JP2012139142A - Hematopoietic stem cell separating material or method for separation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separating material capable of easily and efficiently separating hematopoietic stem cells from a bodily fluid containing hematopoietic stem cells; and a method for separation using the separating material.SOLUTION: The hematopoietic stem cell separating material is made of a non-woven fabric whose average fiber diameter is at least 3 micrometers and not more than 4 micrometers. The method for separating hematopoietic stem cells by using a hematopoietic stem cell separating device 2 filled with the hematopoietic stem cell separating material includes: (A) a step of capturing hematopoietic stem cells into the hematopoietic stem cell separating device by introducing the bodily fluid from an inlet side of the hematopoietic stem cell separating device and extracting the bodily fluid from an outlet side of the hematopoietic stem cell separating device; and (B) a step of introducing a solution for separating the hematopoietic stem cells captured inside the hematopoietic stem cell separating device from the outlet side of the hematopoietic stem cell separating device and extracting the solution from the inlet side of the device. According to the method, hematopoietic stem cells can be efficiently collected and a hematopoietic stem cell-containing liquid with a higher concentration factor can be obtained.

Description

  The present invention relates to a hematopoietic stem cell separation material capable of easily and efficiently separating hematopoietic stem cells from a body fluid containing hematopoietic stem cells, and a separation method using the separation material.

  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. Further, there is a demand for a treatment style that further suppresses side effects by not administering fractions that are not necessary for treatment. 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. Platelet preparations are collected by removing unnecessary cells and components other than platelets by centrifugation.

  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. A hematopoietic stem cell means a CD34-positive cell or a cell capable of forming a colony in an agar-like medium containing a hematopoietic factor. Recently, CD34 negative and CD133 positive cells are considered to be more undifferentiated hematopoietic stem cells. 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 not using a centrifugation method, recently, a method of collecting leukocytes using a filter material that captures only leukocytes without capturing red blood cells and platelets (Patent Document 1, Patent Document 2), leukocytes and red blood cells are collected. An adult stem cell separation method using a cell separation material that can be substantially passed (Patent Document 3) and the like have been reported.

  However, recently, it has been considered that selective enrichment of hematopoietic stem cells is more effective with fewer side effects, and CD34 represented by Isolex, a magnetic cell separation system manufactured by Baxter. A method for selectively separating positive cells is disclosed. However, this separation method uses an antibody and is problematic in terms of cost and safety, and can recover only CD34 positive cells among the hematopoietic stem cells, which is more undifferentiated than the currently discussed CD34 negative. There are various problems such as inability to collect hematopoietic stem cells.

JP-T-2001-518792 International Publication WO 98/32840 JP 2008-22822 A

  An object of the present invention is to provide a separation material and a separation method capable of recovering and concentrating hematopoietic stem cells with high yield from a body fluid containing each blood cell component or a body fluid obtained by roughly separating them.

  The present inventor has intensively studied a separation material and a separation method that can recover and concentrate hematopoietic stem cells in a high yield, which has been difficult to realize in the past.

  As a result, it has been found that the use of a non-woven fabric having specific properties enables more efficient recovery and concentration of hematopoietic stem cells, leading to the completion of the present invention.

  That is, the present invention relates to a separation material capable of concentrating and collecting hematopoietic stem cells described below and a method for separating hematopoietic stem cells using the same.

  (1) A hematopoietic stem cell separation material comprising a nonwoven fabric having an average fiber diameter of 3 micrometers or more and 4 micrometers or less. According to this, hematopoietic stem cells can be efficiently recovered and concentrated.

  (2) The hematopoietic stem cell separation material according to (1), wherein the material constituting the nonwoven fabric is a polyester resin.

  (3) The hematopoietic stem cell separation material according to (2), wherein the polyester resin is a polybutylene terephthalate resin.

  (4) The hematopoietic stem cell separating material according to any one of (1) to (3), wherein the separating material is not subjected to hydrophilic treatment.

  (5) A hematopoietic stem cell separation device in which the separation material according to (1) to (4) is filled in a container having an inlet and an outlet.

(6) A means for containing body fluid on the inlet side of the hematopoietic stem cell separation device according to (5), and a means for containing the solution that has passed through the hematopoietic stem cell separation device on the outlet side of the hematopoietic stem cell separation device,
Furthermore, a solution introducing means for allowing the hematopoietic stem cell separator to flow in the direction opposite to the body fluid in order to desorb and collect the components captured by the hematopoietic stem cell separator on the outlet side of the hematopoietic stem cell separator; A hematopoietic stem cell separation circuit comprising means for containing captured components discharged from the inlet side of the stem cell separation device. According to this, liquid feeding of body fluid and recovery of the captured component can be easily performed.

(7) A hematopoietic stem cell separation device according to (5), or a hematopoietic stem cell separation method using the cell separation circuit according to (6),
(A) a step of capturing hematopoietic stem cells in the hematopoietic stem cell separator by introducing a body fluid from the inlet side of the hematopoietic stem cell separator and leading out from the outlet side;
(B) A method for separating hematopoietic stem cells, comprising a step of introducing a solution for detaching hematopoietic stem cells trapped in the hematopoietic stem cell separation apparatus from the outlet side of the hematopoietic stem cell separation apparatus and deriving from the inlet side.

  (8) The method for separating hematopoietic stem cells according to (7), comprising a step of priming before step (A).

  When this separation material, cell separation device, or cell separation circuit is used, hematopoietic stem cells can be collected and concentrated more efficiently than before.

  According to the present invention, hematopoietic stem cells are efficiently recovered from a body fluid containing each blood cell component such as peripheral blood, umbilical cord blood, bone marrow, tissue extract, menstrual blood, or a body fluid obtained by roughly separating them, and more than other cells. It is possible to achieve a high concentration rate, and it is expected to be widely used in fields such as regenerative medicine.

Example of hematopoietic stem cell separation circuit of the present invention

  The present invention is described in detail below, but the present invention is not limited to the following specific examples.

  As the body fluid in the present invention, whole blood, bone marrow, umbilical cord blood, menstrual blood, and tissue extract can be used. In this case, those roughly separated are also included in the body fluid. 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 according to the purpose for which each fraction is used, the preservation conditions of the body fluid are not questioned at all.

  The hematopoietic stem cell separation material in the present invention preferably has a fiber diameter of 3 micrometers or more and 4 micrometers or less from the viewpoint of separation efficiency of hematopoietic stem cells. The fiber diameter is the width of the fiber in the direction perpendicular to the fiber axis, and the fiber diameter is measured by taking a photograph of a separating material made of a nonwoven fabric with a scanning electron microscope and obtaining it from the scale described in the photograph. It can be obtained by averaging the calculated diameters. That is, the fiber diameter described in the present invention means an average value of the fiber diameters measured as described above, and is an average value of 50 or more, desirably 100 or more. However, when a large number of fibers are overlapped, when other fibers are obstructed and the width cannot be measured, or when fibers having remarkably different diameters are mixed, the fiber diameter is calculated excluding the data. In addition, in the case of a nonwoven fabric composed of a plurality of fibers having greatly different thicknesses, for example, 7 μm or more, the fiber diameter is calculated separately because the smaller the fiber diameter, the greater the influence on the separation efficiency. The thin fiber diameter is defined as the fiber diameter of the nonwoven fabric.

  In the present invention, the material constituting the hematopoietic stem cell separator filled in the hematopoietic stem 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) ), Synthetic polymers such as polyamide, cupra, carbon, phenol, polyester, pulp, hemp, polyurethane, polystyrene, polycarbonate, agarose, cellulose, cellulose Tate, may be used chitosan, natural polymers such as chitin, an inorganic material, metal or the like such as glass.

  Among these, it is particularly preferable to use a polyester resin as a material constituting the hematopoietic stem cell separating material of the present invention from the viewpoint that hematopoietic stem cells can be further purified, and more preferably a polybutylene terephthalate resin. In addition, these separating materials are not subjected to hydrophilic treatment such as hydrophilic coating or graft polymerization on the surface, from the viewpoint of adverse effects on the human body due to peeling of the coating agent and reduction in strength of the separating material due to graft polymerization. ,preferable. In addition, it is also advantageous that the treatment without hydrophilic treatment greatly reduces labor in producing the hematopoietic stem cell separation device.

  The use form of the hematopoietic stem cell separator in the present invention may be used without putting the separator into a container, or the hematopoietic stem cell separator may be used in a container having an inlet and an outlet for body fluid, In consideration of practicality, the latter used in a container is preferred. Further, the blood cell separator may be treated with a body fluid in the form of a flat plate cut to an appropriate size, or may be treated in the form of a roll.

  A container provided with an inlet and an outlet for a body fluid filled with the hematopoietic stem cell separating material of the present invention, and members for performing the method described in the present invention will be described.

  When filling the hematopoietic stem cell separation material of the present invention into a container provided with an inlet and an outlet for body fluid, the container may be compressed and filled, or the container may be filled without being compressed. What is necessary is just to select suitably according to the material etc. of a hematopoietic stem cell isolation | separation material. As a preferred use example of the hematopoietic stem cell separating material, it is preferable to cut the hematopoietic stem cell separating material made of a non-woven fabric into an appropriate size and use it in a single layer or a laminated state in a thickness of about 1 mm to 200 mm. From the standpoint of separation efficiency of each fraction, 1.5 mm to 150 mm is more preferable, and 2 mm to 100 mm is more preferable. Further, when the container is filled, the thickness is preferably about 1 mm to 50 mm and used in a single layer or a laminated state. From the standpoint of separation efficiency of each fraction, 1.5 mm to 40 mm is more preferable, and further preferably 2 mm to 35 mm.

  Further, the hematopoietic stem cell separating material may be wound into a roll and filled into a container. When used in the form of a roll, blood cells may be separated by treating the body fluid from the inside to the outside of the roll, or conversely, the body fluid may be treated from the outside to the inside of the roll. good.

  The form, size, and material of the container filled with the hematopoietic stem cell separating material are not particularly limited. The form of the container may be any form such as a sphere, a container, a cassette, a bag, a tube, or a column. Preferable specific examples include, for example, a translucent cylindrical container having a volume of about 0.1 mL to 400 mL and a diameter of about 0.1 cm to 15 cm, a rectangle or square having a length of about 0.1 cm to 20 cm, and a thickness. However, the present invention is not limited to these examples.

  The container can be made using any structural material. Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride, polyamide, polyimide , Polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene and the like. Of these, polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, and the like are particularly preferable in that they have sterilization resistance.

  On the other hand, when a metal material (biocompatible metal, alloy) is used as the structural material of the container, for example, stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, Examples thereof include cobalt chromium alloy and titanium nitride-coated stainless steel.

  Regarding the hematopoietic stem cell separation circuit of the present invention, in consideration of the fact that body fluid can be easily processed in a closed system, means 3 (means for supplying body fluid) containing body fluid on the inlet side of the hematopoietic stem cell separation device 2, and hematopoiesis Means 5 for storing the solution that has passed through the hematopoietic stem cell separator on the outlet side of the stem cell separator 2 (means for collecting, but if the solution that has passed through the hematopoietic stem cell separator is not required, it can be discarded as is, In addition, on the outlet side of the hematopoietic stem cell separation device 2, in order to desorb and collect the components captured by the hematopoietic stem cell separation device, the hematopoietic stem cell separation device is opposite to the body fluid. It is preferable to have a feature that a solution introduction means 7 for flowing in the direction and a means 6 for accommodating the captured components discharged from the inlet side of the hematopoietic stem cell separation device 2 at this time are provided. The means 3 for storing the body fluid and the means 5 for storing the solution that has passed through the hematopoietic stem cell separator are preferably bag-shaped from the viewpoint of ease of handling, but the present invention is not limited thereto.

  In addition, it is preferable from the viewpoint of ease of use that each storage means is connected by a tube or the like, and each tube is provided with means capable of controlling the flow of the solution such as a clamp, a two-way stopcock, and a three-way stopcock. . The solution introducing means 7 for flowing the component captured by the hematopoietic stem cell separation device 2 in the direction opposite to the body fluid in order to desorb and collect the component is provided in a state where the solution is contained in a bag or the like. Alternatively, it may be provided in a state where the solution is contained in the syringe. It is easy to use the solution introducing means 7 with a branch through a three-way cock between the outlet side of the hematopoietic stem cell separating apparatus 2 and the means 5 for storing the solution that has passed through the hematopoietic stem cell separating apparatus. From this point of view, it is preferable (however, it is also possible to arrange the solution introducing means 7 in place of the means 5 for storing the solution that has passed through the hematopoietic stem cell separating apparatus after the body fluid is first flowed). Further, the trapped component discharged from the inlet side of the hematopoietic stem cell separation device when flowing in the direction opposite to the body fluid to the hematopoietic stem cell separation device in order to desorb and collect the component captured by the hematopoietic stem cell separation device 2 It is preferable that the means 6 for storing the blood flow is arranged between the inlet side of the hematopoietic stem cell separation device 2 and the means 3 for storing the body fluid with a branching through a three-way stopcock or the like (however, the body fluid was first flowed) Later, it is also possible to dispose the means 6 for containing the trapped components discharged from the inlet side of the hematopoietic stem cell separation device instead of the means 3 for containing the body fluid. In addition, the means 6 for storing the captured component discharged from the inlet side of the hematopoietic stem cell separation device is preferably in the shape of a bag because it can be easily obtained and is easy to handle after the operation. Further, a bag capable of cell culture, a bag having cryopreservation resistance, and the like may be selected. Although the cell separation circuit of this invention is illustrated in FIG. 1, this invention is not limited to these.

  Next, a method for separating hematopoietic stem cells will be described.

  The method for separating hematopoietic stem cells in the present invention includes a step of first capturing a hematopoietic stem cell in the hematopoietic stem cell separating apparatus by introducing a body fluid from the inlet side of the hematopoietic stem cell separating apparatus and deriving from the outlet side. A method for separating hematopoietic stem cells, comprising a step of introducing a solution for detaching hematopoietic stem cells trapped in the stem cell separation device from the outlet side of the hematopoietic stem cell separation device and leading out the solution from the inlet side. As the hematopoietic stem cell separating material, those described above can be used.

  Specifically, as a first step, a body fluid is injected into the hematopoietic stem cell separation device from the inlet side, the hematopoietic stem cells are captured by the hematopoietic stem cell separation device, and the red blood cells pass through the hematopoietic stem cell separation device. Next, a washing operation in the hematopoietic stem cell separation device is performed as necessary. Although this washing operation is not always necessary, it is preferably performed to reduce contamination of components other than hematopoietic stem cells in the finally collected hematopoietic stem cells. Next, as a second step, in order to collect hematopoietic stem cells captured in the hematopoietic stem cell separation device from the outlet direction of the hematopoietic stem cell separation device, that is, from the direction opposite to the direction in which the body fluid or the washing solution is passed. The solution can be introduced, and hematopoietic stem cells can be collected and concentrated in a high yield.

  Hereinafter, each step of the method for separating hematopoietic stem cells of the present invention will be described with examples.

1) Body fluid feeding step When passing body fluid from the body fluid inlet side of the hematopoietic stem cell separator filled with hematopoietic stem cell separation material, even if it is sent by natural fall from the container containing the body fluid through the fluid feeding circuit, You may send liquid with a pump. Alternatively, a syringe containing body fluid may be directly connected to the container and the syringe pushed by hand. When the liquid is passed through the pump, the separation efficiency decreases if the liquid feeding speed is too fast, and the processing time is too long if the liquid feeding speed is too slow, and examples include 0.1 mL / min to 100 mL / min, but are not limited thereto. It is not something.

  Moreover, you may implement the process of immersing a separating material with a physiological saline or a buffer solution as pre-processing of a bodily fluid sending process. Although this operation is not always necessary, it is considered that the immersion of the separation material in the above solution may affect the improvement of the separation efficiency and the securing of the blood flow path. Various solutions can be used as this pretreatment solution, but the same solution solution can be shared as long as it is the same as that used in the cleaning process described below. It is preferable. The amount of the pretreatment liquid is practically and preferably about 1 to 100 times that of the container filled with the blood cell separator.

2) Cleaning process (cleaning operation is not always necessary)
The cleaning liquid may be sent by natural fall through the circuit from the same direction as the body fluid feeding process, or may be fed by a pump. The flow rate in the case of feeding with a pump is about the same as that in the body fluid feeding process, and is from 0.1 mL / min to 100 mL / min, but is not limited thereto. The amount of washing differs depending on the volume of the container. However, if the amount of washing is too small, the amount of red blood cell components remaining in the container increases, and if the amount of washing is too large, the separation efficiency is reduced and a great deal of time is required. It is preferable to wash with a volume of about 100 to 100 times.

  As the cleaning solution that can be used, any solution can be used as long as it is possible to wash out red blood cells, suppress contamination of other blood cells in the hematopoietic stem cell-containing solution after separation, and maintain the trapped state of hematopoietic stem cells. Ordinary buffer solutions such as physiological saline, Ringer's solution, medium used for cell culture, and phosphate buffer are preferable.

3) Desorption of hematopoietic stem cells A solution is injected into the hematopoietic stem cell separation device from the direction opposite to the flow of the body fluid (from the body fluid outflow side) to desorb the hematopoietic stem cells. When injecting the solution, the separation solution can be put in a syringe or the like in advance, and the plunger of the syringe can be pushed out vigorously using hands or equipment. The amount of recovered liquid and the flow rate vary depending on the capacity and processing amount of the container, but the capacity is about 0.3 to 100 times that of the container, and the flow rate is preferably 0.5 mL / sec to 20 mL / sec, but is not limited thereto. It is not a thing. Further, the solution may be introduced by natural fall or the solution may be introduced by a syringe pump.

  The solution used in this case is not particularly limited as long as it is a hypotonic solution, and examples thereof include those that have been used as injections such as physiological saline and Ringer's solution, buffer solutions, cell culture media, and the like.

  Further, the viscosity of the collected liquid may be increased in order to increase the collection rate of the captured cells. For this purpose, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethyl cellulose, collagen, hyaluronic acid, gelatin and the like can be added to the above-mentioned separation solution, but are not limited thereto.

  EXAMPLES Hereinafter, although an Example demonstrates in detail regarding this invention, this invention is not limited only to a following example. Since the nonwoven fabrics used in the following examples and comparative examples have different thicknesses per sheet, the number of sheets filled in the containers is different. However, the thickness of the total number or the compression ratio (= thickness when filled in the container / thickness of the number of sheets to be fed) are standardized, and it is carried out under the same conditions.

Example 1
A container having a thickness (inner diameter) of 12 mm and a diameter (inner diameter) of 44 mm is filled with 112 sheets of polybutylene terephthalate nonwoven fabric (fiber diameter 3.8 μm, density K1.3 × 10 ⁵ g / m ³ ) in a laminated state, and cells A separator was prepared, and a hematopoietic stem cell separator as shown in FIG. 1 was produced. Specifically, a three-way stopcock is attached to the upstream side of the cell separator, a blood bag (manufactured by Kawasumi Corp., Kawasumi blood separation bag (capacity 200 mL)) is connected via a tube, and the three-way stopcock is connected downstream of the cell separator. And a bag for collecting the solution that passed through the cell separator was connected via a tube. About 100 mL of physiological saline was introduced into the blood bag, and the three-way stopcock was adjusted so that the physiological saline flowed to the cell separator. After confirming that the physiological saline in the blood bag almost disappeared, the three-way stopcock upstream of the cell separator was adjusted to stop the physiological saline flow. At this time, care was taken so that a slight amount of physiological saline remained in the blood bag and air did not enter the filter. Next, human peripheral blood (100 mL anticoagulated with 28 mL CPD) was introduced into the same blood bag. Similarly, the three-way stopcock was adjusted, and human peripheral blood was introduced into the cell separator and led to the bag. The three-way stopcock was closed immediately before human peripheral blood disappeared from the blood bag and air entered the filter. All but the three-way stopcock on the outlet side were removed from the cell separator. 20 mL of 4% human serum albumin-added sarinhes was taken in a syringe, introduced from a three-way stopcock on the outlet side, led out from the inlet side of the cell separator, and collected in a centrifuge tube. 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. Furthermore, the blood before treatment and the collected solution were hemolyzed with FACS Lysing Solution, then the mononuclear cell positive rate and granulocyte positive rate were obtained with a flow cytometer (BD FACSCanto), and the total number of white blood cells was multiplied by each positive rate. The number of mononuclear cells and the total number of granulocytes were calculated. The ratios obtained by dividing the total number of mononuclear cells and the total number of granulocytes in the collected solution by the total number of mononuclear cells and the total number of granulocytes before treatment were taken as the mononuclear cell recovery rate and granulocyte recovery rate, respectively. Further, according to the ISHAGE guidelines, the number of CD34 positive cells was counted, and the recovery rate of CD34 positive cells was calculated in the same manner. Moreover, the number of colonies by a colony assay was measured, and the recovery rate of colonies was measured. Specifically, the white blood cell concentration was adjusted to 2 × 10 ^ 6, 0.3 mL was added to 3 mL of methylcellulose medium MethoCult H4034 (StemCell Technologies), and then 1.1 mL of the mixed solution was dispensed into a Petri dish. Note and cultured at 37 ° C., 5% CO 2. After 14 days, the number of various colonies consisting of hematopoietic stem cells such as erythroid progenitor cells and leukocyte progenitor cells was counted with a microscope, and the colony recovery rate was calculated from the total number of colonies before and after treatment. The results are shown in Table 1.

(Example 2)
A container with a thickness of 6 mm and a diameter of 18 mm is filled with 28 non-woven fabrics made of polybutylene terephthalate (fiber diameter 3.8 μm, density K1.2 × 10 ⁵ g / m ³ ), and 45 mL of physiological saline is first introduced on the inlet side. Further, the solution was passed by hand using a syringe. Next, 10 mL of CPD anticoagulated fresh human peripheral blood was passed through 2.5 mL / min. Thereafter, 30 mL of 10% FBS-added MEM medium was collected manually by a syringe from the direction opposite to the flow direction. In the same manner as in Example 1, the leukocyte recovery rate, mononuclear cell recovery rate, granulocyte recovery rate, CD34 positive cell recovery rate, and colony recovery rate were calculated. The results are shown in Table 1.

Example 3
A container having a thickness of 6 mm and a diameter of 18 mm is filled with 28 polypropylene non-woven fabrics (fiber diameter 3.5 μm, density K 8.3 × 10 ⁴ g / m ³ ) in a laminated state. First, 45 mL of physiological saline is syringed from the inlet side. The solution was passed by hand using Next, 10 mL of CPD anticoagulated fresh human peripheral blood to which human umbilical cord blood-derived CD34 positive cells were added was passed through 2.5 mL / min, and then 10 mL of physiological saline from the same direction. Thereafter, 30 mL of 10% FBS-added MEM medium was collected manually by a syringe from the direction opposite to the flow direction. In the same manner as in Example 1, the leukocyte recovery rate, mononuclear cell recovery rate, granulocyte recovery rate, CD34 positive cell recovery rate, and colony recovery rate were calculated. The results are shown in Table 1.

(Comparative Example 1)
A container with a thickness of 6 mm and a diameter of 18 mm is filled with 36 nylon non-woven fabrics (fiber diameter 5.0 μm, density K1.3 × 10 ⁵ g / m ³ ) in a laminated state. First, 45 mL of physiological saline is syringed from the inlet side. The solution was passed by hand using Next, 10 mL of CPD anticoagulated fresh human peripheral blood to which human umbilical cord blood-derived CD34 positive cells were added was passed through 2.5 mL / min, and then 10 mL of physiological saline from the same direction. Thereafter, 30 mL of 10% FBS-added MEM medium was collected manually by a syringe from the direction opposite to the flow direction. In the same manner as in Example 1, the leukocyte recovery rate, mononuclear cell recovery rate, granulocyte recovery rate, CD34 positive cell recovery rate, and colony recovery rate were calculated. The results are shown in Table 1.

(Comparative Example 2)
A container with a thickness of 6 mm and a diameter of 18 mm is filled with 30 sheets of polypropylene non-woven fabric (fiber diameter 2.4 μm, density K7.5 × 10 ⁴ g / m ³ ) in a laminated state. First, 45 mL of physiological saline is syringed from the inlet side. The solution was passed by hand using Next, 10 mL of CPD anticoagulated fresh human peripheral blood to which human umbilical cord blood-derived CD34 positive cells were added was passed through 2.5 mL / min, and then 10 mL of physiological saline from the same direction. Thereafter, 30 mL of 10% FBS-added MEM medium was collected manually by a syringe from the direction opposite to the flow direction. In the same manner as in Example 1, the leukocyte recovery rate, mononuclear cell recovery rate, granulocyte recovery rate, CD34 positive cell recovery rate, and colony recovery rate were calculated. The results are shown in Table 1.

(Comparative Example 3)
A container having a thickness of 6 mm and a diameter of 18 mm is filled with 84 sheets of non-woven fabric made of polybutylene terephthalate (fiber diameter 1.8 μm, density K8.6 × 10 ⁴ g / m ³ ), and 45 mL of physiological saline is first introduced on the inlet side. Further, the solution was passed by hand using a syringe. Next, 10 mL of CPD anticoagulated fresh human peripheral blood to which human umbilical cord blood-derived CD34 positive cells were added was passed through 2.5 mL / min, and then 10 mL of physiological saline from the same direction. Thereafter, 30 mL of 10% FBS-added MEM medium was collected manually by a syringe from the direction opposite to the flow direction. In the same manner as in Example 1, the leukocyte recovery rate, mononuclear cell recovery rate, granulocyte recovery rate, CD34 positive cell recovery rate, and colony recovery rate were calculated. The results are shown in Table 1.

1 Chamber 2 Container filled with hematopoietic stem cell separator (hematopoietic stem cell separator)
3 Means for accommodating body fluid 4 Means for accommodating priming fluid 5 Means for accommodating solution that has passed through hematopoietic stem cell separator 6 Means for accommodating hematopoietic stem cell 7 Hematopoietic stem cells captured in hematopoietic stem cell separator Solution introduction means for desorption 8, 9, 10 Three-way stopcock 11, 12, 13, 14, 15, 16, 17 Circuit

Claims (8)

A hematopoietic stem cell separation material comprising a nonwoven fabric having an average fiber diameter of 3 micrometers or more and 4 micrometers or less. The hematopoietic stem cell separation material according to claim 1, wherein the material constituting the nonwoven fabric is a polyester resin. The hematopoietic stem cell separator according to claim 2, wherein the polyester resin is a polybutylene terephthalate resin. The hematopoietic stem cell separator according to any one of claims 1 to 3, wherein the separator is not hydrophilically treated. A hematopoietic stem cell separation device, wherein the hematopoietic stem cell separation material according to claim 1 is filled in a container having an inlet and an outlet. Means for containing body fluid on the inlet side of the hematopoietic stem cell separator according to claim 5, and means for containing the solution that has passed through the hematopoietic stem cell separator on the outlet side of the hematopoietic stem cell separator,
Furthermore, a solution introducing means for allowing the hematopoietic stem cell separator to flow in the direction opposite to the body fluid in order to desorb and collect the components captured by the hematopoietic stem cell separator on the outlet side of the hematopoietic stem cell separator; A hematopoietic stem cell separation circuit comprising means for containing captured components discharged from the inlet side of the stem cell separation device. A hematopoietic stem cell separation device according to claim 5, or a hematopoietic stem cell separation method using the hematopoietic stem cell separation circuit according to claim 6,
(A) a step of capturing hematopoietic stem cells in the hematopoietic stem cell separator by introducing a body fluid from the inlet side of the hematopoietic stem cell separator and leading out from the outlet side;
(B) introducing a solution for detaching hematopoietic stem cells trapped in the hematopoietic stem cell separation device from the outlet side of the hematopoietic stem cell separation device and deriving from the inlet side;
Of isolating hematopoietic stem cells. The method for separating hematopoietic stem cells according to claim 7, comprising a step of priming before step (A).

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