JP5336109B2 - Methods for enriching mononuclear cells and platelets - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for concentrating mononuclear cells and platelets to high concentrations from body fluid containing erythrocytes, nucleated cells and the platelets by a simple operation without using a blood preparation. <P>SOLUTION: The method for concentrating the mononuclear cells and the platelets from the body fluid containing the erythrocytes, the nucleated cells and the platelets by using a filter system having a cell-capturing tool 11 obtained by packing a container having an inlet and an outlet with a cell-capturing filter material, a circuit and a bag 13 includes adding an erythrocyte sedimentation agent to the body liquid stored in the bag, separating the body liquid to a layer inclined to the nucleated cells and the platelets, and a layer inclined to the erythrocytes, introducing only the layer inclined to the nucleated cells and the platelets to the cell-capturing tool to allow the filter material 41 to capture the nucleated cells and the platelets, introducing only the filtrate passing the filter material of &ge;0.2 vol. and &lt;1.0 vol. of the cell-capturing tool to the filter material by extruding the filtrate by a gas, and selectively collecting the mononuclear cells and the platelets captured by the filter material. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for efficiently concentrating target mononuclear cells and platelets to high purity and high concentration by simple operations from a body fluid containing red blood cells, nucleated cells, and platelets.

  In recent years, research and treatment using cells such as regenerative medicine and cell therapy have attracted attention and are being put into practical use. Cells used for such purposes are often collected from bone marrow fluid, umbilical cord blood, peripheral blood, etc., but these contain various cells in addition to those required for research and treatment. Yes. For example, bone marrow fluid, umbilical cord blood, and peripheral blood usually contain a large amount of red blood cells, but the red blood cells prevent the growth of the target cells and reduce the cell engraftment ability in cell therapy. Therefore, how to selectively collect mononuclear cells such as mesenchymal stem cells, adipocytes, hematopoietic stem cells, and mononuclear cells required to reduce erythrocyte contamination was a problem.

  Further, from the viewpoint of therapeutic effect, it is required that the required recovery rate of mononuclear cells is high and the concentration is high. Furthermore, in regenerative medicine, a higher therapeutic effect can be realized by the coexistence of cells containing abundant growth factors, which are one of the three important factors in regeneration, at a high concentration.

Conventionally, specific gravity centrifugation, erythrocyte agglutination, buffy coat method, affinity separation method using antibodies, etc. have been performed as a method for selectively collecting only necessary cells by removing such a large amount of red blood cells. It was.
However, the specific gravity centrifugation method has a high erythrocyte removal rate and a small volume, so that the cell concentration rate is also high. However, on the other hand, the cell recovery rate is low, and it is a complicated operation that requires highly skilled techniques. Furthermore, since the operation is an open system, there is a high risk of contamination and there is a problem in terms of safety.

  In the erythrocyte agglutination method, erythrocytes are agglomerated and precipitated by mixing with hydroxyethyl starch or dextran to collect and sediment the erythrocytes (Non-Patent Documents 1 and 2). If you try to increase the cell recovery rate, the cell recovery rate will decrease, and if you try to increase the cell recovery rate, you will not be able to reduce erythrocyte contamination. There wasn't. Also, with this method, the volume of the liquid cannot be reduced, and a sufficient necessary cell concentration cannot be obtained.

  In the buffy coat method, the bag containing blood is centrifugated to form the upper plasma layer, the buffy coat layer consisting mainly of white blood cells and platelets in the middle layer, and the lower red blood cell layer, and then the upper plasma layer and the lower red blood cell layer The buffy coat layer of the intermediate layer is recovered by expelling the layer from the bag (Non-patent Document 3), but still has the same problem as the hemagglutination method.

  In the affinity separation method using antibodies, the specificity is high and the effect of volume reduction is large. However, in order to recover the separated cells, the bound antibody molecules are treated with an enzyme, so that the cells are damaged, expensive, and complicated. There are problems such as.

  There is also a method of removing red blood cells by hemolysis under low osmotic pressure when the amount of red blood cells is small. However, in such a low osmotic pressure state, even necessary cells are damaged, and red blood cells can be removed while necessary cell functions are lost. Moreover, from the viewpoint of safety in practical use, it is required that all of the components are composed of only their own components and that the operation is simple, without using the ones from other families as much as possible.

  As a method for reducing the contamination of unnecessary cells from a cell-containing solution by a simple operation using a filter device and efficiently separating and recovering nucleated cells, Patent Document 1 discloses that a cell-containing solution is enriched with a layer rich in unnecessary cells. Separate into layers rich in nucleated cells, first introduce into the filter device from the layer rich in unwanted cells, then introduce the layer rich in nucleated cells, then collect the nucleated cells captured by the filter in the recovery solution A method is disclosed. However, this method also introduces a layer rich in unwanted cells such as red blood cells into the filter, so the removal rate of red blood cells is not sufficient, and since a certain amount of recovery solution is used during recovery, the cell concentration is low, and regenerative medicine and cell Erythrocyte removal and cell concentrate suitable for therapeutic use could not be obtained. Furthermore, there is no effect of selectively concentrating mononuclear cells and platelets useful for recovering all nucleated cells and removing platelets as unnecessary cells.

  Moreover, in patent document 2, after introducing a cell-containing liquid into a filter, a method that allows cells to be collected at a high rate without using a commercially available serum protein at the time of collection by leaving a small amount of filtrate that has passed through the filter. It is disclosed. However, since the cell-containing solution is directly introduced into the filter, a large amount of unnecessary cells such as erythrocytes are mixed, and a certain amount of high-viscosity recovery solution is used to increase the cell recovery rate. The operation is complicated and time-consuming because operation such as centrifugation is required. Moreover, there is no description about the effect of concentrating platelets.

  Furthermore, in Patent Document 3, after introducing a cell population into a mononuclear cell capture filter, a rinse solution containing albumin is introduced to wash away erythrocytes remaining in the filter device, and then a recovery solution is introduced into the filter device to filter the filter. A method for recovering target cells captured in an apparatus is disclosed. In this method, since the rinsing solution is introduced, the mixing of red blood cells is reduced. However, since the red blood cells are also introduced into the filter, the red blood cell removal rate is still insufficient. Furthermore, there is no description that concentrates platelets because platelets may be washed away by the action of the rinse solution. In addition, since human serum albumin is used as a recovery solution, there is a high risk of infection. Furthermore, with regard to operability, when the rinsing liquid is passed through, the air in the blood circuit enters the filter device and filtration becomes impossible (air block). The filter device is rinsed before the filtration to prevent air from entering the filter device. In addition, it is necessary to perform priming of the blood circuit and the like, and the cell concentration is not low enough to use a certain amount of the recovered solution.

  In Patent Document 4, after a cell population is filtered with a cell separation filter, a liquid is first introduced from the downstream side of the cell separation filter, and then a gas is introduced so that the cells that need to be collected are not damaged at a high rate. Discloses a separation and recovery method. However, in the method of Patent Document 4, since red blood cells are also introduced into the filter, the amount of red blood cells is large, and the cells that need to be collected cannot be concentrated to a high concentration, and platelets are removed rather than concentrated. I couldn't even do it.

As described above, a cell preparation method for efficiently concentrating necessary cells such as mononuclear cells and platelets from a cell-containing solution containing various cells to high purity and high concentration by a simple operation method is still established. There wasn't.
International Publication No. 2005/035737 Pamphlet JP 2000-325071 A JP 2004-121144 A JP 2001-78757 A Proc. Natl. Acad. Sci. USA, Vol. 92, pp10119-10122 (1995) Indian J. Med. Res. , Vol. 106, pp16-19 (1997) Bone Marrow Transplantation, Vol. 23, pp505-509 (1999)

  An object of the present invention is a method for concentrating mononuclear cells and platelets to a high concentration from a body fluid comprising erythrocytes, nucleated cells and platelets by a simple operation without using a blood product. It is to provide a concentration method with a low contamination rate and a high recovery rate of mononuclear cells and platelets.

  As a result of intensive studies to solve the above problems, the present inventors have added an erythrocyte sedimentation agent to a body fluid containing at least erythrocytes, nucleated cells, and platelets, and thereby applied to nucleated cells and platelet-biased layers and erythrocytes. After separation into the biased layer, only the nucleated cells and platelet-biased layer are passed through the cell trapping filter material, and the filtrate passes through the filter material of 0.2 to 1.0 volume of the cell trapping device. And recovering with gas, it was found that red blood cells can be removed at a high rate by a simple operation, and mononuclear cells and platelets can be efficiently recovered and concentrated to a high concentration, and the present invention has been completed.

That is, the present invention relates to the following.
(1) Mononuclear from a body fluid containing red blood cells, nucleated cells, and platelets using a filter system having a cell trapping device filled with a cell trapping filter material in a container having an inlet and an outlet, a circuit, and a bag. A method of concentrating cells and platelets,
Add an erythrocyte sedimentation agent to the body fluid stored in the bag,
Separated into nucleated cells and a layer biased to platelets and a layer biased to red blood cells,
After introducing only the nucleated cells and platelet-biased layer into the cell trap and capturing the nucleated cells and platelets in the filter material,
Only the filtrate that has passed through the filter material of 0.2 volume or more and less than 1.0 volume of the cell trap, is extruded with gas and introduced into the filter material,
A method for concentrating mononuclear cells and platelets from a body fluid comprising erythrocytes, nucleated cells and platelets, wherein the mononuclear cells and platelets captured by the filter material are selectively collected.
(2) The method of concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells, and platelets according to (1), wherein the gas is air.
(3) The filtrate that has passed through the filter material in a volume of 0.2 to 1.0 volume of the cell trap is the filtrate remaining in the cell trap and the circuit (1) Alternatively, the method for concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets according to (2).
(4) The bodily fluid comprising erythrocytes, nucleated cells and platelets according to any one of (1) to (3), wherein the erythrocyte sedimenting agent is either hydroxyethyl starch or dextran To concentrate mononuclear cells and platelets.
(5) After separating the nucleated cell and the layer biased to platelets and the layer biased to red blood cells, the layer biased to red blood cells is discharged from the bag, and only the layer biased to the nucleated cells and platelets is removed. The method for concentrating mononuclear cells and platelets from a body fluid containing red blood cells, nucleated cells and platelets according to any one of (1) to (4), wherein the method is introduced into the cell trap.
(6) The body fluid containing red blood cells, nucleated cells, and platelets is one body fluid selected from the group consisting of peripheral blood, umbilical cord blood, and bone marrow fluid (1) to (5) A method for concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets according to any one of the above.

  According to the method for concentrating mononuclear cells and platelets according to the present invention, mononuclear cells and platelets can be obtained from a cell-containing solution containing erythrocytes, nucleated cells, and platelets by a simple operation without using blood products. It can be selectively concentrated to a high concentration, the contamination rate of erythrocytes is extremely low, and the recovery rate of mononuclear cells and platelets can be increased.

  The bodily fluid comprising erythrocytes, nucleated cells and platelets as used in the present invention is bone marrow fluid, umbilical cord blood, peripheral blood, etc., and even if these are collected as they are, physiological saline, anticoagulant It may be diluted with a solution or a culture solution. The nucleated cell referred to in the present invention may be any cell as long as it has a nucleus, and examples thereof include leukocytes, mononuclear cells, granulocytes and the like.

  The mononuclear cell in the present invention refers to a mononuclear cell. For example, lymphocytes, monocytes, macrophages, dendritic cells, hematopoietic stem cells, mesenchymal stem cells, osteoblasts and osteoblast precursors. Examples of cells that can be selectively collected as mononuclear cells include hematopoietic stem cells, mesenchymal stem cells, osteoblasts and osteoblast progenitor cells that have the ability to induce proliferation or differentiation. .

  The cell capture filter material as used in the present invention refers to a porous filter material that captures nucleated cells and allows unnecessary cells such as red blood cells to pass through. Capturing nucleated cells here means recovering 60% or more, more preferably 70% or more of nucleated cells from the cell-containing solution. Further, passing unnecessary cells such as erythrocytes means that 90% or more, more preferably 95% or more of erythrocytes are passed from the cell-containing solution without being captured by the filter.

  The cell-capturing filter material used in the present invention can be any material as long as it is a water-insoluble carrier, but examples thereof include materials that can be processed into various shapes, can be sterilized as medical materials, and have high biocompatibility. . For example, natural polymers such as cellulose, dextran, chitin, chitosan, starch, agarose, protein, natural rubber, polystyrene, polyamide, polyester, polyethylene, polyurethane, polyvinyl alcohol, ethylene vinyl alcohol copolymer, isopropylacrylamide polymer, polylactic acid, Examples thereof include synthetic polymers such as polyglycolic acid, lactic acid glycolic acid copolymer, polymethacrylic acid ester, polyvinyl chloride, and polyamino acid, ceramics such as hydroxyapatite, tricalcium phosphate, and alumina, and titanium.

  In addition, a material in which the surface of the cell trapping filter material is processed so as to have affinity with specific cells is also used favorably. For example, an antibody against a specific cell surface antigen, a peptide or a polymer thereof, a cell adhesion molecule such as fibronectin, an extracellular matrix such as collagen and gelatin, which is a modified form thereof, may be fixed or coated on the surface of the filter material. Thus, the target cells can be captured more selectively. These processing agents for the filter surface are selected according to the target cells, and a plurality of types can be used in combination.

  Since the shape of the cell trapping filter material needs to have a sufficient surface area to trap cells, for example, granular, fiber lump, cotton-like, cloth-like, thread-like, hollow fiber-like, thread-bundle-like, woven cloth Shape, non-woven fabric shape, sponge shape, etc. are mentioned, but non-woven fabric and sponge shape are more preferably used from the viewpoint of a large surface area per volume.

  When the cell trapping filter material is a nonwoven fabric, the average 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 more preferably 1.5 μm or more and 10 μm or less. If it is less than 1.0 μm, not only the nucleated cells are firmly captured and it may be difficult to collect, but also the passage of unnecessary cells, especially when they are aggregated, is difficult. On the other hand, if it exceeds 30 μm, the possibility that the nucleated cells pass through without being captured by the fibers increases. In either case, the recovery rate may be reduced, which is not preferable.

  The average fiber diameter here means a value obtained according to the following procedure. That is, a part of the cell trapping filter material recognized as substantially uniform is sampled and photographed at a magnification of 1000 to 3000 using a scanning electron microscope or the like. At the time of sampling, the effective filtration cross-sectional area portion of the cell trapping filter material is divided by a square having a side of 0.5 cm to 1 cm, and 3 or more, preferably 5 or more are randomly sampled. In order to perform random sampling, for example, after specifying an address for each of the above-mentioned sections, it is possible to select more than necessary sections by using a random number table. Also, for each sampled section, take a picture of 3 or more, preferably 5 or more. For the photograph thus obtained, the diameters of all the visible fibers are measured. Here, the diameter means the width of the fiber in the direction perpendicular to the fiber axis. A value obtained by dividing the sum of the diameters of all the measured fibers by the number of fibers is defined as an average fiber diameter. However, when multiple fibers overlap each other and their width cannot be measured behind other fibers, or when multiple fibers melt and become thick fibers, fibers with significantly different diameters If there is a mixture of these, etc., these data are deleted. By the above method, the average fiber diameter is obtained from data of 500 or more, preferably 1000 or more.

In addition, the container of the cell trap used in the present invention is not particularly limited, but has an inlet and an outlet that allow introduction of a cell-containing solution, so that cells can be efficiently trapped in the cell trapping filter material. A structure is preferred. For example, when using a non-woven fabric as the cell trapping filter material, the shape of the container of the cell trap is a flat square in which the inlet and the outlet are located at both ends of the diagonal so that the distance between the inlet and the outlet of the cell-containing liquid is the longest. It is preferable that The effective filtration cross-sectional area of the cell trapping filter material depends on the number of cells to be trapped, but is preferably in the range of 4.0 cm ^{2 to} 45 cm ² , and the cells trapped in the cell trapping filter material are so as not to peel, more preferably from 9.0cm ² ~45cm ^2, to uniformly effective use of the whole cell capture filter material ^is most preferably a 9.0cm ² ~18cm 2.

  As the material for the container of the cell trap, a material that is insoluble in water, excellent in moldability, hermeticity, and sterilization properties and highly biocompatible is preferable. In addition, when the cell-containing liquid passes through the cell trapping filter material or when the mononuclear cells of the cell trapping filter material are collected with the filtrate, it is substantially expanded or leaked by the pressure applied inside the cell trapping device. It is preferable that it is a hard material which does not do. Preferred materials from these viewpoints include polycarbonate, polyethylene, polystyrene, polypropylene, and the like. From the viewpoint of hardness, polycarbonate and polystyrene are more preferable. From the viewpoint of cell loss due to cell adhesion to the container, polycarbonate is the most preferable. Preferably used.

  The erythrocyte sedimenting agent as used in the present invention refers to a substance containing a component that precipitates only erythrocytes from a cell-containing solution containing various cells by recurring formation, and specifically includes hydroxyethyl starch, dextran, and the like. It is done. As the amount of hydroxyethyl starch added to blood, for example, when the average molecular weight of hydroxyethyl starch is 400,000 and a 10% physiological saline solution is used, the amount is preferably 1/3 to 1/10 with respect to blood 1 as the concentration. 0.9 to 2.5% is preferable. Further, the standing time is 10 minutes or more and 80 minutes or less, preferably 20 minutes or more and 50 minutes or less. When the added amount of hydroxyethyl starch is less than 1/10, or when the standing time is less than 10 minutes, the formation of red blood cells is insufficient and the layer separation is insufficient. On the other hand, when the amount of hydroxyethyl starch added is more than 1/3, or when the standing time is longer than 80 minutes, the layer separation proceeds rapidly and the cells settle, which is not preferable. The molecular weight of hydroxyethyl starch is preferably in the range of 70,000 to 400,000, but a molecular weight of 200,000 to 400,000 is more preferably used from the viewpoint of increasing the sedimentation rate and sedimentation of erythrocytes.

  The amount of dextran added to blood is, for example, in the case of 50000 dextran physiological saline with a molecular weight of 70,000, preferably 1.5 to 3.0 with respect to blood 1, and the concentration is 1.0 to 4 .5% is preferred. The standing time is preferably 30 to 75 minutes, and if the amount of dextran added is less than 1.5 to the blood, or the standing time is less than 30 minutes, the red blood cell formation is insufficient and the layer The separation interface is not clear, which is not preferable. On the other hand, if the amount of dextran added is larger than 3.0 or the standing time is longer than 75 minutes, the layer separation proceeds excessively and the cells also settle, which is not preferable. Further, the molecular weight is preferably in the range of 50,000 to 200,000, but those having a molecular weight of 70,000 to 200,000 are preferably used for the same reason as hydroxyethyl starch.

  In the present invention, the layer biased toward erythrocytes contains 60% or more, preferably 70% or more, more preferably 80% or more of erythrocytes in a body fluid containing erythrocytes, nucleated cells and platelets before layer separation. Refers to the layer.

  Further, the nucleated cells and the layer biased to platelets referred to in the present invention are 60% or more, preferably 70% or more, of nucleated cells and platelets in a body fluid containing erythrocytes, nucleated cells and platelets before layer separation. More preferably, it refers to a layer containing 80% or more.

  In the present invention, the selective collection of mononuclear cells and platelets means that when the nucleated cells and platelets captured by the cell capture filter material are collected, the filtrate that has passed through the filter is extruded with a small amount of gas. Mononuclear cells and platelets are preferentially collected from the nucleated cells and platelets captured in the above. Due to the characteristics of the cell surface, mononuclear cells with low adhesion among nucleated cells are preferentially collected over multinucleated cells with high adhesion.

  In the method of the present invention, since the filtrate is used as the recovered liquid, it is not necessary to prepare the recovered liquid separately. In addition, since the filtrate contains plasma components such as plasma proteins, it is not necessary to replenish serum albumin derived from blood products that are usually added as a cell stabilizer to the collected liquid, and there is no risk of infection. The amount of the filtrate used as the recovery liquid needs to be 0.2 volume or more with respect to the volume of the cell trap in order to recover the mononuclear cells and platelets captured by the cell trapping filter material at a high recovery rate. When the amount of the filtrate used for the collection is less than 0.2 volume of the cell trap, foaming is likely to occur and the cell recovery rate decreases. In addition, even if the amount of filtrate used for collection is 1.0 volume or more of the cell trap, the recovery rate of the target mononuclear cells and platelets does not improve. Cell and platelet concentrations decrease. Therefore, in order to obtain a concentrated solution of mononuclear cells and platelets, it is essential that the amount of filtrate used for the collected liquid is 0.2 volume or more and less than 1.0 volume of the cell trap.

  The flow rate of the filtrate used for collection is preferably as high as possible in order to increase the shearing force and collect mononuclear cells at a high rate, but the connection between the cell trap and cell introduction tube due to an increase in internal pressure may cause It is preferable to control the flow rate so as not to cause damage to the cells. In addition, the means for introducing the filtrate used for collection into the cell trap can be obtained by using a device such as a syringe pump, a blood pump, or a peristaltic pump, as a simple method by manually pushing the syringe, For example, a head drop process, and the like. Furthermore, in order to further increase the recovery rate of nucleated cells, vibration may be applied to the filter device, or stopped flow may be performed. The direction in which the filtrate used for recovery is introduced into the filter device is the same or opposite to the direction in which the body fluid is introduced, but the latter is generally preferred because the cell recovery rate is higher.

  The amount of the filtrate used in the present invention is a small amount of 0.2 to 1.0 volume of the cell trap, and the cells cannot be collected only by the filtrate. Therefore, in order to push the filtrate through the cell trapping filter material and push it out to the collection bag. Use gas. The gas may be any gas as long as it is sterile so long as it does not affect the pH of the filtrate and the cell viability, but nitrogen gas, oxygen gas, and air are preferred, and air is most preferred in terms of simplicity. .

  A method of concentrating mononuclear cells and platelets from a body fluid containing red blood cells, nucleated cells, and platelets will be described using the system of FIGS. 1 and 2 as an example. First, the clamp 20 on the red blood cell waste tube 1 is closed, and a body fluid containing red blood cells, nucleated cells, and platelets is introduced into the body fluid storage bag 14 from the injection unit 3 or the red blood cell waste tube 1. An erythrocyte sedimentation agent is introduced from the injection part 3 of the body fluid storage bag 14 and allowed to stand for a certain period of time, so that the body fluid is separated into nucleated cells, a layer biased to platelets, and a layer biased to erythrocytes. Next, the clamp 20 is opened, and the layer biased to red blood cells is discharged from the red blood cell waste tube 1 of the body fluid storage bag 14, and then the clamp 20 is closed and only the layer biased to nucleated cells and platelets is retained. To leave. Discarding the layer biased to red blood cells precipitated by the red blood cell sedimentation agent may be performed before or after connecting the body fluid storage bag 14 and the cell introduction tube 4.

  In FIG. 2, the filter system (concentration system 100) includes a cell trap 11 filled with a cell trapping filter material, a back or drain bag 12, a recovery bag 13 and a body fluid storage bag 14, and a circuit or red blood cell waste tube 1 respectively. The filtrate collection tube 5, the cell collection tube 7, the cell introduction tube 4, the injection tube 6, and the T-shaped tubes 31 and 32 are connected to each other. The injection tube 6 is provided with an injection port 33, an air vent filter 41 is provided between the clamp 21 of the cell introduction tube 4 and the T-shaped tube 31, and clamps 20 to 23 are further arranged. . With all the clamps 21 to 23 of the concentration system 100 closed, the cell introduction tube connecting part 2 of the body fluid storage bag 14 and the cell introduction tube 4 of the concentration system 100 are connected, and the T-shaped tube 31 is connected to the body fluid storage bag 14 and the cells. Only the trap 11 communicates, and the T-shaped tube 32 allows only the cell trap 11 and the drain bag 12 to communicate. The concentration system 100 is suspended on a fixed base such as a stand, and the body fluid storage bag 14, the cell trap 11, and the drain bag 12 are arranged in this order from above in the direction of gravity.

  Next, the clamps 21 and 22 are opened, and the nucleated cells remaining in the body fluid storage bag 14 and the layer biased to platelets are introduced into the cell trap 11 including the cell trapping filter material. By introducing only a layer biased to nucleated cells and platelets into the cell trap 11 without introducing a layer biased to red blood cells into the cell trap, the contamination of red blood cells is reduced as well as capturing in the cell trap. The cells thus obtained are held in a state where they are easily recovered without being embedded in the deep part of the cell trapping filter material due to the shear stress of a large amount of red blood cells. For example, when a body fluid is introduced into the cell trap 11 without layer separation, not only red blood cells are mixed but also the concentration rate of mononuclear cells and platelets is not preferable. In addition, since the same thing occurs when the layer separation efficiency of red blood cells is poor, it is preferable to carefully discharge the layer biased to red blood cells from the body fluid storage bag after sufficient layer separation. If the connecting portion of the body fluid storage bag 14 to which the erythrocyte waste tube is connected is inclined toward the outlet, it is preferable that a layer biased to erythrocytes hardly remains. In addition, there is no particular limitation on the means for introducing the nucleated cell and platelet-biased layer into the cell trap 11, but it may be introduced by crushing a drop, a pump, or a body fluid storage bag. From the viewpoint of operation time, a drop or introduction by a pump is preferable, and a drop is most preferable from the viewpoint of simplicity. The liquid discharged from the cell trap 11 is stored in the drain bag 12. When all the nucleated cells and platelet-biased layers in the body fluid storage bag 14 are introduced into the cell trap 11 and pass through the cell trapping filter material, the clamps 21 and 22 are closed. At this time, in the cell trap 11 and the injection tube 6, the filtrate of 0.2 volume or more and less than 1.0 volume of the cell trap 11 is left.

  Next, only the injection port 33 and the cell trap are in communication with the T-shaped tube 32, and only the cell trap 11 and the recovery solution bag 13 are in communication with the T-shaped tube 31, and the clamp 23 is opened and the injection port is opened. Air is introduced from 33 into the cell trap 11, and mononuclear cells and platelets are collected in the collection bag 13. The amount of air to be injected only needs to be able to collect all the filtrate in the collection bag 13. When all the filtrate is collected in the collection bag 13, the clamp 23 is closed.

Alternatively, without leaving the filtrate in the cell trap 11 and the injection tube 6, air is introduced from the air vent filter 41 using a syringe or the like, and the filtrate remaining in the cell trap 11 and the filtrate collection tube 5 is pushed out to drain bag 12. It may be recovered. In this case, 0.2 to 1.0 volume filtrate of the cell trap 11 is collected from the drain bag 12 using a syringe or the like, introduced from the inlet 33, and the introduced filtrate is pushed out with air. It may be recovered in the recovery liquid bag 13. This method has the advantage that the amount of filtrate introduced can be set accurately.

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

[Example 1]
(1) Production of cell trapping device It consists of an upper container and a lower container, and the inner dimensions after assembly are 43 mm in length, 43 mm in width, and 2.9 mm in height (effective filtration cross-sectional area 18.5 cm ² , internal volume 7 mL) Polyester non-woven fabric having an average fiber diameter of about 12 μm from the inlet side of the container as a nucleated cell capturing material in a polycarbonate container having a liquid inlet and a liquid outlet on the longest diagonal. A nucleated cell-trapping filter material consisting of 14 g, 1.28 g of a polyester nonwoven fabric having an average fiber diameter of 1.7 μm and 0.19 g of a polyester nonwoven fabric having an average fiber diameter of 1.1 μm was filled. Incidentally, the packing density is 0.20 g / cm ³ from the inlet side, respectively, were 0.24 g / cm ³ and 0.20 g / cm ^3.
(2) Erythrocyte layer drainage operation 12 mL of 6% hydroxyethyl starch (Nipropharma “HES40 (erythrocyte sedimentation agent)” was added to the body fluid storage bag 14 storing 58 mL of CPD-added human peripheral blood, and mixed for 10 minutes. Then, after standing for 30 minutes to precipitate red blood cells, a layer biased toward red blood cells settled below the body fluid storage bag 14 was discharged out of the body fluid storage bag 14 through the red blood cell waste tube 1. At this time, Care was taken not to disturb the interface between the erythrocyte-biased layer, the upper-layer nucleated cells, and the platelet-biased layer, and it was slowly drained so as not to drain the upper layer. 21 and 23. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets are divided into multi-item automatic blood cell counts. Apparatus (Sysmex SF3000) measured by using a, 1.3 × ^{10 11} pieces each, were 9.0 × 10 ⁹ pieces.
(3) Cell concentration operation The nucleated cells after discharging the layer biased to erythrocytes and the body fluid storage bag 14 storing the layer biased to platelets and the cell introduction tube 4 are connected, the clamp 21 is opened, and the body fluid storage bag The upper layer nucleated cells stored in 14 and 44 mL of the layer biased to platelets were introduced into the cell trap 11 by a drop, and filtration was started. At this time, a layer biased toward nucleated cells and platelets flowed into the cell trap 11, and the filtered cell suspension was stored in the drain bag 12. All nucleated cells and platelet-biased layers of the body fluid storage bag 14 are introduced into the cell trap 11, the inside of the inlet space of the cell trap 11 is filled with air, and the outlet space of the cell trap 11 is filled. Filtration was completed while the filtrate that passed through the filter material remained in the container. Next, the clamps 21 and 22 were closed. Subsequently, the syringe is filled with 38 mL of air, the clamp 23 is opened, and 38 mL of air is manually pushed out from the inlet 33, and the cells captured by the filter are collected with the filtrate remaining in the outlet side space in the cell trap 11. The cell concentrate was recovered in the recovery bag 13. The amount of the cell concentrate collected at this time was 5.4 mL (0.77 volume of the cell trap 11).
(4) Analysis The erythrocyte removal rate, mononuclear cell recovery rate, mononuclear cell concentration rate, mononuclear cell content rate, and platelet concentration rate of the cell concentrate obtained by this cell concentration operation are respectively calculated by the following formulas. Calculated.
The counts of red blood cell count, mononuclear cell count, mononuclear cell concentration, total cell count, and platelet concentration were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000).
Erythrocyte removal rate (%) = {(number of erythrocytes in CPD-added blood-number of erythrocytes in cell concentrate) / number of erythrocytes in CPD-added blood} × 100
Mononuclear cell recovery rate (%) = (number of mononuclear cells in cell concentrate / number of mononuclear cells in CPD-added blood) × 100
・ Mononuclear cell concentration factor = (Mononuclear cell concentration in cell concentrate / Mononuclear cell concentration in CPD-added blood)
Mononuclear cell content (%) = (number of mononuclear cells in cell concentrate / total number of cells in cell concentrate) × 100
・ Platelet concentration factor = (Platelet concentration in cell concentrate / platelet concentration in CPD-added blood)
(5) Results As a result of this cell concentration operation, the number of red blood cells in the cell concentrate is 7.8 × 10 ⁸ , the red blood cell removal rate is 99.4%, the mononuclear cell recovery rate is 74.6%, and the mononuclear cell concentration is 6.9 × 10 ⁶ cells / mL, mononuclear cells concentration ratio is 8.1 times, the mononuclear cell content 50.2% platelet concentrate ratio was 2.9 times.

[Example 2]
(1) Production of cell trap The same device as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ and 8.9 × 10 ⁹ .
(3) Cell concentration operation The nucleated cells after discharging the layer biased to erythrocytes and the body fluid storage bag 14 storing the layer biased to platelets (cell suspension) and the cell introduction tube 4 are connected, and the clamp 21 is connected. The upper layer nucleated cells stored in the body fluid storage bag 14 and 44 mL of the layer biased to platelets were introduced into the cell trap 11 by a drop, and filtration was started. At this time, the layer biased to nucleated cells and platelets flowed to the cell trap 11, and the filtrate was stored in the drain bag 12. All nucleated cells and platelet-biased layers in the body fluid storage bag 14 are introduced into the cell trap 11, the inlet side space of the cell trap 11 is filled with air, and the outlet side space of the cell trap 11 is in the outlet side space. Filtration was completed with the filtrate passing through the filter material remaining. Next, the clamp 21 is closed, the clamp 23 is opened, 3.5 mL of air is introduced into the air vent filter 41 using a syringe, the filtrate remaining in the outlet side space in the cell trap 11 is extruded, and the drain bag 12 Accumulated. At this time, a small amount of filtrate remained in the outlet side space in the cell trap 11 and the injection tube 6. Subsequent operations were the same as those in Example 1. The amount of the cell concentrate collected at this time was 1.8 mL (0.26 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 1.0 × 10 ⁹ , the red blood cell removal rate was 99.3%, the mononuclear cell recovery rate was 65.1%, and the mononuclear cell concentration was 1.9. × 10 ⁷ cells / mL, mononuclear cell concentration factor was 21.7 times, mononuclear cell content was 49.8%, and platelet concentration factor was 5.5 times.

[Example 3]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ pieces and 1.0 × 10 ¹⁰ pieces.
(3) Cell Concentration Operation In the same manner as in Example 1, a layer biased toward nucleated cells and platelets was introduced into the cell trap 11 by a drop and filtration was completed, and then the clamps 21 and 22 were closed. Next, 1.0 mL of the filtrate that has passed through the filter material that has flowed into the drain bag 12 is collected by connecting a syringe to the filtrate collection port 8, filled with 37 mL of air, manually opened from the inlet 33 by opening the clamp 23. Then, the filtrate and air in the syringe were extruded, the cells captured by the filter were collected with the filtrate that passed through the filter material in the cell trap 11, and the cell concentrate was collected in the collection bag 13. The amount of the cell concentrate collected at this time was 6.7 mL (0.96 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 7.1 × 10 ⁸ , the red blood cell removal rate was 99.5%, the mononuclear cell recovery rate was 72.5%, and the mononuclear cell concentration was 5.4. × 10 ⁶ cells / mL, mononuclear cell concentration factor was 6.3 times, mononuclear cell content was 49.4%, and platelet concentration factor was 2.3 times.

[Example 4]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). They were × 10 ¹¹ and 9.0 × 10 ⁹ .
(3) Cell concentration operation The nucleated cells after discharging the layer biased to red blood cells and the body fluid storage bag 14 storing the layer biased to platelets and the cell introduction tube 4 are connected, the clamp 21 is opened, and the body fluid storage bag The upper layer nucleated cells stored in 14 and 44 mL of the layer biased to platelets were introduced into the cell trap 11 by a drop, and filtration was started. The filtrate was stored in the drain bag 12. All nucleated cells and platelet-biased layers in the body fluid storage bag 14 are introduced into the cell trap 11, the inlet side space of the cell trap is filled with air, and the outlet side space of the cell trap is a filter material. Filtration was completed in a state where the filtrate that passed through remained. Next, the clamp 21 was closed, 30 mL of air was introduced into the air vent filter 41 using a syringe, and the filtrate remaining in the cell trap 11 was completely extruded and stored in the drain bag 12. Subsequently, 6.0 mL of the filtrate stored in the drain bag 12 is collected by connecting a syringe to the filtrate collection port 8, and further filled with 32 mL of air, and the clamp 23 is opened to manually enter the syringe through the injection port 33. The filtrate and air were extruded, the cells trapped on the filter with the filtrate were collected, and the cell concentrate was collected in the collection bag 13. The amount of the cell concentrate collected at this time was 6.1 mL (0.87 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 8.1 × 10 ⁸ , the red blood cell removal rate was 99.4%, the mononuclear cell recovery rate was 68.5%, and the mononuclear cell concentration was 5.6. × 10 ⁶ cells / mL, the mononuclear cell concentration factor was 6.5 times, the mononuclear cell content was 49.1%, and the platelet concentration factor was 2.4 times.

[Example 5]
(1) Production of cell trap The same as in Example 1 was used.
(2) Erythrocyte layer draining operation 30 mL of physiological saline solution in which 5% dextran (Wako Pure Chemical "Dextran 200,000") is dissolved is added to the body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood is stored for 10 minutes. Mixed. Then, after standing for 30 minutes to sediment the red blood cells, the layer biased to the red blood cells settled below the body fluid storage bag 14 was discharged out of the body fluid storage bag 14 through the red blood cell waste tube 1. At this time, care was taken not to disturb the interface between the lower layer of red blood cells and the upper layer of nucleated cells and the layer of platelets, and the cells were slowly discharged so as not to discharge the upper layer. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ and 9.1 × 10 ⁹ .
(3) Cell concentration operation The same operation as in Example 1 was performed. The amount of the cell concentrate collected at this time was 5.0 mL (0.71 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 7.3 × 10 ⁸ , the red blood cell removal rate was 99.4%, the mononuclear cell recovery rate was 71.3%, and the mononuclear cell concentration was 7.1. × 10 ⁶ cells / mL, mononuclear cell concentration factor was 8.3 times, mononuclear cell content was 51.2%, and platelet concentration factor was 2.7 times.

[Comparative Example 1]
(1) Production of cell trap The same device as in Example 1 was used.
(2) Red blood cell layer discharge operation The red blood cell discharge operation was not performed, and the body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood was stored was used as it was. The number of red blood cells in CPD-added human peripheral blood at this time was measured using a multi-item automatic blood cell analyzer (Sysmex Corporation SF3000), and found to be 1.3 × 10 ¹¹ .
(3) Cell Concentration Operation The body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood is stored and the cell introduction tube 4 are connected, the clamp 21 is opened, and the CPD-added human peripheral blood stored in the body fluid storage bag 14 is dropped. It was introduced into the cell trap 11 and filtration was started. The filtrate was stored in the drain bag 12. All the CPD-added human peripheral blood in the body fluid storage bag 14 is introduced into the cell trap 11, the inlet side space of the cell trap is filled with air, and the filtrate remains in the outlet side space of the cell trap. Filtration was completed in the state. Next, the clamps 21 and 22 were closed. Next, 23 mL of liquid added with human serum albumin to a final concentration of 3% and 10 mL of air in a 10% dextran physiological saline solution (Kobayashi Pharmaceutical "Dextran 40 Injection") is filled into a syringe, and the clamp 23 is opened for injection. The liquid and air in the syringe were manually pushed out from the inlet 33, and the cells captured by the filter were collected in the collection bag 13 to obtain a cell concentrate. The amount of the cell concentrate collected at this time was 25.1 mL (3.59 volumes of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 1.6 × 10 ¹⁰ cells, the red blood cell removal rate was 87.7%, the mononuclear cell recovery rate was 77.9%, and the mononuclear cell concentration was 1.3. × 10 ⁶ cells / mL, mononuclear cell enrichment factor is 1.8 times, mononuclear cell content is 39.0%, platelet enrichment factor is 0.5 times, and the concentration of mononuclear cells is high with red blood cell contamination The platelet concentration factor was also low.

[Comparative Example 2]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ and 6.3 × 10 ⁹ .
(3) Cell Concentration Operation Example 2 except that 4.5 mL of air was introduced into the air vent filter 41 using a syringe after filtration and the filtrate remaining in the cell trap was extruded and stored in the drain bag 12. The same operation was performed. The amount of the cell concentrate collected at this time was 1.1 mL (0.16 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 1.6 × 10 ⁹ , the red blood cell removal rate was 98.8%, the mononuclear cell recovery rate was 44.1%, and the mononuclear cell concentration was 2.1. × 10 ⁷ cells / mL, mononuclear cell enrichment factor is 24.5 times, mononuclear cell content is 48.1%, platelet enrichment factor is 5.3 times, and the recovery rate of mononuclear cells is low. There was a lot of loss.

[Comparative Example 3]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ pieces, 5.7 × 10 ⁹ pieces.
(3) Cell concentration operation At the time of collection, 5.5 mL of the filtrate that flowed into the drain bag 12 and passed through the filter material was collected by connecting a syringe to the filtrate collection port 8, and further filled with 32.5 mL of air. Was opened and the same operation as in Example 3 was performed except that the filtrate and air in the syringe were manually pushed out from the injection port 33. The amount of the cell concentrate collected at this time was 12.1 mL (1.73 volumes of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 1.9 × 10 ⁹ , the red blood cell removal rate was 98.4%, the mononuclear cell recovery rate was 75.0%, and the mononuclear cell concentration was 3.1. × 10 ⁶ cells / mL, mononuclear cell concentration rate is 3.6 times, mononuclear cell content is 39.5%, platelet concentration rate is 1.4 times, mononuclear cell concentration and concentration rate are low, In addition, the mononuclear cell content was low, and it was not possible to concentrate mononuclear cells efficiently.

[Comparative Example 4]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The red blood cell discharge operation was not performed, and the body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood was stored was used as it was. The number of red blood cells in CPD-added human peripheral blood at this time was measured using a multi-item automatic blood cell analyzer (Sysmex Corporation SF3000), and was 1.6 × 10 ¹¹ .
(3) Cell Concentration Operation The body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood is stored and the cell introduction tube 4 are connected, the clamp 21 is opened, and the CPD-added human peripheral blood stored in the body fluid storage bag 14 is dropped. It was introduced into the cell trap 11 and filtration was started. The filtrate was stored in the drain bag 12. All the CPD-added human peripheral blood in the body fluid storage bag 14 is introduced into the cell trap 11, the inlet side space of the cell trap is filled with air, and the filtrate that has passed through the filter material in the outlet side space of the cell trap. Filtration was completed with a residual amount remaining. Next, the clamps 21 and 22 were closed. Subsequently, the syringe was filled with 38 mL of air, the clamp 23 was opened, the air in the syringe was manually pushed out from the injection port 33, and the cells captured by the filter with the filtrate that passed through the filter material in the cell trap were collected. The cell concentrate was recovered in the recovery bag 13. The amount of the cell concentrate collected at this time was 3.1 mL (0.44 volume of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 9.1 × 10 ⁹ , the red blood cell removal rate was 94.2%, the mononuclear cell recovery rate was 50.6%, and the mononuclear cell concentration was 7.1. × 10 ⁶ cells / mL, mononuclear cell enrichment factor is 9.6 times, mononuclear cell content is 47.4%, platelet enrichment factor is 2.4 times, there is a lot of red blood cell contamination, and mononuclear cells The recovery rate was also low.

[Comparative Example 5]
(1) Production of cell trap The same as in Example 1 was used.
(2) Erythrocyte layer draining operation The erythrocyte layer draining operation is not performed, and 12 mL of 6% hydroxyethyl starch (Nipropharma “HES40 (erythrocyte sedimenting agent)”) is added to the body fluid storage bag 14 in which 58 mL of CPD-added human peripheral blood is stored. And mixed for 10 minutes. The number of red blood cells in the CPD-added human peripheral blood at this time was measured using a multi-item automatic blood cell analyzer (Sysmex Corporation SF3000), and the result was 1.7 × 10 ¹¹ .
(3) Cell Concentration Operation The CPD-added human peripheral blood and the body fluid storage bag 14 storing 6% hydroxyethyl starch are mixed for 10 minutes and then left to stand for 30 minutes in a state connected to the cell introduction tube 4 to precipitate red blood cells. It was. Next, the clamp 21 is opened, and the layer biased toward the lower red blood cells of the body fluid storage bag 14 is introduced into the cell trap 11. Subsequently, the upper layer nucleated cells and the layer biased toward platelets are similarly applied to the cell trap 11. Introduction and filtration was started. The filtrate was stored in the drain bag 12. All the red blood cell-biased layers, nucleated cells, and platelet-biased layers in the body fluid storage bag 14 are introduced into the cell trap 11, and the inlet side space of the cell trap 11 is filled with air. Filtration was completed with the filtrate passing through the filter material remaining in the outlet side space. Next, a syringe was filled with 23 mL of liquid and 15 mL of air, in which human serum albumin was added to a final concentration of 3% in 10% dextran physiological saline solution (Kobayashi Pharmaceutical "Dextran 40 Injection"), and the clamp 23 was opened for injection. The liquid and air in the syringe were manually pushed out from the inlet 33, and the cells captured by the filter were collected in the collection bag 13 to obtain a cell concentrate. The amount of the cell concentrate collected at this time was 25.8 mL (3.69 volumes of the cell trap 11).
(3) Analysis Calculated with the same calculation formula as in Example 1.
(4) Results The number of red blood cells in the cell concentrate at this time was 1.2 × 10 ¹⁰ cells, the red blood cell removal rate was 92.8%, the mononuclear cell recovery rate was 65.1%, and the mononuclear cell concentration was 1.3. × 10 ⁶ cells / mL, mononuclear cell concentration factor is 1.5 times, mononuclear cell content is 40.2%, platelet concentration factor is 0.5 times, and there is much red blood cell contamination and mononuclear cell concentration Was also low.

[Comparative Example 6]
(1) Production of cell trap The same as in Example 1 was used.
(2) Red blood cell layer discharge operation The same operation as in Example 1 was performed. At this time, the number of red blood cells in the CPD-added human peripheral blood and the number of red blood cells in the upper layer of nucleated cells and platelets were measured using a multi-item automatic blood cell analyzer (Sysmex SF3000). × 10 ¹¹ and 8.9 × 10 ⁹ .
(3) Cell concentration operation The nucleated cells after discharging the layer biased to red blood cells and the body fluid storage bag 14 storing the layer biased to platelets and the cell introduction tube 4 are connected, the clamp 21 is opened, and the body fluid storage bag The upper layer nucleated cells stored in 14 and 44 mL of the layer biased to platelets were introduced into the cell trap 11 by a drop, and filtration was started. The filtrate was stored in the drain bag 12. All nucleated cells and platelet-biased layers in the body fluid storage bag 14 are introduced into the cell trap 11, the inlet side space of the cell trap 11 is filled with air, and the outlet side space of the cell trap 11 is in the outlet side space. Filtration was completed with the filtrate passing through the filter material remaining. Next, a syringe was filled with 23 mL of liquid and 15 mL of air, in which human serum albumin was added to a final concentration of 3% in 10% dextran physiological saline solution (Kobayashi Pharmaceutical "Dextran 40 Injection"), and the clamp 23 was opened for injection. The liquid and air in the syringe were manually pushed out from the inlet 33, and the cells captured by the filter were collected in the collection bag 13 to obtain a cell concentrate. The amount of the cell concentrate collected at this time was 25.8 mL (3.69 volumes of the cell trap 11).
(4) Analysis The calculation was the same as in Example 1.
(5) Results The number of red blood cells in the cell concentrate at this time was 2.6 × 10 ⁹ , the red blood cell removal rate was 98.1%, the mononuclear cell recovery rate was 72.5%, and the mononuclear cell concentration was 1.4. × 10 ⁶ cells / mL, mononuclear cell concentration factor is 1.6 times, mononuclear cell content is 40.1%, platelet concentration factor is 0.6 times, and there is also contamination of red blood cells and mononuclear cell concentration The mononuclear cell content was low and the mononuclear cells could not be concentrated efficiently.

  The method for concentrating mononuclear cells and platelets according to the present invention can easily prepare a mixed solution of mononuclear cells and platelets at a high concentration, and since there is no risk of infection or the like because it is composed solely of complete self-components, Useful in regenerative medicine and cell therapy. Since the concentration method according to the present invention is efficient, it is particularly useful when the amount of body fluid is small.

It is a schematic diagram which shows an example of the system for adding the erythrocyte sedimentation agent of this invention and isolate | separating into the layer biased to the nucleated cell and platelet, and the layer biased to erythrocytes. It is a schematic diagram which shows an example of the concentration system for concentrating the mononuclear cell and platelet of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Red blood cell waste tube 2 ... Cell introduction tube connection part 3 ... Injection | pouring part 4 ... Cell introduction tube 5 ... Filtrate collection tube 6 ... Injection tube 7 ... Cell collection tube 8 ... Filtrate collection port 11 ... Cell trap 12 ... Drain bag 13 ... Recovery bag 14 ... Body fluid storage bags 21, 22, 23 ... Clamps 31, 32 ... T-tube 33 ... Inlet 41 ... Air vent filter 100 ... Concentration system

Claims (6)

Mononuclear cells and platelets from body fluids containing red blood cells, nucleated cells, and platelets using a filter system having a cell trapping device filled with a cell trapping filter material in a container having an inlet and an outlet, a circuit, and a bag A method of concentrating
The filter material is a nonwoven fabric having an average fiber diameter of 1.0 μm to 30 μm,
Add an erythrocyte sedimentation agent to the body fluid stored in the bag,
Separated into nucleated cells and a layer biased to platelets and a layer biased to red blood cells,
After introducing only the nucleated cells and platelet-biased layer into the cell trap and capturing the nucleated cells and platelets in the filter material,
Only the filtrate that has passed through the filter material of 0.2 volume or more and less than 1.0 volume of the cell trap, is extruded with gas and introduced into the filter material,
A method for concentrating mononuclear cells and platelets from a body fluid comprising erythrocytes, nucleated cells and platelets, wherein the mononuclear cells and platelets captured by the filter material are selectively collected.   2. The method of concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets according to claim 1, wherein the gas is air.   The filtrate that has passed through the filter material in a volume of 0.2 to 1.0 volume of the cell trap is the filtrate remaining in the cell trap and the circuit. A method for concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets.   4. The mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets according to any one of claims 1 to 3, wherein the erythrocyte sedimenting agent is either hydroxyethyl starch or dextran. How to concentrate.   After separating the nucleated cells and the layer biased to platelets and the layer biased to erythrocytes, the layer biased to erythrocytes is discharged from the bag, and only the layer that is biased to nucleated cells and platelets is captured. A method for concentrating mononuclear cells and platelets from a body fluid comprising erythrocytes, nucleated cells and platelets according to any one of claims 1 to 4, which is introduced into a vessel.   6. The body fluid comprising the red blood cells, nucleated cells, and platelets is one body fluid selected from the group consisting of peripheral blood, umbilical cord blood, and bone marrow fluid. A method for concentrating mononuclear cells and platelets from a body fluid comprising red blood cells, nucleated cells and platelets.