JP2012120458A - Cell separator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separator and a method for separating cells, which efficiently collect nucleated cells or monocytes from bodily fluid containing hemocyte components such as peripheral blood, cord blood and bone marrow.SOLUTION: The cell separator is composed of a container having an inlet and an outlet, and a nonwoven fabric which substantially captures nucleated cells but which erythrocytes substantially permeate. The nonwoven fabric is layered in the compressed state inside the container. This cell separator for separating nucleated cells or monocytes from the bodily fluid, and the method for separating cells by using the separator are provided. The inlet and the outlet are located opposite from each other with the nonwoven fabric in-between. The distance L from the center of gravity of a surface 1 to a point 1, when the surface on the inlet side of the container is the surface 1 and the point of contact between the inlet and the surface 1 is the point 1, is R/2 or smaller when the distance from the center of gravity of the surface 1 to the furthest point of the surface 1 is R. At the same time, the distance S from the center of gravity of a surface 2 to a point 2, when the surface on the outlet side of the container is the surface 2 and the point of contact between the outlet and the surface 2 is the point 2, is T/2 or smaller when the distance from the center of gravity of the surface 2 to the furthest point of the surface 2 is T.

Description

  The present invention uses a body fluid containing blood cell components such as peripheral blood, umbilical cord blood, bone marrow, tissue extract, menstrual blood, etc., and a cell separator that substantially captures nucleated cells from the body fluid from which they are separated. The present invention relates to a cell separator capable of efficiently collecting captured nucleated cells or mononuclear cells in a means for separating nucleated cells or mononuclear cells, and a cell separation method using the same.

  In recent years, with rapid advances in hematology and scientific technology, only the necessary blood fractions are separated from body fluids such as whole blood, bone marrow, umbilical cord blood, and tissue extracts and administered to patients. A treatment style that further increases and further suppresses side effects by not administering fractions that are not necessary for treatment has become widespread.

  For example, blood transfusion is one of them. An erythrocyte product is a blood product that is used when bleeding and erythrocytes are deficient, or when oxygen is deficient due to reduced function of erythrocytes. Therefore, leukocytes that induce side effects such as abnormal immune responses and graft-versus-host disease (GVHD) are unnecessary and need to be removed. In some cases, platelets may be removed in addition to leukocytes.

  On the other hand, a platelet preparation is a blood preparation used for patients who are bleeding or tend to bleed due to a lack of blood coagulation factors. Centrifugation removes unnecessary cells and components other than platelets and collects only the necessary platelet components.

  In addition, in recent years, hematopoietic stem cell transplantation for leukemia and solid cancer treatment has been actively performed, and cells necessary for treatment (a group of leukocytes containing hematopoietic stem cells) have been separated and administered. As a source of this hematopoietic stem cell, umbilical cord blood is attracting attention in addition to bone marrow and peripheral blood because of its advantages such as low burden on donors and excellent proliferation ability. In recent years, it has been suggested that there are abundant stem cells in menstrual blood, and menstrual blood that has been discarded may be used as a valuable source of stem cells.

  For bone marrow and peripheral blood, it is desirable to separate and purify leukocytes after removing unnecessary cells, but umbilical cord blood is also becoming popular for banking for relatives and is stored frozen until use. From the necessity, leukocytes are separated and purified for the purpose of preventing red blood cell hemolysis due to cryopreservation.

  As a separation method, a centrifugal separation method using a specific gravity liquid using Ficoll and a centrifugal separation method using hydroxyethyl starch, which is an erythrocyte sedimentation agent, have been proposed. There are problems such as mixing and a long time required for processing.

  As a cell separation method that does not use a centrifugal separation method, a method of removing leukocytes using a filter material that captures only white blood cells without capturing red blood cells and platelets has recently been reported (Patent Document 1, Patent Document 2). In the leukocyte removal filters that are currently mainstream (such as “Sepacel” manufactured by Asahi Kasei Medical Co., Ltd., “Pure Cell RC” manufactured by Paul Co., Ltd.), the position of the liquid inlet and outlet from the center of the surface deviates from the center of the surface. A so-called cross flow type is adopted.

  Furthermore, among cross-flow types, shapes that can efficiently capture cells have been studied (Patent Document 3), but there are limited recovery solutions that can be used with these leukocyte removal filters. When used, the problem was that the cell recovery rate decreased. In addition, even when a high-viscosity recovery liquid is used, it is necessary to perform solution replacement by centrifuging in order to use it.

Special table 2001-518792 International Publication Number WO98 / 32840 JP-A-11-266852

  An object of the present invention is to provide a body fluid containing each blood cell component such as peripheral blood, umbilical cord blood, bone marrow, tissue extract, menstrual blood, etc., and a cell separator that substantially captures nucleated cells from the body fluid separated from them. Separating nucleated cells or mononuclear cells by using a cell separator and cell separation method capable of efficiently recovering captured nucleated cells or mononuclear cells and not depending on the recovery solution Is to provide.

  The present inventor has studied a cell separator and a cell separation method that can efficiently recover captured nucleated cells or mononuclear cells, which have not been studied in the past, and that do not depend on the recovery solution. As a result, the following cell separator and cell separation method were found to be effective, and the present invention was completed.

That is, the present invention comprises (1) a container having an inlet and an outlet, and a nonwoven fabric that substantially captures nucleated cells and substantially passes red blood cells, and the nonwoven fabric is laminated in a compressed state in the container. A cell separator for separating nucleated cells or mononuclear cells from the body fluid, wherein the inlet and the outlet are respectively positioned in opposite directions across the nonwoven fabric, and the surface on the inlet side of the container Let point 1 be the point where the entrance and the surface 1 are in contact with each other, the distance L from the center of gravity of the surface 1 to the point 1 is R, and the distance from the center of gravity of the surface 1 to the farthest point of the surface 1 is R L is equal to or less than R / 2, the surface on the outlet side of the container is the surface 2, the point where the outlet and the surface 2 are in contact is the point 2, When the distance S from the center of gravity to point 2 is T and the distance from the center of gravity of surface 2 to the farthest point of surface 2 is T, S is / 2 or less cell separator (2) In the means for separating and collecting the captured nucleated cells, the solution is introduced from the outlet of the body fluid, and the captured nucleated cells are recovered from the inlet (3) The cell separator according to (1) or (2), wherein the angle α of the outlet with respect to the surface 2 of the container is greater than 45 degrees and smaller than 135 degrees (4) The cell separator according to (1) or (2), wherein the outlet angle α with respect to the surface 2 of the container is vertical (5) laminated with a nonwoven fabric that substantially captures platelets ( The cell separator according to any one of 1) to (4).
(6) The cell separator (7) (1) to (6) according to any one of (1) to (5), wherein the separated nucleated cells contain mononuclear cells. A cell separation method according to (7), characterized in that the cell separation method described in (7) is used, and a cell separation method according to (7), wherein the recovery solution is a physiological saline. The cell separation method according to (8), which is a solution containing a cell culture medium, hydroxyethyl starch, and dextran. When the cell separator and the cell separation method of the present invention are used, Nucleated cells or mononuclear cells can be efficiently recovered, and there is little dependence on the recovery solution.

  According to the present invention, the trapped organic substance is separated in a body fluid containing each blood cell component such as peripheral blood, umbilical cord blood, bone marrow, tissue extract, menstrual blood, etc. or a cell separation means for separating nucleated cells from the body fluid roughly separated therefrom. Nucleic cells or mononuclear cells can be recovered with a high yield, and a wide range of recovery liquids can be used.

The figure explaining L and R in plane 1 The figure explaining S and T in plane 2 The figure explaining the angle α of the exit with respect to the surface 2 Shape of container in the present invention Example of a cell separator with a circuit (A) Container used in Examples, (b) Container used in Comparative Examples Leukocyte recovery rate in the examples White blood cell recovery rate in the comparative example

  The present invention will be described in detail below, but the present invention is not limited to the following description.

  The bodily fluid in the present invention means peripheral blood, bone marrow, umbilical cord blood, menstrual blood, and tissue extract, and these may be roughly separated. There are no restrictions on animal species, and any animal may be used as long as it is a mammal such as a human, cow, mouse, rat, pig, monkey, dog, or cat. In addition, regardless of the type of anticoagulant in body fluids, citrate anticoagulation such as ACD (acid-citrate-dextrose), CPD (citrate-phosphate-dextrose), and CPDA (citrate-phosphate-dextrose-adenine) It may be anticoagulated with heparin, low molecular weight heparin, fusan (nafamostat methyl acid) or EDTA. If there is no influence depending on the purpose of use, the preservation conditions of body fluids are not questioned at all.

  In the present invention, the cell separation material filled in the cell separator is not particularly limited, but from the viewpoint of sterilization resistance and cell safety, polyethylene terephthalate, polybutylene terephthalate, polyethylene, high-density polyethylene, low-density polyethylene. , Polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polypropylene, acrylic (polymethyl methacrylate, polyhydroxyethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate), nylon, polyimide, aramid (aromatic polyamide), polyamide, cupra , Synthetic polymers such as carbon, phenol, polyester, pulp, hemp, polyurethane, polystyrene, polycarbonate, agarose, cellulose, cellulose acetate, chitosan, chitin Which natural polymers, inorganic materials and metals, such as glass.

  Preferred are polyethylene terephthalate, polybutylene terephthalate, polypropylene, acrylic, nylon, polyurethane, and glass. These materials are not limited to a single type, and may be combined, mixed, and fused as necessary. Furthermore, if necessary, molecules having affinity for specific cells such as proteins, peptides, amino acids and saccharides may be immobilized.

  The cell separator of the present invention is characterized by having the following points in order to efficiently collect captured cells. (1) A container having an inlet and an outlet, and a nonwoven fabric that substantially captures nucleated cells and substantially passes red blood cells, and the nonwoven fabric is laminated in a compressed state in the container. (2) The said inlet and the said outlet are each located in the reverse direction on both sides of the said nonwoven fabric. (3) A surface on the inlet side of the container is a surface 1, a point where the inlet and the surface 1 are in contact is a point 1, and a distance L from the center of gravity of the surface 1 to the point 1 is from the center of gravity of the surface 1 L is R / 2 or less, where R is the distance to the farthest point on the surface 1. (3) The surface on the outlet side of the container is the surface 2, the point where the outlet and the surface 2 are in contact is the point 2, and the distance S from the center of gravity of the surface 2 to the point 2 is from the center of gravity of the surface 2 S is T / 2 or less, where T is the distance to the farthest point on surface 2.

  When L is greater than R / 2 or S is greater than T / 2, the recovery rate of nucleated cells is significantly reduced when a low-viscosity recovery solution is used, but L is R / 2 or less, When S is T / 2 or less, the recovery rate of nucleated cells does not decrease even with a low viscosity recovery solution. This is thought to be because the recovery rate decreases because the recovery solution does not reach the entire cell separator when the inlet and the outlet are located at the end of each surface.

  Furthermore, the cell separator of the present invention is characterized in that the angle α of the outlet with respect to the surface 2 of the container is larger than 45 degrees and smaller than 135 degrees. When the angle α of the outlet with respect to the surface 2 of the container is 0 degree or more and less than 45 degrees or more than 135 degrees and 180 degrees or less, the recovery rate of nucleated cells is significantly reduced by using a low-viscosity recovery liquid. When the angle α of the outlet with respect to the surface 2 is greater than 45 degrees and smaller than 135 degrees, the recovery rate of cells does not decrease even with a low viscosity recovery solution. This is because when the outlet angle α with respect to the surface 2 of the container is larger than 45 degrees and smaller than 135 degrees, the liquid is diffused throughout the cell separator, but the outlet angle α with respect to the surface 2 of the container is 0 degree or more and 45 degrees. This is considered to be because the recovered liquid flows only locally when the angle is less than or equal to 135 degrees or less than or equal to 135 degrees or less.

  From the distance L from the center of gravity of surface 1 to point 1 defined by the present invention, the distance R from the center of gravity of surface 1 to the farthest point of surface 1, the distance S from the center of gravity of surface 2 to point 2, and the center of gravity of surface 2 The distance T to the farthest point of the surface 2 is as shown in FIG. 1 or FIG. The angle α is as shown in FIG.

In the present invention, the fact that red blood cells substantially pass means that 60% or more of the treated body fluid passes through the nonwoven fabric, and that substantially nucleated cells are substantially captured means that the treated body fluid It means that 60% or more of them are captured by the nonwoven fabric.
Further, the container in the present invention may have any shape such as a cylinder, a quadrangular column, an elliptical column, or a rhombus as shown in FIG. The nonwoven fabric may be cut out according to the shape of the container, or the container may be selected according to the shape of the nonwoven fabric. In addition, the nonwoven fabric may be laminated and filled in a compressed state, that is, the nonwoven fabric may be compressed in all directions in the container, and compressed in one direction, for example, the direction shown by the arrow in FIG. It does not matter. The inlet and outlet positions are preferably positioned in the compressed direction, with the nonwoven fabric sandwiched between the arrows in FIG. 4, but the present invention is not limited to these.

  In order to isolate cells in a closed manner using the cell separator of the present invention, the following circuit can be used. That is, at least one connector that can be connected to the bag on the inlet side, for example, a bottle needle, or at least one bag that contains blood or priming liquid is provided on the inlet side, and a cell separator on the outlet side. A circuit having a bag for containing the liquid that has passed through the container may be attached. It is more preferable to provide a circuit with a syringe connection part for collecting the cells captured on the outlet side and a collection bag for accommodating the cells captured on the inlet side. These circuits are also preferably provided with a three-way stopcock, a roller clamp, a clamp and the like in order to control the flow of the liquid. FIG. 5 illustrates a cell separator having a circuit for processing in a closed system. However, the present invention is not limited to these.

  The present invention also provides a cell separation method using the cell separator of the present invention. In particular, when using a low-viscosity recovery liquid, it is desirable to use the cell separator of the present invention. The low-viscosity recovery liquid means a recovery liquid having a viscosity of 8 mPa · S or less, and examples thereof include a solution containing physiological saline, a cell culture medium, hydroxyethyl starch, and dextran. Preferably it is 7 mPa * S or less, More preferably, it is 6 mPa * s or less. Typical examples of the cell culture medium include Dulbecco MEM (Nissui), α-MEM (GIBCO BRL), MEM (Nissui), IMEM (Nissui), RPMI-1640 (Nissui) medium, and the like. However, it is not limited to these. Furthermore, these recovered liquids may contain proteins such as albumin and FBS.

  EXAMPLES Hereinafter, although an Example demonstrates in detail regarding this invention, this invention is not limited only to a following example.

Example 1
Cells are laminated in a compressed state with 76 sheets of non-woven polybutylene terephthalate (fiber diameter: 2.7 micrometers) cut into a circular shape with a diameter of 32 mm in a cylindrical container having a thickness (inner diameter) of 9 mm and a diameter (inner diameter) of 32 mm. A separator was created. This is a cylindrical container as shown in FIG. 4, and the nonwoven fabric was compressed and filled in the direction indicated by the arrow. The specific shape of the cell separator is as shown in FIG. First, 50 mL of physiological saline was taken in a syringe, introduced by hand from the inlet of the container, and passed through. Next, 80 ml of CPD anticoagulated fresh bovine blood (bovine peripheral blood containing 12% CPD mixed with CPD: blood = 200: 28) at a flow rate of 15 ml / min, and then 15 ml of physiological saline at 15 ml / min in the same manner. Blood was passed using a syringe pump at a flow rate. Then, contrary to liquid flow, 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS was manually pushed from the outlet side and collected from the inlet side. The blood count of the blood before treatment and the blood count of the collected solution were measured with a blood cell counter (Sysmex, K-4500), and the leukocyte recovery rate was calculated. The results are shown in Table 1 and FIG.

(Example 2)
The same procedure as in Example 1 was carried out except that a dextran sugar injection containing 10% ACD-A solution and 4% human serum albumin was used instead of 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS as the recovery solution. did. The results are shown in Table 1 and FIG.

(Example 3)
The same procedure as in Example 1 was carried out except that sarinhes containing 10% ACD-A solution and 4% human serum albumin was used in place of 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS. The results are shown in Table 1 and FIG.

Example 4
The same procedure as in Example 1 was performed except that 10% ACD-A solution-added physiological saline was used in place of 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS as the collected solution. The results are shown in Table 1 and FIG.

(Comparative Example 1)
Laminated 76 sheets of polybutylene terephthalate nonwoven fabric (fiber diameter: 2.7 micrometers) cut into a square with a side of 30 mm in a rectangular column type container with a thickness (inner diameter) of 9 mm and a side (inner diameter) of 30 mm. A cell separator was created. This was a quadrangular prism-shaped container as shown in FIG. 4, and the nonwoven fabric was compressed and filled in the direction indicated by the arrow. The specific shape of the cell separator is as shown in FIG. First, 50 mL of physiological saline was taken in a syringe, introduced by hand from the inlet of the container, and passed through. Next, 80 mL of CPD anticoagulated fresh bovine blood (bovine peripheral blood containing 12% CPD mixed at CPD: blood = 200: 28) was flowed at a flow rate of 15 mL / min, and then 15 mL of physiological saline was similarly added at 15 mL / min. Blood was passed using a syringe pump at a flow rate. Then, contrary to liquid flow, 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS was manually pushed from the outlet side and collected from the inlet side. The blood count of the blood before treatment and the blood count of the collected solution were measured with a blood cell counter (Sysmex, K-4500), and the leukocyte recovery rate was calculated. The results are shown in Table 2 and FIG.

(Comparative Example 2)
The same procedure as in Example 1 was carried out except that sarinhes containing 10% ACD-A solution and 4% human serum albumin was used in place of 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS. The results are shown in Table 2 and FIG.

(Comparative Example 3)
The same procedure as in Example 1 was performed except that 10% ACD-A solution-added physiological saline was used in place of 30 mL of αMEM medium containing 10% ACD-A solution and 10% FBS as the collected solution. The results are shown in Table 2 and FIG.

1. 1. Connector that can be connected to a container containing the priming solution and a container containing the priming solution 2. Connector that can be connected to a syringe Cell separator 4. 4. A connector that can be connected to a container containing body fluid and a container that contains body fluid. Waste liquid bag Collection bag

Claims (9)

A container having an inlet and an outlet, and a non-woven fabric that substantially captures nucleated cells and substantially passes erythrocytes; A cell separator for separating mononuclear cells, wherein the inlet and the outlet are positioned in opposite directions across the nonwoven fabric, the inlet-side surface of the container is a surface 1, and the inlet and the The point where the surface 1 is in contact is point 1, and the distance L from the center of gravity of the surface 1 to point 1 is R, and the distance from the center of gravity of the surface 1 to the farthest point of the surface 1 is R / 2. The surface on the outlet side of the container is a surface 2, the point where the outlet and the surface 2 are in contact is a point 2, and the distance S from the center of gravity of the surface 2 to the point 2 Where S is T / 2 or less, where T is the distance from the center of gravity of surface 2 to the farthest point of surface 2 Cells separator characterized. The cell separator according to claim 1, wherein the means for separating and collecting the captured nucleated cells comprises introducing a solution from the outlet of the body fluid and recovering the nucleated cells captured from the inlet. The cell separator according to claim 1 or 2, wherein the outlet angle α with respect to the surface 2 of the container is larger than 45 degrees and smaller than 135 degrees. The cell separator according to claim 1 or 2, wherein the angle α of the outlet with respect to the surface 2 of the container is perpendicular. The cell separator according to any one of claims 1 to 4, wherein a nonwoven fabric that substantially captures platelets is laminated. The cell separator according to any one of claims 1 to 5, wherein the separated nucleated cells include mononuclear cells. A cell separation method using the cell separator according to any one of claims 1 to 6. The cell separation method according to claim 7, wherein a low-viscosity recovery solution is used. 9. The cell separation method according to claim 8, wherein the recovered solution is a solution containing physiological saline, a cell culture medium, hydroxyethyl starch, and dextran.