filled in a container having a liquid inlet and a liquid outlet, and a cell required for recovery and a cell to be removed, which is connected upstream from the inlet of the filter. A circuit for injecting the cell-containing liquid into the filter, a circuit for introducing the cell recovery liquid to the filter connected downstream from the outlet of the filter or upstream from the inlet, and a circuit upstream or downstream from the inlet of the filter. A cell separation and collection system comprising a circuit for collecting cells captured by the filter, wherein the filter has at least 25% of the entire area of the end face of the filter medium sealed in a liquid-tight manner. It is a cell separation and collection system characterized by the above-mentioned. Hereinafter, the present invention will be described in detail. The cells required for 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 cells that are unnecessary for the above-mentioned purpose or are cells that need to be recovered because they are any pathogenic cells. It is a cell that needs to be actively removed because it causes a problem of contamination. Cell populations 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 fluid, and any treatment such as centrifugation. Or cells obtained by resuspending cells extracted from various organs or tissues in some liquid. The nucleated cell in the present invention refers to a cell having a nucleus in the cell, for example, leukocyte, granulocyte, neutrophil, basophil, eosinophil, myelocyte, erythroblast, lymphocyte, T cell. lymphocytes,
Helper T lymphocytes, cytotoxic T lymphocytes, suppressor T lymphocytes, B lymphocytes, NK cells, NKT cells,
Monocytes, macrophages, dendritic cells, osteoclasts, osteoblasts, osteocytes, hematopoietic stem / progenitor cells (hereinafter abbreviated as hematopoietic stem cells), fibroblasts, chondroblasts, mesenchymal stem / progenitor cells ( stroma stem cell or mesen
chemical stem cells), megakaryocytes and the like.

[0005] The filter medium referred to in the present invention is for capturing cells that need to be collected, and any material can be used as long as it is a commonly used cell capture material, but it has low moldability, sterility and cytotoxicity. Preferred examples in this respect include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, and natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate. Examples thereof include polymers, inorganic materials such as hydroxyapatite, glass, alumina, and titania, and metals such as stainless steel, titanium, and aluminum. Examples of the shape of the filter medium include a woven fabric, a nonwoven fabric, a sponge-like structure (including a membrane), and the like. In the case of a nonwoven fabric, when not immobilizing so-called bioligands specifically binding to specific cells such as antibodies described below on the surface,
Usually, the fiber diameter is 1.0 μm or more and 30 μm or less, preferably 1.0 μm or more and 20 μm or less, and still more preferably 1.5 μm or more and 10 μm or less. 1.
If it is less than 0 μm, cells that need to be collected may be firmly captured, making it difficult to collect. On the other hand, if it exceeds 30 μm, the cells that need to be collected are more likely to pass through without being captured by the fibers. Either case is not preferable because it may lead to a decrease in the recovery rate. When a sponge-like structure is used, the pore size is usually 2.0 μm or more and 25 μm or less, preferably 3.0 μm or more and 20 μm or less,
Still more preferably, it is 4.0 μm or more and 15 μm or less. If it is less than 2.0 μm, the flowability will be remarkably inferior, and it may be difficult to pass the solution itself. If it exceeds 25 μm, the capture rate of cells that need to be recovered will be reduced, and the recovery rate will be reduced, which is not preferable. Filled with a plurality of layers of this filter medium, as a material of a container having a liquid inlet and a liquid outlet, which are excellent in moldability and sterility, and preferred from the viewpoint of low cytotoxicity, polyethylene, polypropylene, Polystyrene, acrylic resin, nylon, polyester,
Polycarbonate, polyacrylamide, polyurethane, synthetic polymers such as vinyl chloride, hydroxyapatite, glass, alumina, inorganic materials such as titania, stainless steel, titanium, metals such as aluminum,
It is not limited to these. Examples of the structure of the container include a rectangular parallelepiped, a cube, a columnar shape, an elliptical columnar shape, and the like, but any shape may be used. As for the positions of the liquid inlet and the liquid outlet, the liquid inlet may be any position at which liquid can be introduced into the uppermost layer of the filter medium, and the liquid outlet may be a position at which liquid can be extracted from the lowermost layer of the filter medium. Good. In addition, these filter media can be used as they are, but may be those that have been subjected to surface modification as necessary, such as selective passage or capture of 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 enhance platelet permeability can be mentioned. In the case of selectively capturing cells, a ligand having affinity for a specific cell such as an amino acid, a peptide, a saccharide, or a glycoprotein (including a bioligand such as an antibody or an adhesion molecule) can be obtained by, for example, a method described in JP-A-2-261833. And a method of fixing by the haloacetamide method proposed in Japanese Patent Application Publication No.

In the present invention, the end faces of the laminated filter media are sealed in a liquid-tight manner, but the ratio must be at least 25% of the total area. If it is less than 25%, the effect of improving the cell recovery rate may not be exhibited. As a method for sealing the end face of the filter medium in a liquid-tight manner, when the filter medium is a thermoplastic polymer substance, heat fusion is suitably used. Examples of the method of heat fusion include a method in which the end face is brought into contact with a metal plate heated to a high temperature, a method in which a laser cutter is used for cutting and fusing the filter medium, a method using a high-frequency welder, and an ultrasonic welder, but are not limited thereto. It is not something to be done. In addition, as a method other than heat fusion, a method of sealing the end face with an adhesive, a method of bonding or fusing a liquid-impermeable sheet or plate to the end face, and the like, but are not limited thereto. Absent. The present inventors found that by sealing the end face of the filter medium in a liquid-tight manner, the recovery rate is improved by some effect on the flow of the recovered liquid at the time of collection by sealing the end face. Although it is speculated that the mechanism has been improved, the exact mechanism has not been clarified. Regarding the position of the end face sealing, when the side face of the square filter medium is end face sealed, for example, when the end face is sealed at 50%, for example, when the cell containing liquid is passed through the filter medium, Tend to produce better results when the furthest point from the point of first contact with the filter media is sealed, for reasons that are not clear.
The cell separation / collection system according to the present invention includes at least the above-described cell separation / collection filter, a circuit connected upstream from an inlet of the filter, and a circuit for injecting a cell-containing liquid containing cells to be collected and cells to be removed into the filter, A circuit for introducing a cell recovery liquid to the filter connected downstream of the outlet or upstream of the inlet of the filter, and recovering cells trapped by the filter connected upstream of the filter or downstream of the outlet of the filter Circuit. Here, the circuit that is connected upstream from the inlet of the filter and that injects the cell-containing liquid containing the cells requiring recovery and the cells to be removed into the filter may be connected to a container or the like in which the cell-containing liquid is stored. Or that can be connected to a living tissue in which a cell-containing liquid is present. In the former specific example, for example, if the container storing the cell-containing liquid is a blood bag, a connection with a tube with a spike, a tube with a luer adapter, or a sterile connector (hereinafter referred to as “SCD connection”) is performed. If so, select as appropriate such as a simple tube. If the container containing the cell-containing liquid is a syringe with a needle, a tube with a septum that can be punctured, and if the syringe without a needle and has a luer opening, a luer adapter is appropriately selected. As a specific example of the latter, for example, when umbilical cord blood is targeted, the living tissue is the umbilical cord and / or the placenta, and a tube with a metal needle capable of piercing them is mentioned. In the case of a tube, a clamp for opening and closing, a roller clamp for adjusting the flow rate, a mesh chamber for removing agglomerates, a syringe for providing a flow rate (including a flow path switching means), and the like are provided on the way. Is also good. In the case of a syringe, it may be connected directly to the inlet of the filter without passing through a tube.

[0007] A circuit for introducing a liquid for cell recovery into the filter, which is connected downstream of the outlet of the filter or upstream of the inlet, may be connected in advance to a container containing the liquid to be injected into the filter, or may be connected later. It is classified as follows depending on whether it is possible or not and according to the liquid injection means.
That is, when a container containing the liquid to be injected into the filter is connected in advance, the circuit includes a tube with a bag, a syringe, and the like. Examples of a method of injecting the liquid into the filter include a method using a head, a method of crushing a bag, and a method of manually or mechanically pushing a plunger of a syringe. When a container containing the liquid to be injected into the filter is connected later, when a syringe is used, a tube with a piercable septum, a tube with a luer adapter, a tube with a three-way stopcock, etc., to which a syringe can be connected, can be mentioned. When a bag is used, a circuit that can be connected to the bag, that is, a tube with a spike, a tube with a luer adapter, or a simple tube if an SCD connection is performed, is appropriately selected. In the case of a syringe, the syringe may be directly connected to the inlet or outlet of the filter without using a tube. Any liquid may be used as the cell recovery liquid to be injected into the filter, as long as it does not adversely affect the cells. Some examples include physiological saline, D-PBS (Dulbecoline buffer), HBSS (Hanks) Liquid) and a culture medium such as RPMI-1640. If necessary, albumin, globulin, glucose, etc. may be added to these liquids for the purposes of protecting cells, supplementing nutrition, providing anticoagulant properties, preventing frost damage during cryopreservation, and improving viscosity (which may be effective in improving recovery rate). Saccharose, trehalose, citrate compounds, EDTA, dimethyl sulfoxide, dextran, polyvinylpyrrolidone, glycerin, chitin derivatives, hydroxyethyl starch, gelatin and the like may be added.

[0008] The circuit for collecting cells trapped in the filter, which is connected upstream from the inlet of the filter or downstream from the outlet, is classified as follows according to the type of container used to collect the cells flowing out of the filter. Is done. That is, when collecting in a bag, the bag is connected in advance, or a circuit connectable to the bag, that is, a tube with a spike, a tube with a luer adapter,
Alternatively, if an SCD connection is to be made, an appropriate selection is made, such as a simple tube. Also, when collecting in a conical tube, it is sufficient that the circuit has an open end, and when collecting with a syringe with a luer mouth, a luer adapter,
Use a three-way stopcock. In the case of a syringe, the syringe may be directly connected to the inlet or outlet of the filter without using a tube. The positional relationship between the circuit that introduces liquid into the filter and the circuit that collects cells trapped in the filter is such that when injecting liquid in the same direction as the flow direction during filtration, the circuit that introduces liquid must be at the filter inlet. Further upstream, a circuit for collecting cells is connected downstream of the filter outlet, and when injecting a liquid in a direction opposite to the flow direction during filtration, a circuit for introducing a liquid is provided downstream of the filter outlet to remove cells. The recovery circuit is connected upstream from the filter inlet. It is more preferable to inject the cell-recovery liquid in the direction opposite to the flow direction during filtration because a high recovery rate tends to be obtained.

[0009]

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.

Embodiment 1 Cell separation and recovery material Polyester nonwoven fabric with average fiber diameter of 2.3 μm (approximately 6
0 g / m ² , bulk height about 0.3 mm) and 18 polyester non-woven fabrics having an average fiber diameter of 12 μm (basis weight: about 100 g / m ²⁾
, About 0.47 mm in height), and cut into 35 mm squares with a push cutter. This end face (one face) was pressed against a high-temperature heated (about 250 ° C.) stainless steel plate and continuously fused (hereinafter referred to as an end face sealing rate of 25%) to obtain a cell separation / collection material. 2. Cell separation and collection filter and cell separation and collection device The cell separation / recovery material prepared in step 4 is a container outer dimension (length × width × thickness) of 41 × 41 × 18 mm, a polyester nonwoven fabric having an average fiber diameter of 12 μm on the outlet side of a polycarbonate container having a liquid outlet and a liquid inlet on a diagonal line. A cell separation / recovery filter was prepared by filling the cells. In addition, the position of the sealed end face was set to the position farthest from the liquid inlet. A tube having a three-way stopcock 4 having a branch to a cell collection bag 6 in the middle was connected to the inlet side of the cell separation and collection filter with a spike 2 at the tip. In addition, a three-way cock 5 was provided on the outlet side of the cell separation / collection filter 1 and a tube connected to the drain bag 3 at the end was connected to the cell separation / collection device shown in FIG. 3. Raw blood Three types of about 100 ml of umbilical cord blood (including 28 ml of anticoagulant) were each divided into two equal parts, placed in a 200 ml blood bag, and used in this example and comparative examples later. 4. Cell separation and collection operation 2. Spike 2 of the cell separation and recovery device prepared in 3. Was connected. The three-way cock 4 is set in a direction in which only the cord blood containing bag and the cell separation / collection filter 1 communicate with each other, and the three-way stopcock 5 is set in a direction in which only the cell separation / collection filter 1 and the drain bag communicate with each other, and the cord blood is dropped to the cell separation / collection filter. The solution was passed and filtered. Next, stop cock 5
A 30 ml syringe (Lure lock port) containing 25 ml of Dextran 40 Injection (trade name of Dextran 40-Yoshitomi manufactured by Yoshitomi Pharmaceutical Co., Ltd.) is connected. The stopcock 4 includes the cell separation and collection filter 1 and the cell collection bag 6
Only the direction of communication. Next, the cells trapped in the cell separation / collection filter 1 were collected in the collection bag 6 by manually pushing the plunger of the syringe. 5. Analysis The number of white blood cells and the number of red blood cells were measured using a multi-item automatic blood cell analyzer (“SF3000” manufactured by Sysmex Corporation), and the leukocyte recovery rate and red blood cell removal rate were calculated. The recovery rate of CD34-positive cells was measured using SSC using FITC-labeled anti-CD34 antibody.
-A known flow cytometry method developed on FITC (Miyazaki et al .: Daily Practice and Blood, Vol. 5, No. 2, 21-24)
Page, 1995) using a Coulter flow cytometer "EPICS-Elite". 6. Results Table 1 summarizes the results of the three types of cord blood.

[Table 1]

[0010]

Comparative Example 1 The same experiment as in Example 1 was performed except that a cell separation / collection material whose end face was not heat-sealed was used. Table 2 summarizes the results.

[Table 2] Table 3 summarizes the leukocyte recovery rate and the CD34-positive cell recovery rate of Example 1 and Comparative Example 1. It can be seen that the cell collection rate is improved by sealing the end face.

[Table 3]

[0011]

Embodiment 2 Cell separation / collection material Using the same nonwoven fabric as in Example 1, not only one end face continuously fused but also two adjacent faces continuously fused (hereinafter referred to as an end face sealing ratio of 50 %) And a product obtained by fusing all the end faces (hereinafter referred to as an end face sealing rate of 100%) was prepared as a cell separation / collection material. 2. Cell separation and collection filter and cell separation and collection device A cell separation / collection filter and a cell separation / collection device similar to those in Example 1 were prepared using the cell separation / collection material prepared in the above. Here, the position of the sealed end face was set to the position farthest from the liquid inlet. 3. Raw blood Approximately 200 ml of fresh whole blood (including 28 ml of anticoagulant) was divided into six equal parts, placed in a 200 ml blood bag, and used in this example and later comparative examples. 4. Cell separation and collection operation The same cell separation and collection operation as in Example 1 was performed. 5. Analysis The same analysis as in Example 1 was performed except that CD34-positive cells were not measured. 6. Results Table 4 summarizes the results.

[Table 4]

[0012]

Comparative Example 2 A cell separation and collection material in which 10% and 20% of the side area of the end face was continuously sealed (hereinafter referred to as an end face sealing rate of 10%, 2
The same experiment as in Example 2 was performed except that a cell separation and collection material whose end face was not sealed (hereinafter referred to as an end face sealing rate of 0%) was used. Table 5 summarizes the results.

[Table 5] Table 6 summarizes the leukocyte recovery rates of Example 2 and Comparative Example 2. It can be seen that the cell recovery rate is improved when the end face sealing rate is 25% or more. In addition, although there is a tendency to drop slightly at 100%, this is not considered to be a significant decrease.

[Table 6]

[0013]

As described above, according to the present invention, the cells required to be recovered can be recovered at a high rate from a mixture of the cells required to be recovered and the cells to be removed by a simple operation. It is great to contribute.

[Brief description of the drawings]

FIG. 1 is a schematic view of a cell separation and collection material of Example 1.

FIG. 2 is a schematic diagram of the cell separation and collection device of Example 1.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Cell separation and collection filter 2 Spike 3 Drain bag 4 Three-way cock 5 Three-way cock 6 Cell collection bag

Continuation of the front page F term (reference) 4B029 AA09 BB11 HA06 4B065 AA90X BD18 CA46 4D019 AA03 BA13 BB05 BB10 BC06 BC13 CB04

Claims (4)

[Claims] 1. A method for separating and recovering cells required for recovery from a cell population containing cells required for recovery and cells to be removed, wherein the stacked filter media is filled in a container having a liquid inlet and a liquid outlet, and the cells are separated and recovered. A filter for separating and collecting cells, wherein at least 25% of the entire area of the end surface of the filter medium in the filter is sealed in a liquid-tight manner. 2. The cell separation / collection filter according to claim 1, wherein the cells requiring collection are nucleated cells. 3. 25% of the total area of the end face of the laminated filter media
A cell separation / collection material characterized by being sealed in a liquid-tight manner as described above. 4. A filter for cell separation and collection wherein at least a stacked filter medium is filled in a container having a liquid inlet and a liquid outlet, and connected upstream of an inlet of the filter.
A circuit for injecting a cell-containing liquid containing cells requiring recovery and cells to be removed into the filter, a circuit for introducing a cell recovery liquid to the filter connected downstream from the outlet of the filter or upstream from the inlet, And a circuit connected to the filter upstream of the inlet or downstream of the outlet to collect cells trapped by the filter. A cell separation / collection system, which is sealed.