WO2012070622A1 - 白血球または単核球の分離方法、分離材 - Google Patents
白血球または単核球の分離方法、分離材 Download PDFInfo
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- WO2012070622A1 WO2012070622A1 PCT/JP2011/077067 JP2011077067W WO2012070622A1 WO 2012070622 A1 WO2012070622 A1 WO 2012070622A1 JP 2011077067 W JP2011077067 W JP 2011077067W WO 2012070622 A1 WO2012070622 A1 WO 2012070622A1
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- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
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- A—HUMAN NECESSITIES
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Definitions
- the present invention relates to a separation material and a separation method for selectively collecting leukocytes or mononuclear cells from a body fluid containing each blood cell component.
- 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.
- Red blood cell preparations do not require leukocytes that induce side effects such as abnormal immune reactions or graft-versus-host disease (GVHD), and it is necessary to remove leukocytes with a filter.
- GVHD graft-versus-host disease
- platelets may be removed in addition to leukocytes.
- 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.
- unnecessary cells and components other than platelets are removed by centrifugation, and only the necessary platelet components are collected.
- hematopoietic stem cell transplantation for the treatment of leukemia and solid cancer has been actively performed, and a method of separating and administering leukocyte groups containing hematopoietic stem cells necessary for treatment has been taken.
- 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.
- menstrual blood 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.
- leukocytes 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.
- Patent Document 1 a method of collecting leukocytes using a filter material that captures only leukocytes without capturing red blood cells and platelets.
- Patent Document 2 a method of collecting leukocytes using a filter material that captures only leukocytes without capturing red blood cells and platelets.
- the fiber diameter needs to be less than 3 ⁇ m in order to allow the filter to capture leukocytes (Non-Patent Document 1), and the fiber diameter of the separating material used in the reports of each document. Is less than 2.5 ⁇ m (Patent Document 1, Patent Document 2, Patent Document 3).
- the conventional leukocyte removal filter is intended to remove leukocytes to the utmost limit, and while the leukocyte capture rate is high, it tends to clog at the time of body fluid treatment, and causes an increase in pressure during the collection operation. There were problems such as inducing declines and technical problems.
- An object of the present invention is to provide a highly efficient separation material for recovering leukocytes or mononuclear cells with high efficiency while avoiding an increase in pressure from a body fluid containing each blood cell component, and to use the separation material.
- Cell separation method is to provide a highly efficient separation material for recovering leukocytes or mononuclear cells with high efficiency while avoiding an increase in pressure from a body fluid containing each blood cell component, and to use the separation material.
- the present inventors have intensively studied a separation material that can efficiently separate white blood cells or mononuclear cells from a body fluid and that does not easily cause an increase in pressure, and a cell separation method. As a result, it has been found that by using a specific separating material, leukocytes or mononuclear cells can be separated with high efficiency, and the present invention has been completed.
- the present invention provides a leukocyte or a mononuclear from a body fluid characterized by comprising a nonwoven fabric having an average fiber diameter of 2.0 ⁇ m or more and 6.0 ⁇ m or less and an air permeability coefficient M of 6.2 or more and 35 or less.
- the present invention relates to a separating material for separating a sphere.
- the nonwoven fabric is preferably made of at least one selected from the group consisting of polyolefin, polyamide, and polyester.
- the body fluid is preferably at least one selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, menstrual blood, and tissue extract.
- the present invention also relates to a cell separation container obtained by filling the above-described separation material into a container having a body fluid inlet and outlet.
- a cell separation container obtained by filling the above-described separation material into a container having a body fluid inlet and outlet.
- the cell separation container it is preferable that one or more separation materials are stacked and filled in the direction in which the body fluid flows, and preferably packed in a compressed state in the direction in which the body fluid flows.
- the cell separation container is preferably a column type.
- the present invention also relates to a cell separation method including a step of separating a white blood cell or a mononuclear cell by bringing a body fluid into contact with the separation material.
- the present invention also includes a first step of contacting a body fluid with the cell separation container to capture leukocytes or mononuclear cells on the separation material, and removing the leukocytes or mononuclear cells from the separation material using a separation solution.
- the present invention relates to a cell separation method for leukocytes or mononuclear cells, which comprises a second step of recovery.
- the first step is a step of introducing body fluid from the inlet of the cell separation container and discharging it from the outlet
- the second step is to introduce a peeling solution from the outlet of the cell separation container.
- the step of collecting leukocytes or mononuclear cells from the entrance is preferred.
- a step of removing contaminant components in the cell separation container by introducing physiological saline or a buffer solution from the inlet after the first step and before the second step.
- a step of bringing the separation material into contact with physiological saline or a buffer solution before the first step it is preferable to include a step of fixing the body fluid inlet of the cell separation container at a lower height than the body fluid outlet of the cell separation container before the first step.
- leukocytes and platelets are substantially captured by the separation material, and red blood cells are not substantially captured.
- the isolated leukocytes or mononuclear cells preferably include hematopoietic stem cells and / or mesenchymal stem cells.
- the present invention also relates to a cryopreservation method characterized in that the cells obtained by the above cell separation method are placed in a liquid nitrogen environment. In a liquid nitrogen environment, it is preferably ⁇ 196 ° C. to ⁇ 30 ° C. In this cryopreservation method, it is preferable to use at least one cryoprotectant selected from the group consisting of dimethyl sulfoxide, dextran, albumin, and hydroxyethyl starch.
- the survival rate of cryopreserved stem cells is preferably 80% or more.
- the present invention also relates to leukocytes, mononuclear cells, or stem cells obtained by the cell separation method described above.
- Stem cells are CD34 positive cells, CD133 positive cells, CD34 negative and CD133 positive cells, CD34 positive and CD133 positive cells, CD34 positive and CD133 negative cells, CD45 negative and CD44 positive and CD73 positive and CD90 positive cells, CD45 negative and CD235a negative And CD33 negative and CD7 negative cells, CD45 positive and CD133 positive and CD117 positive cells, CD45 positive and CD133 negative and CD117 positive cells, CD45 positive and CD133 positive and CD164 positive cells, CD45 positive and CD133 negative and CD164 positive cells, and CD45 It is preferred to include any cell selected from the group consisting of negative and CD309 positive cells.
- the leukocytes preferably include CD45 positive and CD164 positive cells, or CD45 positive and CD117 positive cells.
- leukocytes or mononuclear cells are separated efficiently and easily from body fluids such as whole blood, bone marrow, umbilical cord blood, menstrual blood, and tissue extracts, and are less likely to cause clogging and increased pressure. can do.
- the separation material and cell separation method of the present invention has many advantages in that clogging is less likely to occur than previously reported, and the recovery rate of leukocytes and mononuclear cells is high. Furthermore, this technique can be used after pretreatment such as buffy coat, but basically, without such pretreatment, peripheral blood, umbilical cord blood, bone marrow, menstrual blood, tissue It is possible to separate white blood cells or mononuclear cells from the extract.
- the filter obtained by filling the separation material of the present invention into a container can be used for processing in a sterile closed system.
- the collected leukocyte-containing solution or mononuclear cell-containing solution is a group of cells rich in hematopoietic stem cells and mesenchymal stem cells, and is a filter for preparing therapeutic cells for regenerative medicine such as leukemia treatment, myocardial regeneration and vascular regeneration.
- the leukocytes obtained by using the separation material of the present invention have a very low red blood cell contamination rate, and even if cryopreserved until use, there is very little influence due to hemolysis or the like.
- the separation material of the present invention is very useful as a filter for preparing a cell source for regenerative medicine in addition to the use for preparing a preparation for blood transfusion. By using the separation material of the present invention, it is possible to prepare a therapeutic cell with few side effects and high safety.
- the present invention provides a leukocyte or a mononuclear cell from a body fluid characterized by comprising a nonwoven fabric having an average fiber diameter of 2.0 ⁇ m or more and 6.0 ⁇ m or less and an air permeability coefficient M of 6.2 or more and 35 or less. It is related with the separating material for isolate
- the separating material is made of a nonwoven fabric that is fibrous and easily manufactured and available from the viewpoint of the contact time between the material and the body fluid.
- Nonwoven fabric production methods can be broadly classified into wet and dry types, and resin bonds, thermal bonds, spunlaces, needle punches, stitch bonds, spunbonds, melt blows, etc., but are limited to these production methods. There is no. In the case of a nonwoven fabric having a fiber diameter of 10 ⁇ m or less, a melt blow method or a spun lace method is suitable.
- the nonwoven fabric may be calendered or plasma treated.
- split fibers obtained by splitting a composite single yarn into a plurality of fibers can also be suitably used. This is because the fibers are intertwined in a complicated manner and the blood cell separation efficiency is good.
- the separating material may be used without being put in the container, or may be used by putting the separating material in a container having an inlet and an outlet for body fluid. Among these, considering the practicality, the latter embodiment used in a container is preferable.
- the average fiber diameter of the fibers constituting the nonwoven fabric is preferably 2.0 ⁇ m or more and 6.0 ⁇ m or less, more preferably 2.5 ⁇ m or more and 5.7 ⁇ m or less, and 3.5 ⁇ m or more and 5.0 ⁇ m or less. More preferably.
- the average fiber diameter is smaller than 2.0 ⁇ m, clogging tends to occur and the recovery rate tends to decrease.
- the average fiber diameter is larger than 6.0 ⁇ m, the ability to capture leukocytes into the separating material tends to be lowered.
- the average fiber diameter is the fiber width perpendicular to the fiber axis.
- the measurement of the fiber diameter is obtained by taking a photograph of a separating material made of a nonwoven fabric with a scanning electron microscope and averaging the calculated values of the fiber diameter obtained from the scale described in the photograph. That is, it means an average value of measured fiber diameters, and is an average value of 50 or more, preferably 100 or more. However, if there are many overlapping fibers, other fibers are in the way and the width cannot be measured, or if fibers with significantly different diameters are mixed, the fiber diameter is calculated excluding the data. To do.
- the air permeability coefficient M of the separating material is preferably 6.2 or more and 35 or less, more preferably 7.0 or more and 14.2 or less, and further preferably 9.2 or more and 10.0 or less.
- the air permeability coefficient is smaller than 6.2, the cells are densely captured by the separation material, and thus the recovery performance tends to be lowered.
- the air permeability coefficient is larger than 35, cells tend to be hardly captured by the separation material.
- the air permeability coefficient M is a value defined by the product of the air permeability (cc / cm 2 ⁇ sec) and the thickness (mm) of the separation material, and is an essential parameter excluding the influence of the thickness of the separation material. is there.
- the air permeability is a parameter that depends on the pore size of the separation material, but even if the air permeability is the same, the smaller the thickness of the separation material, the smaller the essential air permeability. The degree becomes smaller. Therefore, by multiplying the air permeability and the thickness, it becomes a parameter representing the essential pore diameter of the separating material.
- the air permeability can be easily measured in accordance with or in accordance with the fragile method described in JIS L1096-1999.
- the thickness can be measured with various devices including a digital caliper.
- the measuring method of air permeability is not limited to these measuring methods.
- the average fiber diameter of the separation material is 2.0 ⁇ m or more and 6.0 ⁇ m or less and the air permeability coefficient M of the separation material is 6.2 or more and 35 or less, leukocytes or mononuclear cells can be efficiently separated. it can.
- polyolefin As a material used for the separating material, polyolefin, polyamide, polyester, and the like are desirable from the viewpoint of sterilization resistance and cell safety.
- the polyolefin include polypropylene, polyethylene, high-density polyethylene, and low-density polyethylene
- examples of the polyamide include nylon
- examples of the polyester include polyethylene terephthalate and polybutylene terephthalate.
- polyethylene terephthalate polybutylene terephthalate, polypropylene, acrylic, nylon, polyurethane, and glass are preferable.
- these materials are not limited to a single type, and may be combined, mixed, and fused as necessary.
- molecules having affinity for specific cells such as proteins, peptides, amino acids, and sugars may be fixed to the material.
- the body fluid means whole blood, peripheral blood, bone marrow, umbilical cord blood, menstrual blood, tissue extract, and combinations thereof, and may be roughly separated.
- animal species from which body fluids are collected include mammals such as humans, cows, mice, rats, pigs, monkeys, dogs, and cats.
- the body fluid may have been previously treated with an anticoagulant.
- Anticoagulants include citric acid anticoagulants such as ACD (acid-citrate-dextrose), CPD (citrate-phosphate-dextrose), and CPDA (citrate-phosphate-dextrose-adenine), heparin, and low molecular weight heparin.
- Anti-coagulants such as Fusan (Nafamostat methyl acid) and EDTA.
- Body fluid storage conditions are not particularly limited as long as the purpose of use of each fraction is not affected.
- leukocytes or mononuclear cells that can be separated by the separation material include lymphocytes, monocytes, CD3 positive cells, CD14 positive cells, CD19 positive cells, hematopoietic stem cells, and mesenchymal stem cells. It is done.
- the present invention also relates to a cell separation container obtained by filling the above separation material into a container having an inlet and an outlet for body fluid.
- the form, size, and material of the container filled with the separation 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 capacity of about 0.1 mL to 400 mL and a diameter of about 0.1 cm to 15 cm.
- a rectangular column container having a length of about 0.1 cm to 20 cm and a thickness of about 0.1 cm to 5 cm can be used, but the present invention is not limited thereto.
- Examples of the container type include a cross flow type and a column type. Either the cross flow type or the column type can be used, and the type of the container is not particularly limited, but the column type is more preferable from the viewpoint that the recovered liquid can be introduced uniformly.
- a cross flow type is used from the viewpoint of efficiently recovering cells, but there is a limitation that only a high-viscosity stripping solution can be used.
- the separation material of the present invention is combined with a column type container, the cell recovery rate does not decrease even with a low viscosity stripping solution, and high separation performance can be obtained.
- the column type means, for example, a container having a liquid inlet and outlet near the center of the surface with respect to the filter surface, or a container in which the inlet and outlet portions are positioned perpendicular to the filter surface, or the filter surface.
- the container is characterized in that the liquid flows in a direction perpendicular to the above, or the container characterized in that the liquid flows in parallel to the compression direction of the separating material.
- FIG. 7 shows an example of the column type.
- the cross-flow type refers to the position of the liquid inlet and outlet with respect to the filter surface.
- the container which is deviated from the center of the container and in which the inlet part and the outlet part are located parallel to the filter surface is inserted.
- the inlet portion and the outlet portion being positioned perpendicular to the filter surface means that the angle (acute angle) between the inlet and the outlet from the surface is 45 degrees or more and less than 90 degrees.
- That the portion and the outlet portion are positioned in parallel means that the angle (acute angle) between the inlet and the outlet from the surface is 0 degree or more and less than 45 degrees.
- the container can be made using any structural material.
- the structural material include non-reactive polymers, biocompatible metals, alloys, and glass.
- Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides and polyimides , Polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene and the like.
- structural materials having sterilization resistance are preferred, and specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, and polymethylpentene.
- the separating material is preferably used by cutting a separating material made of a non-woven fabric into a suitable size and laminating one or more sheets in the direction of body fluid flow to a thickness of about 1 mm to 200 mm. From the viewpoint of separation efficiency of each fraction, the thickness of the laminate is more preferably from 1.5 mm to 150 mm, and further preferably from 2 mm to 100 mm.
- the separation material When the separation material is filled in the container, it can be used in the state in which the body fluid flows in a state where one or more sheets are laminated, and the thickness is preferably about 1 mm to 50 mm. From the viewpoint of separation efficiency of each fraction, it is more preferably from 1.5 mm to 40 mm, and further preferably from 2 mm to 35 mm.
- the separating material may be wound into a roll and filled into a container.
- blood cells When used in the form of a roll, blood cells may be separated by treating body fluid from the inside to the outside of the roll, or conversely, body fluid may be treated from the outside to the inside of the roll. Good.
- the container When filling the container with the separating material, the container may be compressed in the direction in which the body fluid flows, or the container may be filled without being compressed.
- the presence or absence of compression can be appropriately selected according to the material of the separating material.
- the separation material may be filled in a flat plate shape cut to an appropriate size, or may be filled in a rolled shape. Furthermore, two or more types may be used in combination, or a separation material other than the above-described separation material may be used in combination, and it is only necessary to constitute a separation system that substantially captures and collects leukocytes.
- To substantially capture leukocytes means that 60% or more of leukocytes contained in the body fluid in contact with the separation material are captured by the separation material. Furthermore, it is preferable that the leukocytes captured by the separation material of the present invention account for 60% or more of the leukocytes captured by the entire cell separation container.
- the cell separation container is characterized by substantially not capturing red blood cells but substantially capturing white blood cells.
- substantially not capturing red blood cells means that 60% or more of the red blood cells contained in the body fluid in contact with the separating material has a property of passing through the separating material.
- the separation material of the present invention may be a material that substantially captures platelets, but is not limited thereto. The fact that platelets are substantially captured means that 50% or more of the platelets contained in the body fluid in contact with the separating material is captured by the separating material. More preferably, 60% or more is captured by the separating material.
- the present invention also relates to a cell separation method including a step of separating a white blood cell or a mononuclear cell by bringing a body fluid into contact with the separation material described above. Furthermore, the present invention separates the captured leukocytes or mononuclear cells using a peeling solution, and a first step of capturing the leukocytes or mononuclear cells on the separation material by contacting a body fluid with the cell separation container described above.
- the present invention relates to a cell separation method comprising a second step of recovering from a material.
- a body fluid is injected into a container filled with a separation material from the inlet side, and leukocytes or mononuclear cells are captured and erythrocytes are allowed to pass.
- a high yield of leukocytes or mononuclear cells captured by the separation material is obtained by flowing a stripping solution from the outlet direction of the container, that is, from the direction opposite to the direction in which the body fluid or the washing solution is passed Can be separated and recovered.
- the erythrocytes accumulated in the container can be efficiently collected and separated by passing the washing liquid from the same direction. You can also.
- the body fluid inlet When flowing body fluid into a cell separation container having a body fluid inlet and outlet, the body fluid inlet may be set higher than the body fluid outlet and the body fluid may flow in the same direction as gravity. May be set to be lower than the outlet of the body fluid, and the body fluid may flow in the direction opposite to the gravity. By flowing the body fluid in the direction opposite to the gravity, the body fluid flows uniformly in the container, so that the separation efficiency can be further improved.
- a blood cell component separation system can be constructed using the cell separation container.
- the blood cell component separation system is practically and preferably provided with an inlet and an outlet for a washing solution and a peeling solution, a red blood cell collection bag, a white blood cell collection bag, and the like simultaneously.
- the cell separation container has an inlet through which the body fluid flows in and an outlet through which the body fluid flows, and is further independent of the body fluid inlet and the body fluid inlet, and the inflow part of the washing solution that flows the red blood cells accumulated in the container.
- the body fluid outlet and the body fluid outlet have a washing liquid outflow part, and the stripping solution is introduced independently of the body fluid and the washing liquid outflow part or the outflow part.
- the inlet and outlet of the washing liquid attached to the container may function as an inlet and outlet of body fluid, and the blood bag and the washing solution bag may be connected to the inlet side circuit via a three-way cock.
- the separation solution introduction port may function as a body fluid outlet.
- recovery side of peeling solution may function as an inlet_port
- 9 and 10 show an example of a blood cell component separation system.
- the blood cell component separation system further includes a body fluid storage bag, a separation solution collection bag for collecting separated leukocytes, a red blood cell collection bag, and the like.
- a body fluid storage bag By connecting these bags to the inlets and outlets of the solutions described above, it becomes possible to separate body fluids in a sterile closed system.
- Each bag is preferably used after being used, and may be shaped like a commonly used blood bag, but may be a flat cartridge type.
- a bag capable of cell culture, a bag having cryopreservation resistance, and the like may be selected as necessary.
- the separation method of the present invention will be specifically described.
- 1) Body fluid feeding process When the body fluid is passed from the body fluid inlet side of the container filled with the separating material, even if the body fluid is sent from the container containing the body fluid through the liquid feeding circuit by natural fall, it is sent by the pump. May be.
- the syringe containing the body fluid may be directly connected to the container and pushed by hand.
- the separation efficiency tends to decrease if the liquid feeding speed is too fast, and the processing time tends to be longer if it is too slow.
- Examples of the liquid feeding speed include a speed of 0.1 mL / min to 100 mL / min, but are not limited thereto.
- the pretreatment solution does not need to be the same as the solution used in the following cleaning step, but if it is the same, the solution bag can be shared.
- the amount of pretreatment liquid is practically and preferably about 1 to 100 times that of the container filled with the separation material.
- a buffer solution which can be used, Common buffer solutions, such as a Ringer's solution, the culture medium used for a cell culture, and a phosphate buffer solution, are preferable.
- This step is not always necessary, but may be performed to increase the removal efficiency of impurities.
- impurities that can be removed by this step include non-blood cell components such as red blood cells and plasma.
- the cleaning liquid When passing the cleaning liquid from the cleaning liquid inflow side in the same direction as the body fluid supplying step, the cleaning liquid may be supplied by natural fall through a circuit or may be supplied 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 the speed is 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.
- any solution may be used as long as it is possible to wash out only red blood cells, can suppress the mixing of other blood cells in the collected white blood cells, and can maintain the trapped state of the blood cells.
- general buffer solutions such as physiological saline, Ringer's solution, medium used for cell culture, and phosphate buffer are preferable.
- Peeling solution is injected into the container filled with leukocyte or mononuclear cell exfoliation separation material in the direction opposite to the direction of body fluid flow (in the body fluid outflow side) to exfoliate leukocytes.
- the stripping solution can be put in a syringe or the like in advance, and the plunger of the syringe can be pushed out using hands or equipment.
- the amount of recovered liquid and the flow rate vary depending on the capacity and throughput of the container, but the liquid amount is about 1 to 100 times that of the container, and a flow rate of 0.5 mL / sec to 20 mL / sec is preferable, but is not limited thereto. It is not something.
- the stripping solution is not particularly limited as long as it is a hypotonic solution, but those that have been used as injections such as physiological saline, Ringer's solution, dextran sugar injection, hydroxyethyl starch, buffers, cell culture media, etc. Can be mentioned.
- the viscosity of the collected liquid may be increased in order to increase the recovery rate of the captured cells.
- 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.
- the viscosity of the stripping solution is not particularly limited. However, if the viscosity is too high, the recovery operation tends to be difficult, so 20 mPa ⁇ s or less is more preferable.
- the leukocytes collected by the cell separation method preferably include hematopoietic stem cells, mesenchymal stem cells, and CD34 positive cells.
- the present invention also relates to a cryopreservation method characterized in that the cells obtained by the above cell separation method are placed in a liquid nitrogen environment. Since the above cell separation method has a very low stress on the cells compared to the conventional centrifugation method, the cells obtained by this cell separation method have a very high activity after cryopreservation.
- cryoprotectant When cells are cryopreserved, a cryoprotectant is added for the purpose of protecting the cells during freezing.
- the type of cryoprotectant added is not particularly limited, and dimethyl sulfoxide, dextran, albumin, hydroxyethyl starch, and the like can be used.
- the cryoprotectant may be used alone or in combination of several kinds.
- the cells may be stored in a liquid nitrogen environment, stored in a state immersed in liquid nitrogen, or stored in a liquid nitrogen gas.
- the temperature at the time of storage is not particularly limited, but is preferably ⁇ 196 ° C. to ⁇ 30 ° C. in order to prevent a decrease in cell activity. It is more preferably ⁇ 196 ° C. to ⁇ 50 ° C., and further preferably ⁇ 196 ° C. to ⁇ 70 ° C.
- the present invention also relates to leukocytes, mononuclear cells, and stem cells obtained by the above-described cryopreservation method.
- the stem cell obtained by the above cryopreservation method may be any stem cell as long as it is a cell having a self-replicating ability and a differentiation ability contained in a body fluid. Specifically, hematopoietic stem cells, mesenchymal stem cells, Embryonic-like Stem Cell, vascular endothelial progenitor cells and the like can be mentioned.
- hematopoietic stem cells include CD34 positive cells, CD133 positive cells, CD34 negative and CD133 positive cells, CD34 positive and CD133 positive cells, CD34 positive and CD133 negative cells, CD45 positive and CD133 positive and CD117 positive cells, CD45 positive and CD133 Negative and CD117 positive cells, CD45 positive and CD133 positive and CD164 positive cells, CD45 positive and CD133 negative and CD164 positive cells.
- Common hematopoietic stem cells include CD34 positive cells or CD133 positive cells.
- hematopoietic stem cells are changed into CD34 negative and CD133 positive cells, CD34 positive and CD133 positive cells, CD34 positive and CD133 negative cells. It is thought to differentiate. If the separating material of the present invention is used, these hematopoietic stem cells can be separated, and even if the cells after separation are frozen, the activity is less decreased.
- CD117-positive cells that are receptors for hematopoietic growth factors (Stem Cell Factor) and CD164-positive cells involved in cell adhesion, which are said to be particularly involved in engraftment at the time of transplantation are also separated by the present invention. There is little decrease in activity even after freezing after separation with the material.
- CD45 negative, CD44 positive, CD73 positive, and CD90 positive cells which are considered to be cord blood mesenchymal stem cells, show little decrease in activity even when frozen after separation with the separation material of the present invention. Although there are only a few mesenchymal stem cells in the cord blood and there is a problem that the loss is large and difficult to separate by centrifugation, the separation material of the present invention can efficiently separate them.
- CD45 positive and CD164 positive cells and CD45 positive and CD117 positive cells in leukocytes is less decreased even when frozen after separation with the separation material of the present invention.
- the cell survival rate after cryopreservation is preferably 80% or more, and more preferably 85% or more.
- the nonwoven fabrics shown in the following examples and comparative examples are filled by adjusting the number of sheets so that the thickness when uncompressed is in a certain range.
- the container filled with the separation material is a column type as shown in FIG. 7, and the liquid inlet and outlet are located at the center with respect to the filter surface. The liquid inlet and outlet are located vertically.
- 45 mL of physiological saline was manually passed from the inlet side using a syringe.
- CPD CPD anticoagulated fresh bovine blood
- Example 5 A similar operation was performed using the same separation material as in Example 1 except that 10 mL of CPD anticoagulated fresh bovine blood was used instead of 20 mL of CPD anticoagulated fresh bovine blood. Furthermore, the blood before treatment and the collected separated solution are hemolyzed by FACS Lysing Solution, then the mononuclear cell positive rate is obtained by a flow cytometer (BD FACSCanto), and the total mononuclear is obtained by multiplying the white blood cell count and the mononuclear cell positive rate. The number of balls was calculated. The ratio obtained by dividing the total number of mononuclear cells in the collected solution by the total number of mononuclear cells before treatment was taken as the mononuclear cell recovery rate. The results are shown in Table 2, FIG. 2 and FIG.
- Example 6 A similar operation was performed using the same separation material as in Example 2 except that 10 mL of CPD anticoagulated fresh bovine blood was used instead of 20 mL of CPD anticoagulated fresh bovine blood. The results are shown in Table 2, FIG. 2 and FIG.
- Example 7 A similar operation was performed using the same separation material as in Example 3 except that 10 mL of CPD anticoagulated fresh bovine blood was used instead of 20 mL of CPD anticoagulated fresh bovine blood. Furthermore, the blood before treatment and the collected separated solution are hemolyzed by FACS Lysing Solution, then the mononuclear cell positive rate is obtained by a flow cytometer (BD FACSCanto), and the total mononuclear is obtained by multiplying the white blood cell count and the mononuclear cell positive rate. The number of balls was calculated. The ratio obtained by dividing the total number of mononuclear cells in the collected solution by the total number of mononuclear cells before treatment was taken as the mononuclear cell recovery rate. The results are shown in Table 2, FIG. 2 and FIG.
- the operation was performed in the same manner as in Example 7 except that it was filled.
- the results are shown in Table 2, FIG. 2 and FIG.
- Comparative Example 4 A similar operation was performed using the same separation material as in Comparative Example 1 except that 10 mL of CPD anticoagulated fresh human blood was used instead of 20 mL of CPD anticoagulated fresh bovine blood. A little resistance was applied during the recovery operation, suggesting that the column internal pressure was high and clogged. The results are shown in Table 2, FIG. 2 and FIG.
- Comparative Example 5 A similar operation was performed using the same separation material as in Comparative Example 2 except that 10 mL of CPD anticoagulated fresh human blood was used instead of 20 mL of CPD anticoagulated fresh bovine blood. The results are shown in Table 2, FIG. 2 and FIG.
- Example 9 Separation material similar to Example 3 except that 20 mL of fresh bovine blood of CPD anticoagulated was used and 10 mL of fresh porcine bone marrow anticoagulated with heparin (final concentration 50 units / mL) and CPD (final concentration 12%) was used. The same operation was performed using The results are shown in Table 3, FIG. 3 and FIG.
- Comparative Example 6 The same operation as in Example 9 was performed except that the separation material of Comparative Example 2 was used. There was very resistance to pushing out with a syringe during the recovery operation, and it was not possible to push out smoothly, suggesting that the column internal pressure was high and clogged. The results are shown in Table 3, FIG. 3 and FIG.
- Example 11 Using the separation material of Example 3, 25 mL of fresh bovine blood containing 12% CPD, the recovery operation was performed with 30 mL of ⁇ MEM medium (viscosity 2.9 mPa ⁇ s) containing 10% ACD-A and 10% FBS. The results are shown in Table 4.
- Example 12 Experiments were carried out in the same manner as in Example 11 except that 10% ACD-A, 4% human serum albumin-containing low molecular weight dextran sugar injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) (viscosity 7.3 mPa ⁇ s) was used. Carried out. The results are shown in Table 4.
- Example 13 The experiment was carried out using the same separator and the same method as in Example 11 except that 6% ACD-A-containing Sarinhes infusion solution 6% (manufactured by Fresenius Kirby Japan) (viscosity 2.3 mPa ⁇ s) was used. The results are shown in Table 4.
- Example 14 Experiments were carried out in the same manner as in Example 11 except that 10% ACD-A, 6% Sarinhes infusion containing 4% human serum albumin (Fresenius Kirby Japan) (viscosity 4.3 mPa ⁇ s) was used. Carried out. The results are shown in Table 4.
- Example 15 The experiment was carried out using the same separation material and the same method as in Example 11 except that 20% sucrose containing 10% ACD-A was used. The results are shown in Table 4.
- Example 16 The experiment was carried out using the same separating material and the same method as in Example 11 except that 10% ACD-A-containing physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) (viscosity 1.1 mPa ⁇ s) was used. The results are shown in Table 4.
- leukocytes can be collected at a high collection rate regardless of the type of the collected liquid.
- Example 17 The cells collected in Example 8 were prepared to a leukocyte concentration of 2 ⁇ 10 6 , 0.3 mL was added to 3 mL of methylcellulose medium MethoCult H4034 (manufactured by StemCell Technologies), and then 1.1 mL of the mixed solution was added. The aliquot was dispensed into a petri dish and cultured at 37 ° C. under 5% CO 2 . When observed with a microscope after 14 days, it was confirmed that various colonies composed of hematopoietic stem cells such as erythroid progenitor cells and leukocyte progenitor cells were formed, and the recovered cells contained CD34 positive cells and hematopoietic stem cells. A photograph of a colony composed of hematopoietic stem cells is shown in FIG.
- Example 18 3 mL of the cell solution collected in Example 9 and Example 10 was made up to 10 mL in total with 10% FBS-containing ⁇ MEM medium, seeded in a 10 cm petri dish, and cultured at 37 ° C. under 5% CO 2 . After exchanging the medium every 3 days and culturing for 9 days, a colony of mesenchymal stem cells adhering to the petri dish was confirmed, and the recovered cells were confirmed to contain mesenchymal stem cells.
- the cell separation container was set so that the inlet was lower than the outlet as described in FIG. First, about 50 mL of physiological saline was passed so as to flow from the body fluid inlet to the outlet. Next, 80 mL of CPD anticoagulated human peripheral blood was passed in the same direction, and leukocytes were captured in the cell separation container.
- Example 20 4% human serum albumin-containing sarinhes infusion solution 6% (manufactured by Fresenius Kirby Japan) (viscosity 4.3 mPa ⁇ s), except that physiological saline (viscosity 1.1 mPa ⁇ s) is used instead of saline 19 The operation was performed. The results are shown in Table 5.
- Example 21 The same operation as in Example 19 was performed except that CPD anticoagulated human umbilical cord blood was used instead of CPD anticoagulated human peripheral blood. The results are shown in Table 5.
- the cell separation container was set so that the inlet was higher than the outlet as shown in FIG. First, about 50 mL of physiological saline was passed so as to flow from the body fluid inlet to the outlet. Next, 80 mL of CPD anticoagulated human umbilical cord blood was passed in the same direction, and leukocytes were captured in the cell separation container.
- Example 23 The same operation as in Example 19 was performed except that 150 mL of CPD anticoagulated bovine peripheral blood was used instead of 80 mL of CPD anticoagulated human peripheral blood. The results are shown in Table 5.
- Example 24 The same operation as in Example 22 was performed except that 150 mL of CPD anticoagulated bovine peripheral blood was used instead of 80 mL of CPD anticoagulated human umbilical cord blood. The results are shown in Table 5.
- Example 25 The cells separated in Example 21 and Example 22 were cryopreserved, and the activity of the cells after cryopreservation was evaluated.
- the separated cells were transferred to Cryobag (manufactured by Pharmacophama), cooled to 4 ° C., and a mixed solution of DMSO and dextran 40 was added as a cryoprotectant so that the final concentration of DMSO was 10%. Thereafter, the temperature was gradually lowered by a program freezer and stored in a frozen state in a liquid nitrogen tank (minus 196 ° C.). After 14 days, the cryopreserved cells were thawed in a 37 ° C. warm bath and transferred to a mixture of dextran and albumin.
- the mixture was centrifuged to remove the supernatant, and then the cells were suspended again in a mixture of dextran and albumin, and the cells were counted.
- the recovery rate after separation the ratio of the number of cells contained in the treated liquid after separation to the number of cells before separation was calculated.
- the recovery rate after freezing the ratio of the number of cells contained in the treated solution after freezing to the number of cells before freezing was calculated.
- leukocytes ILs, monocytes, CD34 positive cells, CD133 positive cells, CD34 negative and CD133 positive cells, CD34 positive and CD133 positive cells, CD45 positive and CD133 positive and CD117 positive cells, CD45
- the recovery rate after separation, the recovery rate after cryopreservation, and the survival rate after cryopreservation were calculated for positive and CD133 negative and CD164 positive cells, and CD45 positive and CD117 positive cells. The results are shown in Table 6.
- the cells obtained in Example 22 were leukocytes, mononuclear cells, CD34 positive cells, CD133 positive cells, CD34 negative and CD133 positive cells, CD34 positive and CD133 positive cells, CD34 positive and CD133 negative cells, CD45 negative and CD44.
- Positive and CD73 positive and CD90 positive cells CD45 negative and CD235a negative and CD33 negative and CD7 negative cells, CD45 positive and CD133 positive and CD117 positive cells, CD45 positive and CD133 negative and CD117 positive cells, CD45 positive and CD133 positive and CD164 positive After separation for cells, CD45 positive and CD133 negative and CD164 positive cells, CD45 negative CD309 positive cells, CD45 positive and CD164 positive cells, CD45 positive and CD117 positive cells Recovery, the recovery rate after cryopreservation was calculated survival rate after cryopreservation. The results are shown in Table 7.
- the cell separation method of the present invention since the cell separation method of the present invention has a very low stress on the cells, the cells obtained by this cell separation method maintain high activity even after cryopreservation.
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Abstract
Description
1)体液送液工程
分離材を充填した容器の体液入口側より体液を通液する際には、体液を入れた容器から送液回路を通じて自然落下で送液しても、ポンプにより送液してもよい。また、体液を入れたシリンジを直接、容器に接続し、手でシリンジを押してもよい。ポンプにより通液する場合には、送液速度が速過ぎると分離効率が落ち、遅過ぎると処理時間がかかる傾向がある。送液速度としては、例えば0.1mL/minから100mL/minの速度が挙げられるが、これに限定されるものではない。
本工程は必ずしも必要ではないが、夾雑物の除去効率を高める場合には実施してもよい。この工程により除去できる夾雑物として、赤血球、血漿等の非血球成分が挙げられる。洗浄液の流入側より体液送液工程と同方向より洗浄液を通液する際には、洗浄液は回路を通じて、自然落下で送液しても、ポンプにより送液してもよい。ポンプにより送液する場合の流速は、体液送液工程と同程度であり、0.1mL/minから100mL/minの速度が挙げられるが、これに限定されるものではない。洗浄量は容器の容量によって異なるが、洗浄量が少なすぎると容器に残存する赤血球成分が多くなり、洗浄量が多すぎると分離効率が落ちるとともに多大な時間を要することから、容器の0.5倍から100倍程度の液量で洗浄することが好ましい。
分離材を充填した容器に体液の通液とは逆方向(体液流出側)より剥離溶液を注入し、白血球を剥離させる。剥離溶液を注入する際には、剥離溶液を予めシリンジ等に入れておき、シリンジのプランジャーを手や機器を用いて勢い良く押し出すことにより実行できる。回収液量や流速は、容器の容量や処理量により異なるが、容器の1倍から100倍程度の液量で、流速0.5mL/secから20mL/secの流速が好ましいが、これらに限定されるものではない。
厚さ6mm直径18mmの、ポリカーボネートからなるカラム状の容器に、ポリブチレンテレフタレート製不織布(平均繊維径2.0μm、通気度係数M=7.0)28枚を、積層状態で、図7に示した態様となるよう充填した。まず生理食塩水45mLを入口側よりシリンジを用いて手押しで通液した。次にCPD抗凝固の新鮮ウシ血液20mL(CPD:血液=200:28で混合した12%CPDを含むウシ末梢血)を2.5mL/minの速度で通液し、次に同方向より生理食塩水10mLを通液した。その後、逆方向から10%FBS添加αMEM培地30mL(粘度2.9mPa・s)をシリンジを用いて手押しで通液することにより白血球を回収した。剥離溶液はスムーズに導入することができた。処理前血液の血算、回収した溶液の血算を血球カウンター(シスメックス社製、K-4500)により測定し、白血球の回収率を算出した。結果は表1、図1、および図4に示した。
ポリプロピレン製不織布(平均繊維径3.5μm、通気度係数M=9.6)を28枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。結果は表1、図1、および図4に示した。
ポリブチレンテレフタレート製不織布(平均繊維径2.9μm、通気度係数M=10.0)を28枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。結果は表1、図1、および図4に示した。
ナイロン製不織布(平均繊維径5.0μm、通気度係数M=9.2)を32枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。結果は表1、図1、および図4に示した。
ポリブチレンテレフタレート製不織布(平均繊維径1.7μm、通気度係数M=5.9)を84枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。回収操作時に少し抵抗がかかっていたことから、カラム内圧が高く詰まっていることが示唆された。結果は表1、図1、および図4に示した。
ポリプロピレン製不織布(平均繊維径2.1μm、通気度係数M=6.0)を30枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。結果は表1、図1、および図4に示した。
ポリプロピレン製不織布(平均繊維径4.9μm、通気度係数M=39.6)を24枚積層状態で充填したこと以外は実施例1と同様の操作を実施した。結果は表1、図1、および図4に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、CPD抗凝固の新鮮ヒト血液10mLを用いた以外は実施例1と同様の分離材を用い、同様の操作を実施した。さらに、処理前の血液、回収した分離溶液をFACS Lysing Solutionで溶血後、フローサイトメーター(BD FACSCanto)により単核球陽性率を求め、白血球数と単核球陽性率を掛けあわせて総単核球数を算出した。回収した溶液中の総単核球数を処理前の総単核球数で割った割合を単核球回収率とした。結果は表2、図2、および図5に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、CPD抗凝固の新鮮ヒト血液10mLを用いた以外は実施例2と同様の分離材を用い、同様の操作を実施した。結果は表2、図2、および図5に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、CPD抗凝固の新鮮ヒト血液10mLを用いた以外は実施例3と同様の分離材を用い、同様の操作を実施した。さらに、処理前の血液、回収した分離溶液をFACS Lysing Solutionで溶血後、フローサイトメーター(BD FACSCanto)により単核球陽性率を求め、白血球数と単核球陽性率を掛けあわせて総単核球数を算出した。回収した溶液中の総単核球数を処理前の総単核球数で割った割合を単核球回収率とした。結果は表2、図2、および図5に示した。
ポリブチレンテレフタレート製不織布(平均繊維径2.9μm、通気度係数M=10.0)の代わりにポリプロピレン製不織布(繊維径5.7μm、通気度係数M=14.2)を40枚積層状態で充填したこと以外は実施例7と同様に操作を実施した。結果は表2、図2、および図5に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、CPD抗凝固の新鮮ヒト血液10mLを用いた以外は比較例1と同様の分離材を用い、同様の操作を実施した。回収操作時に少し抵抗がかかっていたことから、カラム内圧が高く詰まっていることが示唆された。結果は表2、図2、および図5に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、CPD抗凝固の新鮮ヒト血液10mLを用いた以外は比較例2と同様の分離材を用い、同様の操作を実施した。結果は表2、図2、および図5に示した。
CPD抗凝固の新鮮ウシ血液20mLの代わりに、ヘパリン(最終濃度50単位/mL)とCPD(最終濃度12%)で抗凝固した新鮮ブタ骨髄10mLを用いた以外は実施例3と同様の分離材を用い、同様の操作を実施した。結果は表3、図3、および図6に示した。
ポリブチレンテレフタレート製不織布(平均繊維径5.3μm、通気度係数M=20.0)を24枚積層状態で充填したこと以外は実施例9と同様の操作を実施した。結果は表3、図3、および図6に示した。
比較例2の分離材を用いた以外は実施例9と同様の操作を実施した。回収操作時にシリンジで押し出すのに非常に抵抗があり、スムーズに押し出すことができなかったことから、カラム内圧が高く詰まっていることが示唆された。結果は表3、図3、および図6に示した。
実施例3の分離材、12%CPDを含む新鮮ウシ血液25mLを用い、10%ACD-A、10%FBS含有αMEM培地30mL(粘度2.9mPa・s)で回収操作を行った。結果は表4に示した。
10%ACD-A、4%ヒト血清アルブミン含有低分子デキストラン糖注(大塚製薬社製)(粘度7.3mPa・s)を用いること以外は実施例11と同様の分離材、同様の方法で実験を実施した。結果は表4に示した。
10%ACD-A含有サリンヘス輸液6%(フレゼニウスカービジャパン社製)(粘度2.3mPa・s)を用いること以外は実施例11と同様の分離材、同様の方法で実験を実施した。結果は表4に示した。
10%ACD-A、4%ヒト血清アルブミン含有サリンヘス輸液6%(フレゼニウスカービジャパン社製)(粘度4.3mPa・s)を用いること以外は実施例11と同様の分離材、同様の方法で実験を実施した。結果は表4に示した。
10%ACD-A含有20%スクロースを用いること以外は実施例11と同様の分離材、同様の方法で実験を実施した。結果は表4に示した。
10%ACD-A含有生理食塩水(大塚製薬社製)(粘度1.1mPa・s)を用いること以外は実施例11と同様の分離材、同様の方法で実験を実施した。結果は表4に示した。
実施例8で回収された細胞を白血球濃度2×106になるように調製し、メチルセルロース培地MethoCult H4034(StemCell Technologies社製)3mLに対して0.3mLを添加した後、混合液1.1mLをペトリディッシュに分注し、37℃、5%CO2下で培養した。14日後に顕微鏡にて観測したところ、赤血球系前駆細胞や白血球前駆細胞等の造血系幹細胞から成る各種コロニーが形成され、回収された細胞がCD34陽性細胞や造血幹細胞を含むことが確認された。造血系幹細胞からなるコロニーの写真を図8に示す。
実施例9、実施例10で回収された細胞溶液の3mLを10%FBS含有αMEM培地で合計10mLとし、10cmのシャーレに播種し、37℃、5%CO2下で培養した。3日毎に培地交換を行い、9日間培養した結果、シャーレに接着する間葉系幹細胞のコロニーを確認し、回収された細胞が間葉系幹細胞を含むことが確認された。
厚さ12mm直径44mmの、ポリカーボネートからなるカラム状の容器に、ポリブチレンテレフタレート製不織布(平均繊維径3.5μm、通気度係数M=8.9)112枚を、積層状態で、図7に示した態様となるよう充填した。図10に記載したように入口が出口より低くなるように細胞分離容器をセットした。まず生理食塩水約50mLを体液の入口から出口へ流れるように通液した。次にCPD抗凝固ヒト末梢血80mLを同方向へ通液し、白血球を細胞分離容器に捕捉させた。最後にラインを切り替え、通液とは逆方向つまり体液の出口から入口に流れるように、4%ヒト血清アルブミン含有サリンヘス輸液6%(フレゼニウスカービジャパン社製)(粘度4.3mPa・s)を、シリンジを用いて手押しで導入し、細胞を細胞回収バッグに回収した。結果は表5に示した。
4%ヒト血清アルブミン含有サリンヘス輸液6%(フレゼニウスカービジャパン社製)(粘度4.3mPa・s)の代わりに生理食塩水(粘度1.1mPa・s)を用いること以外は実施例19と同様の操作を行った。結果は表5に示した。
CPD抗凝固ヒト末梢血の代わりにCPD抗凝固ヒト臍帯血を用いること以外は実施例19と同様の操作を行った。結果は表5に示した。
厚さ12mm直径44mmの、ポリカーボネートからなるカラム状の容器に、ポリブチレンテレフタレート製不織布(平均繊維径3.5μm、通気度係数M=8.9)112枚を、積層状態で、図7に示した態様となるよう充填した。図9に示したように入口が出口より高くなるように細胞分離容器をセットした。まず生理食塩水約50mLを体液の入口から出口へ流れるように通液した。次にCPD抗凝固ヒト臍帯血80mLを同方向へ通液し、白血球を細胞分離容器に捕捉させた。最後にラインを切り替え、通液とは逆方向つまり体液の出口から入口に流れるように、4%ヒト血清アルブミン含有サリンヘス輸液6%(フレゼニウスカービジャパン社製)(粘度4.3mPa・s)を、シリンジを用いて手押しで導入し、細胞を細胞回収バッグに回収した。結果は表5に示した。
CPD抗凝固ヒト末梢血80mLの代わりにCPD抗凝固ウシ末梢血150mLを用いること以外は実施例19と同様の操作を行った。結果は表5に示した。
CPD抗凝固ヒト臍帯血80mLの代わりにCPD抗凝固ウシ末梢血150mLを用いること以外は実施例22と同様の操作を行った。結果は表5に示した。
実施例21、および実施例22で分離した細胞を凍結保存し、凍結保存後の細胞の活性を評価した。分離した細胞をCryobag(Macopharma社製)に移し4℃に冷やした後、DMSOの最終濃度が10%になるように凍結保護剤としてDMSOとデキストラン40の混合液を添加した。その後プログラムフリーザーで少しずつ段階的に温度を下げ、液体窒素タンクの中(マイナス196℃)で、凍結状態で保存した。14日間後、凍結保存した細胞を37℃の温浴中で解凍し、デキストランとアルブミンの混合液に移した。その混合液を遠心し上清を除いた後、細胞をデキストランとアルブミンの混合液に再度懸濁し、細胞のカウントを行った。分離後回収率として、分離前の細胞数に対する、分離後の処理液中に含まれる細胞数の割合を算出した。また、凍結後回収率として、凍結前の細胞数に対する、凍結後の処理液中に含まれる細胞数の割合を算出した。
2 体液の出口
3 分離材
4、5 分離材を圧縮させるためのワッシャー
6 容器
7 血球分離カラム
8 チャンバー
9 血球分離材の充填された容器
10 体液バッグ
11 プライミング液バッグ(兼洗浄液バッグ)
12 赤血球回収バッグ
13 白血球回収バッグ(単核球回収バッグ)
14 回収ポート
15、16、17 三方活栓
18~24 回路
Claims (22)
- 平均繊維径が2.0μm以上6.0μm以下であり、かつ、通気度係数Mが6.2以上35以下である不織布からなることを特徴とする、体液から白血球または単核球を分離するための分離材。
- 不織布が、ポリオレフィン、ポリアミド、およびポリエステルからなる群から選択される少なくとも1つからなる、請求項1に記載の分離材。
- 体液が、末梢血、臍帯血、骨髄、月経血、および組織抽出物からなる群から選択される少なくとも1つである、請求項1または2に記載の分離材。
- 請求項1~3のいずれかに記載の分離材を、体液の入口と出口を有する容器に充填して得られる細胞分離用容器。
- 分離材が、体液の流れる方向に1枚または複数枚積層して充填されていることを特徴とする、請求項4に記載の細胞分離用容器。
- 分離材が、体液の流れる方向に圧縮された状態で充填されていることを特徴とする、請求項4または5に記載の細胞分離用容器。
- カラムタイプであることを特徴とする、請求項4~6のいずれかに記載の細胞分離用容器。
- 請求項1~3のいずれかに記載の分離材に体液を接触させて白血球または単核球を分離する工程を含む、細胞分離方法。
- 請求項4~7のいずれかに記載の細胞分離用容器に体液を接触させて白血球または単核球を分離材に捕捉させる第一の工程、および、剥離溶液を用いて、捕捉した白血球または単核球を分離材から回収する第二の工程からなる、細胞分離方法。
- 第二の工程が、細胞分離用容器の出口から剥離溶液を導入し、入口から白血球または単核球を回収する工程である、請求項9に記載の細胞分離方法。
- さらに、第一の工程の後であって第二の工程の前に、入口から生理食塩水または緩衝液を導入することにより、細胞分離用容器内の夾雑成分を除去する工程を含む、請求項9または10に記載の細胞分離方法。
- さらに、第一の工程の前に、分離材を生理食塩水または緩衝液と接触させる工程を含む、請求項9~11のいずれかに記載の細胞分離方法。
- さらに、第一の工程の前に、細胞分離容器の体液の入口を細胞分離容器の体液の出口より低い高さに固定する工程を含む、請求項9~12のいずれかに記載の細胞分離方法。
- 白血球と血小板が実質的に分離材に捕捉され、赤血球が実質的に捕捉されないことを特徴とする、請求項8~13のいずれかに記載の細胞分離方法。
- 分離された白血球または単核球が、造血幹細胞および/または間葉系幹細胞を含むことを特徴とする、請求項8~14のいずれかに記載の細胞分離方法。
- 請求項8~15のいずれかに記載の細胞分離方法に、さらに、液体窒素環境下にする工程を含むことを特徴とする、細胞の凍結保存方法。
- 液体窒素環境下が-196℃から-30℃であることを特徴とする請求項16に記載の凍結保存方法。
- ジメチルスルホキシド、デキストラン、アルブミン、およびヒドロキシエチルスターチからなる群から選択される少なくとも1つの凍結保護剤を使用することを特徴とする請求項16または17に記載の凍結保存方法。
- 凍結保存した幹細胞の生存率が80%以上であることを特徴とする請求項16~18のいずれかに記載の凍結保存方法。
- 請求項8~15のいずれかに記載の細胞分離方法によって得られた白血球、単核球、または幹細胞。
- CD34陽性細胞、
CD133陽性細胞、
CD34陰性且つCD133陽性細胞、
CD34陽性且つCD133陽性細胞、
CD34陽性且つCD133陰性細胞、
CD45陰性且つCD44陽性且つCD73陽性且つCD90陽性細胞、
CD45陰性且つCD235a陰性且つCD33陰性且つCD7陰性細胞、
CD45陽性且つCD133陽性且つCD117陽性細胞、
CD45陽性且つCD133陰性且つCD117陽性細胞、
CD45陽性且つCD133陽性且つCD164陽性細胞、
CD45陽性且つCD133陰性且つCD164陽性細胞、および
CD45陰性且つCD309陽性細胞
からなる群から選択される
いずれかの細胞を含む請求項20に記載の幹細胞。 - CD45陽性且つCD164陽性細胞、または
CD45陽性且つCD117陽性細胞を含む請求項20に記載の白血球。
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2010
- 2010-11-25 US US13/989,523 patent/US20130323712A1/en not_active Abandoned
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2011
- 2011-11-24 WO PCT/JP2011/077067 patent/WO2012070622A1/ja active Application Filing
- 2011-11-24 CN CN201180056749.2A patent/CN103228780B/zh active Active
- 2011-11-24 SG SG2013040043A patent/SG190874A1/en unknown
- 2011-11-24 EP EP11843415.8A patent/EP2644689A4/en not_active Withdrawn
- 2011-11-24 KR KR1020137016332A patent/KR20140004110A/ko active Application Filing
- 2011-11-24 JP JP2012545792A patent/JP5944832B2/ja active Active
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Also Published As
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EP2644689A4 (en) | 2017-05-03 |
JPWO2012070622A1 (ja) | 2014-05-19 |
KR20180063346A (ko) | 2018-06-11 |
CN103228780B (zh) | 2015-04-15 |
US20180002663A1 (en) | 2018-01-04 |
JP5944832B2 (ja) | 2016-07-05 |
SG190874A1 (en) | 2013-07-31 |
EP2644689A1 (en) | 2013-10-02 |
KR20140004110A (ko) | 2014-01-10 |
CN103228780A (zh) | 2013-07-31 |
US20130323712A1 (en) | 2013-12-05 |
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