JP2005336080A - Method and system for separating and collecting cell for therapeutic angiogenesis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and collecting a large amount of cells for therapeutic angiogenesis, usable for carrying out the therapy by the angiogenesis by injection to a disease site of a human body from a raw material cell sap; and to provide a system for the method. <P>SOLUTION: The method for separating and collecting the cells for the therapeutic angiogenesis comprises a capturing step for capturing the cells effective for the angiogenesis by a cell-capturing filter by flowing the raw material sap containing the cells effective for the angiogenesis through the cell-capturing filter obtained by packing a cell-capturing material substantially not capturing erythrocytes but capturing the cells effective for the angiogenesis, and a collecting step for collecting the cells captured by the cell-capturing filter, and repeatedly carries out the capturing and collecting operations including the capturing step and the collecting step by using the same filter. The system for separating and collecting the cells for the angiogenesis is used for carrying out the method. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and system for separating and recovering cells effective for angiogenesis from a raw material cell solution containing cells effective for angiogenesis and contaminated cells.

  Hyperlipidemia or diabetic patients are rapidly increasing due to the influence of westernization and aging of the diet, and as a result, diseases caused by vascular disorders are increasing. In particular, cardiovascular or cerebrovascular disorders often result in direct death or have serious sequelae, and are becoming serious problems with the progress of an aging society.

  As a means of recovering organs and tissues that have become dysfunctional, the concept of regenerative medicine, in which organs or tissues themselves are regenerated in the body by transplantation of cells before differentiation into organs or tissues, has attracted worldwide attention. Is being researched. Among regenerative medicine, angiogenesis by vascular endothelial progenitor cells (EPC) is an area that has recently made significant progress. Prof. Asahara proposed a new mechanism of angiogenesis (see Non-Patent Document 1), tried treatment based on it, and published clinical effects one after another (see Non-Patent Document 2). In addition, angiogenesis therapy has spread to the area of organ regeneration, and for example, it has been reported that stem cells are activated by vascularization of ischemic sites in response to pancreatic dysfunction and promotes organ regeneration. (Refer nonpatent literature 3).

  A field where angiogenesis therapy is currently pioneering is obstructive arteriosclerosis of the lower limbs. As vascular disorders of the lower limbs, there are ulcers and gangrenes associated with ischemia of the extremities and toes called so-called Buerger's disease, but recently there has been a marked increase in the incidence of diabetic leg occlusive arteriosclerosis. The estimated number of patients with obstructive arteriosclerosis in Japan is 100,000, and is expected to reach 1 million in the future (see Non-Patent Document 4).

  In particular, the number of patients who are forced to undergo amputation due to obstructive arteriosclerosis of the lower limbs is increasing year by year, and is said to be 20,000 per year. It has been reported that the prognosis of lower limb amputation due to lower limb obstructive arteriosclerosis is poor, half die after 5 years, and 30% leads to amputation of the contralateral leg (see Non-Patent Document 5). ).

  As a means to avoid amputation of the lower extremity, a method of treating obstructive arteriosclerosis by collecting the patient's own mononuclear cells and injecting them into the ischemic site was tried, and obtained approval from the Ministry of Health, Labor and Welfare as highly advanced medical treatment It came to. The treatment method that has been approved by the Ministry of Health, Labor and Welfare as a highly advanced medical treatment is a treatment method in which bone marrow fluid is collected and mononuclear cells are separated and transfused, while granulocyte colony stimulating factor (G-CSF), etc. A method of recovering mononuclear cells in peripheral blood, such as administering bone marrow stimulants by intravenous injection, mobilizing a large amount of mononuclear cells containing CD34 positive cells in the bone marrow into the peripheral blood, Has achieved results. In particular, the latter method with G-CSF administration has been published with many clinical effects (see Non-Patent Document 6), and mononuclear cells present in peripheral blood without stimulation such as G-CSF administration. There is also an example in which a sphere is collected by continuous centrifugation and is applied to treatment by transfusion to a blood vessel occlusion site (see Non-Patent Document 7). This method is also expected to promote a new mechanism of mononuclear cell movement from bone marrow to peripheral blood by collecting mononuclear cells in peripheral blood and depleting mononuclear cells in peripheral blood. There is a possibility that cells expressing angiogenesis can be collected without administration of CSF. The treatment method using these peripheral blood mononuclear cells has an advantage that the burden on the patient is lighter than that of bone marrow collection and treatment can be performed frequently.

  The range of angiogenesis therapy referred to in the present invention includes all areas where therapeutic effects can be expressed by angiogenesis as described above, in addition to obstructive arteriosclerosis.

  Angiogenesis therapy begins with the treatment of lower limb obstructive arteriosclerosis, develops into the treatment of diseased sites due to various vascular disorders in the human body, and has the potential for organ regeneration induced by angiogenesis It can be said to be an effective therapy. However, many problems remain in the separation and concentration of cell compositions for angiogenesis therapy, and angiogenesis therapy can be used effectively in the early stages of the disease or even more prophylactically. To solve this, many problems must be solved.

  Currently, continuous centrifugation is mainly used for the separation and concentration of mononuclear cells. The continuous centrifugation method is a method in which blood is centrifuged while extracorporeally circulating and separated into red blood cells, platelets, white blood cells, plasma, and the like, and necessary components are collected and the rest are returned to the donor. However, the anticoagulant used in the continuous centrifugation method is citrate ACD-A or the like, and cannot cope with, for example, heparin.

  Commonly used anticoagulants include ACD-A, CPD, fusan, heparin, etc., which are preferably selected according to the purpose and conditions, but citrate anticoagulants are used in large quantities. It is known to cause poisoning symptoms rarely. In addition, patients with vascular disorders such as obstructive arteriosclerosis often have diabetes as a primary disease, and often have diabetic nephropathy. In these cases, dialysis treatment has already been performed, and if it can be connected to a dialysis machine and mononuclear cell separation and recovery can be performed at the same time, it can be effectively treated and the burden on the patient can be reduced. Since the anticoagulant used is heparin, it is difficult to separate and recover mononuclear cells simultaneously by connecting a continuous centrifuge to a dialysis machine. Realization of a mononuclear cell separation and recovery system that can use heparin anticoagulants not only solves the problem of side effects in extracorporeal circulation using citrate anticoagulants, but also in the application of angiogenesis therapy in dialysis patients The expectation in the clinical field is great because the burden on the patient and the operation time can be reduced.

  Furthermore, when mononuclear cells are separated by some filter operation instead of centrifugation, it is possible to activate the mononuclear cells by the interaction between the mononuclear cells and the filter surface and promote the production of cytokines. As a result, there is a possibility that cells or components having a high therapeutic effect can be efficiently recovered from peripheral blood even in patients who are not administered G-CSF.

  A new blood treatment system that exceeds the limits of the current centrifugation method is expected to play a major role in the expansion of treatment methods by angiogenesis.

  Until now, several attempts have been made to collect cells using filters. The concept of collecting leukocytes using a material in which fibers are packed in a column and the fiber surface is coated is disclosed in Patent Literature 1 and Patent Literature 2, and Patent Literature 3 showing those systems is Patent Literature 3. . In particular, Patent Document 4 and Patent Document 5 and Patent Document 6 and Patent Document 7 show the systems for the purpose of recovering nucleated cells including mononuclear cells. In either case, a technique for effectively recovering target cells while preventing clogging of the raw material liquid is disclosed. Further, there are Patent Documents 8 and 9 as leukocyte removal systems based on extracorporeal circulation. However, in the design philosophy so far, studies on processing a large amount of blood by extracorporeal circulation and collecting cells captured by the cell trapping filter by blood processing and using them for specific treatment are insufficient. In view of the number of cells necessary for treatment, the amount of cells obtained by blood collection, for example, 500 mL or less, often cannot meet the required number of cells. Therefore, it is necessary to consider the introduction of an extracorporeal circulation system. In the extracorporeal circulation system, it is necessary to avoid a filter blockage for processing a lot of blood with a small priming volume, to secure a capacity capable of capturing a predetermined amount of cells, or When considering the burden on patients, heparin can be used as an anticoagulant, and it is necessary to add to the design philosophy how to effectively recover captured cells. is there.

  The method of processing a large amount of peripheral blood using a cell trapping filter to capture cells that are effective for angiogenesis and further recovering them is an idea that could not be realized so far. In addition, the selective capture performance of specific cells by the filter and the interaction of promoting activation to the captured cells by the filter surface are functions that can only be realized when a filter device is realized. It was not obtained. By configuring such a device, it is possible to realize a treatment method that can expect excellent effects even in a treatment method that does not perform G-CSF administration and has a low patient burden.

Isner, J. & Asahra, T. J.Clin.Invest., 103: 1231-1236 Tateishi-Yuyama E., Matsubara H., et.al.THE LANCET, vol360, Aug. 10, 2003 Sakano, S., et.al., Nature Biotechnology, July issue (2003) “Lower limb obstructive arteriosclerosis” supervised by Hiroshi Shigematsu (2001) Management of Peripheral Arterial Disease J Vasc Surg 31,2000 Taku Horie Journal of Japanese Society for Regenerative Medicine vol2 Suppl Po-058 (2003) Takayoshi Asai, Naomi Shimizu et al. Journal of Japanese Society of Blood Transfusion vol.49 No.2, Page259 (2003) JP 55-129755 A Japanese Unexamined Patent Publication No. 57-145562 JP-A-9-56813 Japanese Patent Laid-Open No. 11-9270 JP-A-11-56351 JP 2001-136955 A JP 2001-198214 A Japanese Patent Laid-Open No. 62-243561 JP 2001-161812 A

  An object of the present invention is to provide a method for separating and recovering a large amount of angiogenic therapy cells for treatment by angiogenesis by injecting into a diseased part of a human body from peripheral blood, and a system for the method. And

  The inventors of the present invention have made extensive studies in order to solve such problems, and applied to a cell trapping filter formed by filling a cell trapping material that does not substantially trap red blood cells but traps angiogenic cells. For angiogenesis therapy, comprising a step of causing a cell capture filter to capture cells effective for angiogenesis by flowing a raw material cell solution containing effective cells, and a step of recovering the cells captured by the cell capture filter In the method for separating and recovering cells, if a large amount of raw material cell fluid is passed through a certain amount of cell capture material, it becomes difficult to collect the captured cells, and the cell capture material is kept in contact with the raw material cell solution for a long time. We discovered that cells adhere firmly to the trapping material and are difficult to recover, and that it is necessary to recover cells little by little in a short time in order to separate and recover cells with high efficiency. It was. Furthermore, when making this knowledge a system that can actually be used, it has been found that by performing repeated capture and collection little by little using the same filter, the cell recovery efficiency can be increased and the system can be simplified. It has come to be completed.

  That is, according to the present invention, a raw material cell solution containing cells effective for angiogenesis is allowed to flow through a cell capture filter that is filled with a cell capture material that does not substantially capture red blood cells but traps cells effective for angiogenesis. In the method for separating and recovering cells for angiogenesis therapy, comprising a capturing step for capturing cells effective for angiogenesis by the cell capturing filter, and a recovering step for recovering the cells captured by the cell capturing filter. There is provided a method for separating and recovering angiogenic therapy cells, which comprises repeatedly performing a capturing / recovering operation comprising a capturing step and the recovering step using the same filter.

Preferably, the cells effective for angiogenesis include nucleated cells.
Preferably, the cells effective for angiogenesis include mononuclear cells.

Preferably, the total number of cells effective for angiogenesis in the raw material cell fluid to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is effective for angiogenesis. The cell recovery rate is set to 50% or more.
Preferably, the total number of cells effective for angiogenesis in the raw material cell fluid to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is 1 × 10 ⁷ to 1 × 10 ^Nine .

Preferably, the contact time between the cell capture material and the raw material cell solution for one capture / collection operation including the capture step and the recovery step is such that the recovery rate of cells effective for angiogenesis is 50% or more. Set.
Preferably, the contact time between the cell capture material and the raw material cell liquid for one capture / collection operation including the capture step and the recovery step is 5 to 50 minutes.

Preferably, the recovery rate of cells that are effective for angiogenesis is 50% or more when the raw material cell liquid that is flown per 1 m ² of the surface area of the cell capture material per capture / recovery operation including the capture step and the recovery step is 50% or more. Set as follows.
Preferably, the raw material cell solution to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is 10 to 60 cm ³ .

Preferably, the recovery rate of cells effective for angiogenesis is 50% or more.
Preferably, the total amount of mononuclear cells to be collected is 10 ¹⁰ or more.
Preferably, the cell capture filter is incorporated in series or in parallel with a part of the blood circuit of the blood extracorporeal circulation system.

  According to another aspect of the present invention, there is provided an angiogenesis therapy cell obtained by the above-described method for separating and recovering an angiogenesis cell.

  According to yet another aspect of the present invention, the cell trapping material is filled with a cell trapping material that traps cells effective for angiogenesis, but does not substantially trap red blood cells, upstream conduits, and red blood cells. The filter, the downstream pipe line, and the return port for the raw material cell liquid are connected in this order, and further, the recovery liquid introducing means is provided upstream or downstream of the cell trapping filter, and the part where the recovery liquid introducing means is provided is upstream. An angiogenesis characterized in that a cell recovery means is provided on the downstream side if it is on the downstream side, and on the upstream side if it is on the downstream side, and further, a pipeline bypassing the upstream pipeline and the downstream pipeline is connected A system for separating and collecting therapeutic cells is provided.

  According to still another aspect of the present invention, there is provided a cell trapping material that captures cells that are effective for angiogenesis but do not substantially capture red blood cells, but the outlet of the raw material cell fluid, the upstream duct branched from the middle, The same number of cell trapping filters as the number of branches in the upstream conduit, the downstream conduit having the same number of branches as the cell trapping filter, and the return port for the source cell fluid are connected in this order, and the cells A recovery liquid introduction means connected to each branched pipe line upstream or downstream of the capture filter is provided, and if the part where the recovery liquid introduction means is provided is upstream, it is downstream, and if it is downstream There is provided an angiogenesis cell separation and collection system characterized in that cell collection means for collecting cells from individual branched branches on the upstream side is provided.

Preferably, the above-described cell separation and recovery system for angiogenesis therapy is incorporated in series or in parallel with a part of the blood circuit of the extracorporeal blood circulation system.
Preferably, the extracorporeal blood circulation system is a hemodialysis system.

Preferably, a cell concentrator is further connected to the cell recovery means.
Preferably, the cell concentrator is a hollow fiber membrane type cell concentrator.

  By using the present invention, a large number of cells for angiogenesis therapy can be obtained with a simple system and with high recovery efficiency.

Hereinafter, the present invention will be described in detail.
The cells effective for angiogenesis referred to in the present invention include nucleated cells and platelets. Among nucleated cells, mononuclear cells are particularly effective cells for angiogenesis. Moreover, the cell composition for angiogenesis therapy means all the compositions which contain the said cell effective for angiogenesis as a component.

  In the cell composition for angiogenesis therapy, examples of components contained in addition to the cells effective for angiogenesis include erythrocytes mixed in the raw material cell fluid. Red blood cells are cells that are not particularly required in angiogenesis therapy because they do not have the ability to differentiate into blood vessels and do not have the function of producing humoral factors and the like effective for angiogenesis. However, since it is difficult to completely remove cells effective for angiogenesis from the raw material cell solution in the process of separation and recovery, they may be included in the composition as contaminated cells in the cell composition for angiogenesis therapy.

  The source cell fluid containing cells effective for angiogenesis referred to in the present invention may be any cell suspension containing at least cells effective for angiogenesis, and examples thereof include peripheral blood, bone marrow fluid, and cord blood.

  As described above, the scope of angiogenesis therapy includes all areas that can exhibit therapeutic effects by angiogenesis in addition to obstructive arteriosclerosis. Atherosclerosis is one example, and coronary artery occlusion or lower limb obstructive arteriosclerosis.

  The cell trapping filter referred to in the present invention is obtained by filling a cell trapping material into a container having a source cell fluid inlet and a source cell fluid outlet.

  A porous material is preferably used as the cell trapping material. The porous body mentioned here is preferably at least one selected from the group consisting of a porous body made of fibers, a porous body made of particles, and a sponge-like porous body. In order to effectively capture cells, it is preferable to effectively contact the surface of the material constituting the cell trapping material while the source cell fluid passes through the cell trapping material. A shape that passes through the pores of the body is preferred. The porous body is not limited to a general nonwoven fabric or sintered body, but may be any porous body that is formed into a porous body shape during the treatment of the raw material cell solution. Those filled in can also be employed as the porous body. Sponge-like porous materials include porous materials obtained by phase separation, porous materials obtained by foaming, and porous materials opened by ionizing radiation irradiation or etching. .

  Any material can be used as a material for these porous materials as long as it does not substantially capture erythrocytes and can capture cells that are effective in angiogenesis, but it has excellent moldability and sterilization properties and low cytotoxicity. In view of the above, preferred examples include polyolefin resins such as polyethylene, polypropylene, and poly-4-methylpentene, fluororesins such as polyvinylidene fluoride resin, ethylene / tetrafluoroethylene resin, and polychlorotrifluoroethylene resin, polysulfone, and polystyrene. , Acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, etc., various other synthetic polymers, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, alginate, hydroxyapatite, glass, alumina, titani Inorganic material such as stainless steel, titanium, and metals such as aluminum.

  Among these, non-woven fabric filters made of polyester or polypropylene have many achievements as leukocyte removal filters, and can be suitably used in the present invention. For example, a non-woven fabric having an average fiber diameter of 1.0 to 30 μm is a material suitable for capturing nucleated cells or platelets including mononuclear cells, and is preferably used as a component of a cell trapping filter, more preferably an average. The fiber diameter is 1.0 to 10 μm. If the thickness is less than 1.0 μm, cells effective for angiogenesis are firmly captured and may be difficult to recover. If the thickness exceeds 30 μm, cells effective for vascular sacredness are more likely to pass through without being captured by the fibers. In either case, the recovery rate may be lowered, which is not preferable.

  Depending on the use conditions and the purpose of use, it is possible to select those in which the surface inside the pores of the cell trapping material is appropriately modified. For example, a cell-trapping material having a hydrophilic material surface is effective for uniformly flowing the raw material cell fluid through the filter, and is preferably selected. The surface modification method is basically based on the construction of a hydrophilic surface, and a known method can be employed depending on the purpose. For example, the surface can be hydrophilized by treatment with hot water after irradiation with ionizing radiation. Surface modification is also possible by coating an amphiphilic polymer. For example, hydrophilization with hydroxyacrylic or methacrylic polymers such as hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, amine polymers, polyethylene glycol polymers, or copolymers thereof. is there. Among these, hydroxyethyl methacrylate and dimethylaminoethyl methacrylate copolymer have a track record as a coating agent having excellent performance of selectively adsorbing leukocytes, and are preferably employed (Japanese Patent Laid-Open No. 10-234361). . In addition, the graft polymerization method is a method of binding a chemically hydrophilic polymer to the cell trapping material, and has an advantage such as no fear of elution. The method described in JP 2000-185094 A is preferably employed.

  Examples of the material of the container filled with the cell capturing material and having the inlet and outlet for the raw material cell solution are preferable in terms of excellent moldability and sterility, and low cytotoxicity. Polyethylene, polypropylene, Polyolefin resin such as poly-4-methylpentene, polyvinylidene fluoride resin, ethylene / tetrafluoroethylene resin, fluorine-based resin such as polychlorotrifluoroethylene resin, polysulfone, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, Other synthetic polymers such as polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, alginate, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel, titanium, aluminum Like metal etc. is.

  Examples of the structure of the container include a rectangular parallelepiped, a cube, a cylinder, and an elliptic cylinder, but any shape may be used. In addition, the position of the inlet and outlet of the raw material cell solution may be a position where the liquid can be introduced into the uppermost layer of the filter medium, and the outlet may be a position where the liquid can be led out from the lowermost layer of the filter medium.

  In the method of flowing the raw material cell solution to the cell trapping filter described above, the liquid may be introduced into the filter and passed in a certain direction. The means for introducing the raw material cell fluid into the cell trapping filter can be achieved by using devices such as syringe pumps, blood pumps, peristaltic pumps, etc., as a simple method of manually pushing the syringe, or by crushing the bag storing the liquid to induce liquid flow However, when a filter is incorporated into an extracorporeal circulation system, a blood pump or a peristaltic pump is preferably used.

  The differential pressure before and after the flow of the raw material cell solution through the cell trapping filter is preferably 0.01 or more and less than 50 kPa, more preferably 0.1 or more and less than 20 kPa. When the source cell solution contains high concentration of red blood cells, if the differential pressure is too large, blood cell degeneration cannot be avoided. Within the above differential pressure range, blood cell degeneration can be avoided, and a constant treatment speed can be maintained.

When the raw material cell liquid is allowed to flow through the cell trapping filter, the total number of cells effective for angiogenesis in the raw material cell liquid to be flown per 1 m ² of the surface area of the packed cell trapping material is calculated as the recovery rate of cells effective for angiogenesis. It is preferable to set it to be 50% or more. For example, the total number of cells effective for angiogenesis in the raw material cell fluid to be flown per 1 m ² of the surface area of the cell trapping material is more preferably 1 × 10 ⁷ to 1 × 10 ⁹ . The surface area of the cell trapping material here refers to the material surface area of the trapping material through which cells effective for angiogenesis, mainly mononuclear cells, and erythrocytes pass. For example, when a nonwoven fabric is used as the cell trapping material, Calculated by the formula.

Cell capture material surface area (m ² ) = (4 × A × 10 ⁻³ ) / (B × C)
A: Weight of cell trapping material (mg)
B: Average fiber diameter (μm) of the cell trapping material
C: Specific gravity of the material of the cell trapping material (g / cm ³ )

If the total number of cells effective for angiogenesis is less than 1 × 10 ⁷ cells per 1 m ² of the surface area of the cell trapping material, a sufficient amount of raw material cell fluid cannot be flowed, making it difficult to collect cells and angiogenesis. When the total number of effective cells is more than 1 × 10 ⁹ per 1 m ² of the surface area of the cell trapping material, the blood flow rate is too high and the treatment itself becomes difficult, and even if it can be treated, the recovery rate is significantly reduced. For this reason, it is not preferable, and further, cells are laminated on the surface of the capturing material, and cells are buried in the pores of the cell capturing material.

Further, when the raw material cell liquid is allowed to flow through the cell trapping filter, the contact time between the cell trapping material and the raw material cell liquid is preferably set so that the recovery rate of cells effective for angiogenesis is 50% or more. For example, the contact time between the cell capture material and the raw material cell liquid for one capture / collection operation including the capture step and the recovery step is 5 minutes to 50 minutes, more preferably 5 minutes to 30 minutes. be able to. The contact time here refers to the time from the introduction of the raw material cell solution into the cell capture filter until the introduction of the filter washing solution as described below. If the washing operation is not added to the process, the raw material cell solution is introduced. It refers to the time from the beginning of the process until the recovery liquid is introduced. When the contact time is less than 5 minutes, for example, when a filter is incorporated in the extracorporeal circulation system, the flow rate of blood to flow is appropriate to be about 30 to 80 cm ³ / min (the cause of coagulation in the extracorporeal circuit when the flow rate is too slow) If the speed is too high, the burden on the donor is large and blood flow cannot be obtained.) Since the amount of blood to be processed is small, a sufficient amount of cells may not be obtained at the time of recovery, which is not preferable. On the other hand, if the contact time exceeds 50 minutes, the cells captured by the filter stick firmly to the surface of the filter medium, which is not preferable because the recovery rate is reduced.

Further, when the raw material cell fluid is allowed to flow through the cell trapping filter, the amount of the raw material cell fluid to be flown per 1 m ² of the surface area of the cell trapping material may be set so that the recovery rate of cells effective for angiogenesis is 50% or more. preferable. For example, it is preferable that the amount of the raw material cell fluid to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is 10 to 60 cm ³ . When the amount of the raw material cell fluid is less than 10 cm ³ , a sufficient amount of cells effective for angiogenesis cannot be flowed, and it becomes difficult to collect the cells. When the amount of the raw material cell fluid is larger than 60 cm ³ The cells are laminated on the surface of the capturing material, and the cells are buried in the pores of the cell capturing material, which is not preferable because the recovery rate is lowered.

  According to the method of the present invention, the recovery rate of cells effective for angiogenesis is preferably 50% or more, and more preferably 60% or more.

  The method for recovering captured cells as referred to in the present invention is to collect the cells captured by the cell capture filter by sending the recovery solution to the cell capture filter in the same direction as the flow direction of the raw material cell fluid or in the opposite direction. Say. Means for introducing the recovered liquid into the cell trapping filter are those using a device such as a syringe pump, a blood pump, a peristaltic pump, etc., a method of pushing the syringe by hand as a simple method, and crushing the bag storing the liquid to induce a liquid flow Although a method, a drop treatment, etc. can be mentioned, it is preferable to apply a certain amount of shear stress to the captured cells in order to improve the recovery rate, and it is desirable to perform the recovery operation in a region higher than the differential pressure at the time of cell capture.

  Examples of recovered liquids that have little cell degeneration effective for angiogenesis include buffer solutions such as physiological saline, Dulbecco's phosphate buffer solution (D-PBS), Hanks solution (HBSS), and culture media such as RPMI1640 Is preferably employed. Moreover, in order to collect | recover efficiently, it is more preferable to have a fixed viscosity, and it is preferable to adjust a viscosity in the range with few bad influences on the cell effective for angiogenesis. By adding dextran, hydroxyethyl starch, albumin, globulin, gelatin, glucose, saccharose, trehalose, etc., the benefits of cell protection effective for angiogenesis are obtained, and the recovery rate is improved by adjusting the viscosity of the recovery solution. Is possible. A chelating agent such as EDTA may be contained for the purpose of removing divalent cations and facilitating cell detachment.

  In the method for separating and recovering cells for angiogenesis therapy, the amount of erythrocytes can be reduced by adding a step of washing the cell capture filter after flowing the raw material cell solution through the cell capture filter. It is preferable to add a washing step.

  As a method for washing the cell trapping filter, the washing solution may be sent out to the filter from the same direction or the reverse direction of the flow of the raw material cell solution. At this time, the direction in which the washing solution is sent out is more preferably the same direction as the direction in which the raw material cell solution is passed, because it is more effective in preventing the outflow of cells trapped on the filter and effective for angiogenesis. The washing solution is not particularly limited as long as it does not damage cells and allows erythrocytes to flow out. Preferred examples include buffer solutions such as physiological saline, PBS (phosphate buffer solution) and HBSS (Hanks solution). , RPMI1640 and other cell culture media are included in these liquids for the purpose of cell protection, nutritional supplementation, anticoagulant imparting, viscosity improvement, etc., as necessary, plasma, serum, albumin, globulin, glucose, saccharose, Trehalose, a citrate compound, EDTA, dextran, gelatin or the like may be added. In addition, the means for introducing the washing solution into the cell trapping filter is a device using a syringe pump, a blood pump, a peristaltic pump or the like, a method of pushing the syringe by hand as a simple method, or crushing a bag storing the liquid to cause a liquid flow And the head processing.

  Cells for angiogenesis therapy obtained by the above method are infused directly or after applying some kind of treatment. The term “some treatment” as used herein refers to a further concentration operation or dilution operation, a temperature history such as freezing and warming, and the like, and these treatment methods are appropriately selected depending on the treatment procedure. That is, when infused in small amounts over a long period of time, when infused to several locations of scattered disease sites, when infused to a specific organ, the infusion of the cell composition depends on the size of the infusion area and the degree of disease. The best method is selected. Moreover, it is preferable that the patient who receives the treatment by infusion and the person who provides cells effective for angiogenesis in the blood are the same person. By being the same person, rejection associated with transplantation can be basically avoided.

In order to collect a large amount, particularly 10 ¹⁰ or more mononuclear cells by the method for separating and collecting cells for angiogenesis therapy in the present invention, it is efficient to repeat the above-described capture and recovery step using the same filter. A lot of mononuclear cells can be obtained well. As described above, it is desirable that the total number of nucleated cells in the raw material cell fluid to flow per 1 m ² of the surface area of the cell trapping material packed in the cell trapping filter is 1 × 10 ⁷ to 1 × 10 ⁹ , 10 in order to recover the ¹⁰ monocyte not must shed material cell solution containing at least 10 ¹⁰ or more mononuclear, when the raw material cell fluid is peripheral blood, the single nucleated cells Since the content of nuclei is 30 to 40%, the cell trapping material needs to be 30 m ² or more. For example, when a nonwoven fabric is used as a cell trapping material, if the filter is filled with a cell trapping material with a surface area of 30 m ² or more, the volume of the filter becomes 500 to 1000 cm ² , which is realistic for daily use in medical settings. Not. Therefore, for example, if the amount of the cell capture material is reduced to 1/10, the raw cell fluid is divided into 10 equal parts, and the capture and recovery operation is repeated 10 times using the same filter, the cell capture material is collected per capture and recovery operation. the number of nucleated cells flowing through 1 m ² becomes 10 ^9, be recoverable mononuclear with high efficiency, and the volume of the filter can also be in the 50 to 100 cm ² and a compact, it is a filter system sufficient for practical use.

The method for separating and collecting angiogenesis cells in the present invention is preferably performed using an extracorporeal circulation system. In order to increase a certain therapeutic effect by angiogenesis, it is preferable that a predetermined amount or more of cells effective for angiogenesis are concentrated, for example, cells necessary for obstructive arteriosclerosis of severe lower limbs due to diabetes The number is said to be 10 ¹⁰ in a mononuclear sphere. However, this also depends on the area of the disease site to which angiogenesis therapy is applied, and if the site to be infused is limited, 10 ¹⁰ mononuclear cells are not necessarily required, and in some cases 10 ⁸ mononuclear cells. There may be a case where a certain therapeutic effect can be expected from the nuclear sphere. However, in practice, it is said that 10 ⁹ or more, preferably 10 ¹⁰ or more mononuclear cells are necessary for effective treatment at a diseased site, and in this case, extracorporeal circulation is assumed. In addition, it is more preferable to incorporate a system for repeatedly capturing and collecting using the same filter as described above, because the filter can be made compact, so that the amount of extracorporeal blood can be reduced, and mononuclear cells can be collected with high efficiency. .

  The anticoagulant in the case of extracorporeal circulation is appropriately selected. In addition to citrate anticoagulants such as ACD-A and CPD, fusan containing nafamostat mesylate is assumed, but heparin anticoagulants are commonly used for hemodialysis and have side effects It is suitably employed as an anticoagulant with a low content. In particular, by using heparin, it is possible to connect to a hemodialysis circuit for processing, and for dialysis patients, there is a great advantage that an act other than the usual dialysis treatment is not necessary for collecting mononuclear cells. be able to.

  The method for separating and recovering cells for angiogenesis therapy according to the present invention includes a cell capture material that captures cells effective for angiogenesis, but does not substantially capture red blood cells, upstream ducts, and red blood cells. The cell trapping filter, the downstream pipe line, and the return port of the raw material cell liquid are connected in this order, and the recovery liquid introducing means is further provided upstream or downstream of the cell capturing filter, and the recovery liquid introducing means is provided. If the part is upstream, it can be carried out by using a cell separation means for angiogenesis therapy provided with cell collection means on the downstream side, and if it is on the downstream side, it can be carried out. Moreover, it can carry out using the system by which the pipe line which bypasses an upstream pipe line and a downstream pipe line is connected to the said system. Furthermore, the outlet tube of the raw material cell liquid, the upstream pipe branching into a plurality from the middle, the upstream pipe which is filled with a cell capture material that captures red blood cells but does not substantially capture red blood cells but is effective for angiogenesis The same number of cell trapping filters as the number of branches in the channel, the downstream pipe with the same number of branches as the cell trapping filter, and the return port for the source cell fluid are connected in this order, and further upstream or downstream of the cell trapping filter Recovery liquid introduction means connected to the individual branched pipes, provided that the portion provided with the recovery liquid introduction means is on the downstream side, and on the downstream side, the individual pipes on the upstream side are branched. It can be carried out using a cell separation / collection system for angiogenesis therapy provided with a cell collection means for collecting cells from the path.

  The system of the present invention will be described below with reference to the drawings. FIG. 1 shows an example of the structure of the cell trapping filter. The cell-trapping material laminate 1 is a laminate of two or more cell-trapping materials having different structures or materials. In FIG. 1, as the cell trapping material laminate 1, a cell trapping material A (5), a cell trapping material B (6), and a cell trapping material C (7) are stacked. FIG. 1 a shows a set of cell trapping material laminates 1 housed in a cell trapping filter case 4. The raw material cell fluid flows in from the cell trapping filter inlet 2, passes through the cell trapping material laminate 1, and flows out from the cell trapping filter outlet 3. The cell trapping material laminate 1 shows a structure in which three layers of cell trapping materials having different structures or materials are stacked. The type, the number of layers, and the combination of the cell trapping materials to be stacked avoid clogging and are efficient. A structure that captures and collects cells is selected.

  FIG. 1 b shows a cell trapping filter case 4 filled with two sets of cell trapping material laminates 1. The principle is the same as in FIG. 1a, but a large membrane area in the cell trapping filter case can be secured by introducing two sets of cell trapping material laminates into one container. In FIG. 1b, the raw material cell liquid introduced from the cell trapping filter inlet 2 is first introduced into the external space 9 of the cell trapping material laminate, passes through the cell trapping material laminate 1, and is sandwiched between the cell trapping material laminates. It flows into the space 8 and then flows out from the cell capture filter outlet 3. The structure of the cell trapping filter can be configured in the same way as in FIG. 1a.

  FIG. 1c shows that the raw material cell fluid introduced from the cell trapping filter inlet 2 is first introduced into the space 8 sandwiched between the cell trapping material laminates, permeates the cell trapping material laminate, and passes through the cell trapping material laminate. It flows into the external space 9 and subsequently flows out from the cell capture filter outlet 3. Again, the structure of the cell trapping filter can be configured in the same way as in FIG. 1a.

  2a and 2b are views of FIGS. 1a and 1b as viewed from the front. The shape is a rhombus, FIG. 2a shows a set of cell trapping material stacks housed in a cell trapping filter case, and FIG. 2b shows a set of two cell trapping material stacks. . The cell trapping material adhesion portion 10 outlined in black on the outer periphery of the cell trapping material laminate in FIG. 2b is a portion where two sets of cell trapping material laminates are adhered. The front view corresponding to FIG. 1c is basically the same as that of FIG. 2c and 2d are rectangular shapes with respect to FIGS. 2a and 2b. The shape can be selected as appropriate from the viewpoint of ease of molding, flow uniformity, and the like.

  As a system for using the cell trapping filter in a closed system, the configuration described in Japanese Patent Application Laid-Open No. 2001-198214 can be suitably employed. The outline is shown in FIG.

  In FIG. 3 a, 11 is a source cell fluid outlet, and 12 is a source cell fluid return port. Moreover, the collection liquid introduction means 14 and the cell collection means 16 are added downstream and upstream of the cell trapping filter, respectively. A portion where these various liquid introduction means or recovery means are connected is upstream or downstream of the cell trapping filter 20 and is branched from the middle of the tube. As the branch portions 13, 15 and 17, a combination of a T-shaped tube or a Y-shaped tube with a clamp or the like, or a three-way cock is appropriately employed. In addition, it is not necessary to connect various liquid introduction means or recovery means from the beginning, and by installing spikes, lures, metal needles, etc. at the ends of each branched tube, or by sealing the tubes with the ends sealed By using, for example, aseptic connection, it is possible to connect while maintaining the closed system when using the means for introducing and collecting various liquids.

  As an example of the method of use, the case where it is incorporated into an extracorporeal circulation system will be described. First, the branching section 15 is connected to the cell capture filter 20 and the raw material so that only the outlet 11 of the raw material cell fluid communicates with the cell capture filter 20 Only the cell fluid return port 12 communicates. Cells are connected from the source cell fluid outlet 11 so that blood flows from the patient's veins, and further provided with means for introducing heparin as an anticoagulant, blood is introduced into the cell capture filter 20, and cells containing mononuclear cells Is trapped in the cell trapping material laminate, and the processed blood is returned to the patient through the return port 12 of the raw cell fluid.

  After processing a predetermined amount of the raw material cell liquid, the syringe 14 serving as the collection liquid introduction means containing the collection liquid is communicated only with the cell capture filter 20 through the branch section 13, and the branch section 15 is connected to the cell capture filter 20 and the cell collection. After only the blood bag 16 serving as the means is communicated, when the plunger of the syringe 14 is pushed with hand, the cells captured by the cell capturing filter 20 are collected in the blood bag 16.

  In order to introduce an operation of replacing the remaining blood inside the cell capture filter with the collected liquid prior to cell collection with the collected liquid, that is, a washing step, it is preferable to perform as follows. In FIG. 3 a, a washing solution is introduced in advance into the infusion bag 16 serving as a cell collection means, and after the cells containing mononuclear cells are captured by blood treatment in the cell trapping filter 20, the washing solution is passed through the branch portion 15. The remaining raw material cell fluid is allowed to permeate through the filter unit 20 and is discharged from the cell capture filter. Thereafter, only the cell capture filter 20 and the syringe 14 are communicated with each other in the branching section 13 and the plunger of the syringe 14 is pushed with force by hand, and the cells captured by the cell capture filter 20 are collected in the infusion bag 16. FIG. 3 b shows an example in which a blood bag 18 as a cleaning liquid introducing means is connected via a branch portion 17. The usage is the same as above. After the treatment with the raw material cell solution, the washing solution is passed through the cell capture filter 20 from the infusion bag 18, and then the collection solution is introduced from the syringe 14, and the cells containing mononuclear cells are collected in the infusion bag 16 serving as a cell collection means. . FIG. 3c shows an example in which the syringe 14 serving as a collection liquid introduction unit is installed upstream of the cell capture filter 20, and the infusion bag 16 serving as a cell collection unit is installed downstream of the cell capture filter. In FIG. 3c, after the cells containing mononuclear cells are captured by the cell capture filter 20 by the raw material cell solution treatment, the washing solution is introduced from the infusion bag 18 serving as the washing solution introduction means, and subsequently the collected solution is introduced from the syringe 14; Cells containing mononuclear cells can be collected in the infusion bag 16 serving as a cell collection unit.

  In FIG. 3 d, a concentration filter 19 is installed between the branch portion 15 and the infusion bag 16. The concentration filter 19 has a function of removing a part of the liquid component and increasing the concentration of the cells when collecting the cells captured by the cell capturing material laminate. Further, as shown in FIG. 3e, the collected cells may be further concentrated using a concentration filter 19. At that time, the concentrated recovered liquid is recovered in the infusion bag 21.

  FIG. 4 shows a configuration in which two filters are connected in parallel as an example when a plurality of cell separation filters are connected. Similarly to the above, the raw cell fluid may be flowed through the cell capture filter and recovered from each of them may be performed at one time. However, while the raw cell fluid is flowed through one filter, the conduit connected to the other filter is Stop the filter with a clamp or the like, and let the source cell fluid flow through the other filter while collecting this filter. When the method of collecting cells while alternately using a plurality of filters, such as flowing the raw material cell solution again, it is possible to efficiently separate and collect cells. At this time, the cell recovery means may be installed for each individual filter (FIG. 4a), but as shown in FIG. 4b, each cell recovery means can be connected to a line from each filter and stored in one infusion bag. You may do it.

  FIG. 5 shows an example in which a cell trapping material is introduced into a cylindrical shape in a cell trapping filter case. The specific shape of the cylindrical shape is simply a stack of various cell trapping materials on a cylinder as shown in FIG. 5a, a pleat type as shown in FIG. 5b, or a spiral type as shown in FIG. 5c. It is possible to select as appropriate.

FIG. 6 shows a case where it is incorporated in a hemodialysis system. 22 shows a dialyzer, FIG. 6a shows a case where the dialyzer is connected in series, and FIG. 6b shows a case where it is connected in parallel. The bypass line 23 is appropriately installed as necessary.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

[Example 1]
1. Cell capture filter The cell capture filter shown in FIG. One set of cell trapping material laminates was introduced into a polycarbonate container having a container outer dimension (length × width × thickness) of 26 × 26 × 15 mm and having a liquid outlet and a liquid inlet on a diagonal line. The cell-trapping material laminate was prepared by laminating 12 polyester nonwoven fabrics with an average fiber diameter of 33 μm, 12 polyester nonwoven fabrics with an average fiber diameter of 12 μm, and 9 polyester nonwoven fabrics with an average fiber diameter of 1.7 μm in this order. The filter was filled so that a nonwoven fabric with an average fiber diameter of 33 μm came to the filter inlet side. Each nonwoven fabric had a cut size of 21 × 21 mm, a filtration cross-sectional area of 4.6 cm ² , and a fiber surface area of 0.5 m ² . The filter was coated with a hydrophilic polymer. That is, a 1% ethanol solution of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate copolymer (97: 3 molar ratio of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate) was passed from the inlet side of the filter, and then nitrogen gas was passed through. Dried.

2. Cell separation and collection system for angiogenesis therapy and operation The cell capture filter prepared in 1 above was incorporated into a circuit to constitute a system corresponding to FIG. As the raw material cell fluid, human fresh peripheral blood to which heparin 5 U / cm ³ was added as an anticoagulant was used. An infusion bag containing blood in the source cell fluid outlet 11 and an infusion bag for holding the processed blood in the source cell fluid return port 12 are connected, and further, a syringe 14 as recovery fluid introduction means and an infusion fluid as cell recovery means A bag 16 and an infusion bag 18 as a cleaning liquid introducing means were connected. The collected liquid was introduced into the syringe 14 in advance. Physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was used as the washing liquid, and dextran 40 injection (manufactured by Kobayashi Pharmaceutical Co., Ltd.) was used as the recovery liquid.

From the infusion bag containing the blood, 9.2 cm ³ of blood was introduced into the filter at a flow rate of 0.46 cm ³ / min for 20 minutes using a peristaltic pump. At this time, the number of nucleated cells contained in 9.2 cm ³ of blood was 4.7 × 10 ⁷ , and 9.4 × 10 ⁷ nucleated cells were allowed to flow per 1 m ² of the surface area of the filled nonwoven fabric. The number of mononuclear cells was 1.7 × 10 ⁷ . The blood that has passed through the cell trapping filter flows into the treated blood bag as it is, and after the filtration is completed, 5 cm ³ of the washing solution is transferred from the infusion bag 18 serving as a washing solution introducing means, and the cell is trapped at a flow rate of 0.46 cm ³ / min using a peristaltic pump. The sample was poured into the filter unit 20 and the plunger of the syringe 14 was pushed by hand. The collected liquid 9 cm ³ was introduced into the cell trapping filter, and the cells containing mononuclear cells were collected in the blood bag 16 serving as a cell collecting means. . Thereafter, using a peristaltic pump from the infusion bag containing the blood again, 9.2 cm ³ of blood is introduced into the filter at a flow rate of 0.46 cm ³ / min for 20 minutes, and 5 cm ³ of washing solution is allowed to flow, followed by the recovery operation. went. A total of 92 cm ³ of blood was processed in a total of 10 steps of this capture and recovery process. The number of cells and the content of each cell fraction were measured using a multi-item automatic blood cell counter (Microcell Counter, manufactured by Sysmex Corporation) for all the blood and collected liquid that flowed into the processed blood bag through the raw blood and the cell trapping filter. .

The recovery rate of mononuclear cells was calculated by the following formula.
Mononuclear cell recovery rate (%) = (number of mononuclear cells contained in the collected liquid obtained by one capture and recovery operation) / (number of mononuclear cells contained in the original blood flowed by one capture and recovery operation) ) × 100
The average value of the recovery rate of mononuclear cells after 10 operations was 64.0%, and the total number of recovered mononuclear cells was 1.1 × 10 ⁸ .

[Example 2]
1. Cell capture filter The cell capture filter shown in FIG. The outer dimensions of the container (length x width x thickness) are 105 x 105 x 14 mm, and a pair of cell capture material laminates is placed in a styrene / butadiene block copolymer container having a liquid outlet and a liquid inlet on a diagonal line. Introduced. The cell-trapping material laminate was prepared by laminating 4 polyester nonwoven fabrics with an average fiber diameter of 33 μm, 4 polyester nonwoven fabrics with an average fiber diameter of 12 μm, and 9 polyester nonwoven fabrics with an average fiber diameter of 1.7 μm in this order. The filter was filled so that a nonwoven fabric with an average fiber diameter of 33 μm came to the filter inlet side. The cut size of each nonwoven fabric was 96.5 × 96.5 mm, the filtration cross-sectional area was 93 cm ² , and the surface area of the fiber was 9.9 m ² . The filter was coated with a hydrophilic polymer. That is, a 1% ethanol solution of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate copolymer (97: 3 molar ratio of hydroxyethyl methacrylate and dimethylaminoethyl methacrylate) was passed from the inlet side of the filter, and then nitrogen gas was passed through. Dried.

2. Cell separation and collection system for angiogenesis therapy and operation The cell capture filter prepared in 1 above was incorporated into a circuit to constitute a system corresponding to FIG. As the raw material cell solution, bovine daily stored blood to which ACD was added as an anticoagulant so that blood: ACD = 8: 1 was used. An infusion bag containing blood in the source cell fluid outlet 11 and an infusion bag for holding the processed blood in the source cell fluid return port 12 are connected, and further, a syringe 14 as recovery fluid introduction means and an infusion fluid as cell recovery means A bag 16 and an infusion bag 18 as a cleaning liquid introducing means were connected. The collected liquid was introduced into the syringe 14 in advance. Physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was used as the washing liquid, and dextran 40 injection (manufactured by Kobayashi Pharmaceutical Co., Ltd.) was used as the recovery liquid.

From the infusion bag containing blood, 400 cm ³ of blood was introduced into the filter at a flow rate of 20 cm ³ / min for 20 minutes using a peristaltic pump. At this time, the number of nucleated cells contained in 400 cm ³ of blood was 2.4 × 10 ⁹ , and 2.4 × 10 ⁸ nucleated cells flowed per 1 m ² of the surface area of the filled nonwoven fabric. The number of mononuclear cells was 1.2 × 10 ⁹ . The blood that has passed through the cell trapping filter flows into the treated blood bag as it is, and after completion of filtration, the cell trapping filter unit is fed with 70 cm ³ of the washing solution from the infusion bag 18 serving as a washing solution introducing means at a flow rate of 20 cm ³ / min. Next, the plunger of the syringe 14 was pushed by hand vigorously and 70 cm ³ of the recovery solution was introduced into the cell trapping filter, and cells containing mononuclear cells were recovered in the blood bag 16 serving as a cell recovery means. Thereafter, using a peristaltic pump from the infusion bag containing the blood again, 400 cm ³ of blood was introduced into the filter at a flow rate of 20 cm ³ / min for 20 minutes, and a washing solution of 70 cm ³ was allowed to flow, followed by a recovery operation. A total of 4000 cm ³ of blood was processed in a total of 10 times of this capture and recovery process. The raw blood and the blood that has flowed into the treated blood bag through the cell capture filter and the collected liquid are all counted with a multi-item automatic blood cell counter (Microcell Counter, manufactured by Sysmex Corporation) using a flow cytometer (Beckman Coulter). The content of each cell fraction was measured. The calculation formula for the recovery rate of mononuclear cells is the same as in Example 1.
The average value of the recovery rate of mononuclear cells after 10 operations was 90.8%, and the total number of recovered mononuclear cells was 1.0 × 10 ¹⁰ .

[Comparative Example 1]
1. Cell capture filter Same as Example 1

2. Cell separation and collection system for angiogenesis therapy and operation The same system as in Example 1 was used, and the raw material cell fluid used was human fresh peripheral blood to which heparin 5 U / cm ³ was added as an anticoagulant.

From the infusion bag containing blood, 92 cm ³ of blood was introduced into the filter at a flow rate of 0.46 cm ³ / min for 200 minutes using a peristaltic pump. At this time, the number of nucleated cells contained in 92 cm ³ of blood was 3.5 × 10 ⁸ , and 7.0 × 10 ⁸ nucleated cells were flowed per 1 m ² of the surface area of the filled nonwoven fabric. The number of mononuclear cells was 1.7 × 10 ⁸ . The blood that has passed through the cell trapping filter flows into the treated blood bag as it is, and after the filtration is completed, 5 cm ³ of the washing solution is transferred from the infusion bag 18 serving as a washing solution introducing means, and the cell is trapped at a flow rate of 0.46 cm ³ / min using a peristaltic pump. The sample was poured into the filter unit 20 and the plunger of the syringe 14 was pushed by hand. The collected liquid 9 cm ³ was introduced into the cell trapping filter, and the cells containing mononuclear cells were collected in the blood bag 16 serving as a cell collecting means. . The number of cells and the content of each cell fraction were measured using a multi-item automatic blood cell counter (Microcell Counter, manufactured by Sysmex Corporation) for all the blood and collected liquid that flowed into the processed blood bag through the raw blood and the cell trapping filter. The calculation formula for the recovery rate of mononuclear cells is the same as in Example 1.
The mononuclear cell recovery rate was 21.3%, and the total number of recovered mononuclear cells was 3.6 × 10 ⁷ .

[Comparative Example 2]
1. Cell capture filter Same as Example 2

2. Cell separation and collection system for angiogenesis therapy and operation The same system as in Example 2 was used, and the raw material cell fluid was ACD as an anticoagulant and was added to blood: ACD = 8: 1. Was used.

From the infusion bag containing blood, 400 cm ³ of blood was introduced into the filter at a flow rate of 20 cm ³ / min for 200 minutes using a peristaltic pump. At this time, the number of nucleated cells contained in 400 cm ³ of blood was 2.4 × 10 ¹⁰ , and 2.4 × 10 ⁹ nucleated cells were flowed per 1 m ² of the surface area of the filled nonwoven fabric. The number of mononuclear cells was 1.2 × 10 ¹⁰ . The blood that has passed through the cell trapping filter flows into the treated blood bag as it is, and after completion of filtration, the cell trapping filter unit is fed with 70 cm ³ of the washing solution from the infusion bag 18 serving as a washing solution introducing means at a flow rate of 20 cm ³ / min. Next, the plunger of the syringe 14 was pushed by hand vigorously and 70 cm ³ of the recovery solution was introduced into the cell trapping filter, and cells containing mononuclear cells were recovered in the blood bag 16 serving as a cell recovery means. The raw blood and the blood that has flowed into the treated blood bag through the cell capture filter and the collected liquid are all counted with a multi-item automatic blood cell counter (Microcell Counter, manufactured by Sysmex Corporation) using a flow cytometer (Beckman Coulter). The content of each cell fraction was measured. The calculation formula for the recovery rate of mononuclear cells is the same as in Example 1.
The mononuclear cell recovery rate was 12.7%, and the total number of recovered mononuclear cells was 1.5 × 10 ⁹ .

[Comparative Example 3]
1. Cell capture filter Same as Example 2

2. Cell separation and collection system for angiogenesis therapy and operation The same system as in Example 2 was used, and the raw material cell fluid was ACD as an anticoagulant and was added to blood: ACD = 8: 1. Was used.

From the infusion bag containing the blood, 1000 cm ³ of blood was introduced into the filter at a flow rate of 5 cm ³ / min for 200 minutes using a peristaltic pump. At this time, the number of nucleated cells contained in 1000 cm ³ of blood was 5.9 × 10 ⁹ , and 6.0 × 10 ⁸ nucleated cells flowed per 1 m ² of the surface area of the filled nonwoven fabric. The number of mononuclear cells was 2.9 × 10 ⁹ . The blood that has passed through the cell trapping filter flows into the treated blood bag as it is, and after completion of filtration, the cell trapping filter unit is fed with 70 cm ³ of the cleaning solution from the infusion bag 18 serving as a means for introducing the cleaning solution at a flow rate of 5 cm ³ / min. Next, the plunger of the syringe 14 was pushed by hand vigorously and 70 cm ³ of the recovery solution was introduced into the cell trapping filter, and cells containing mononuclear cells were recovered in the blood bag 16 serving as a cell recovery means. The raw blood and the blood that has flowed into the treated blood bag through the cell capture filter and the collected liquid are all counted with a multi-item automatic blood cell counter (Microcell Counter, manufactured by Sysmex Corporation) using a flow cytometer (Beckman Coulter). The content of each cell fraction was measured. The calculation formula for the recovery rate of mononuclear cells is the same as in Example 1.
The mononuclear cell recovery rate was 35.5%, and the total number of recovered mononuclear cells was 1.1 × 10 ⁹ .

Table 1 shows the characteristics of the cell separation methods of Examples 1 and 2 and Comparative Examples 1 to 3, the mononuclear cell recovery rate, and the number of recovered mononuclear cells.

According to the present invention, it is possible to provide a simple and effective technique for processing a large amount of blood by extracorporeal circulation, adsorbing specific useful components, and efficiently recovering them. The method of the present invention is greatly characterized in that the patient burden in extracorporeal circulation is small and the patient himself can be a provider of active ingredients. The present invention is not limited to a treatment method based on angiogenesis, but has the potential to be applied to regenerative medicine using stem cell transplantation or cell therapy using transplantation between the same tumors, and is very useful industrially.

It is a figure which shows a cell capture filter structure. It is a figure which shows the front view of a cell capture filter. It is a figure which shows the circuit of a cell capture filter. It is a figure which shows the structure of a cell capture filter. It is a figure which shows the structure of a cell capture filter. It is a figure which shows the circuit of the cell capture filter integrated in the hemodialysis system.

Explanation of symbols

1 Cell capture material laminate 2 Cell capture filter inlet 3 Cell capture filter outlet 4 Cell capture filter case 5 Cell capture material A
6 Cell capture material B
7 Cell capture material C
8 Space between cell trapping material laminate 9 External space of cell trapping material laminate 10 Cell trapping material adhesion portion 11 Raw material cell liquid outlet 12 Raw material cell fluid return port 13 Branching portion 14 Collected liquid introduction means 15 Branching Portion 16 Cell recovery means 17 Branching portion 18 Rinse solution introduction means 19 Concentration filter 20 Cell capture filter 21 Cell recovery means 22 Dialysis device 23 Bypass line

Claims (19)

Cells that are effective for angiogenesis by flowing a raw material cell solution containing cells that are effective for angiogenesis through a cell capture filter that is filled with a cell capture material that captures cells that do not substantially capture red blood cells but that are effective for angiogenesis. In the method for separating and recovering angiogenic cells for angiogenesis, comprising a capture step of capturing the cell capture filter by the cell capture filter, and a recovery step of recovering the cells captured by the cell capture filter. The method for separating and recovering angiogenic therapy cells is characterized by repeatedly performing the capture and recovery operation using the same filter. The method for separating and collecting angiogenesis therapy cells according to claim 1, wherein the cells effective for angiogenesis include nucleated cells. The method for separating and collecting cells for angiogenesis therapy according to claim 1 or 2, wherein the cells effective for angiogenesis include mononuclear cells. The total number of cells effective for angiogenesis in the raw material cell fluid to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is recovered. The method for separating and collecting angiogenic therapy cells according to any one of claims 1 to 3, wherein the rate is set to be 50% or more. The total number of cells effective for angiogenesis in the raw material cell fluid flowing per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is 1 × 10 ⁷ to 1 × 10 ^9. The method for separating and collecting cells for angiogenesis therapy according to any one of claims 1 to 4, wherein the cells are individual. The contact time between the cell capture material and the raw material cell solution for one capture / collection operation including the capture step and the recovery step is set so that the recovery rate of cells effective for angiogenesis is 50% or more. The method for separating and recovering angiogenesis therapy cells according to any one of claims 1 to 5. The angiogenesis therapy according to any one of claims 1 to 6, wherein a contact time between the cell capture material and the raw material cell liquid for one capture / collection operation including the capture step and the recovery step is 5 to 50 minutes. Cell separation and recovery method. The raw material cell liquid that flows per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is set so that the recovery rate of cells effective for angiogenesis is 50% or more. The method for separating and collecting cells for angiogenesis therapy according to any one of claims 1 to 7. The blood vessel according to any one of claims 1 to 8, wherein the raw material cell fluid to be flown per 1 m ² of the surface area of the cell capture material per capture / collection operation including the capture step and the recovery step is 10 to 60 cm ^3. A method for separating and collecting cells for neoplastic therapy. The method for separating and recovering angiogenesis therapy cells according to any one of claims 1 to 9, wherein the recovery rate of cells effective for angiogenesis is 50% or more. The method for separating and recovering angiogenic therapy cells according to any one of claims 1 to 10, wherein the total amount of mononuclear cells to be recovered is 10 ¹⁰ or more. The method for separating and collecting angiogenic therapy cells according to any one of claims 1 to 11, wherein the cell trapping filter is incorporated in series or in parallel with a part of a blood circuit of a blood extracorporeal circulation system. Angiogenesis therapy cells obtained by the method for separating and recovering angiogenesis cells according to any one of claims 1 to 12. Source cell fluid outlet, upstream conduit, cell capture filter that is filled with a cell capture material that does not substantially capture red blood cells but traps angiogenic cells, downstream conduit, source cell fluid The return ports are connected in this order, and further, a recovery liquid introduction means is provided upstream or downstream of the cell trapping filter. If the part where the recovery liquid introduction means is provided is upstream, it is downstream, and if it is downstream, A system for separating and collecting angiogenic therapy cells, characterized in that a cell recovery means is provided on the upstream side, and further, a pipeline bypassing the upstream pipeline and the downstream pipeline is connected. Source cell fluid outlet, upstream pipe branching from the middle, and upstream pipe branch filled with cell capture material that captures red blood cells but does not trap red blood cells effectively The same number of cell trapping filters, downstream pipes with the same number of branches as the cell trapping filter, and the return port of the raw material cell fluid are connected in this order, and the upstream or downstream branch of the cell trapping filter is further branched A recovery liquid introduction means connected to each pipe line is provided, and if the part where the recovery liquid introduction means is provided is upstream, the cells are taken from the individual pipes branched from the upstream side. A system for separating and collecting cells for angiogenesis therapy, wherein cell collecting means for collecting the cells is provided. 16. An isolation / recovery system for angiogenesis therapy cells, wherein the isolation / recovery system for angiogenesis therapy cells according to claim 14 or 15 is incorporated in series or in parallel with a part of a blood circuit of a blood extracorporeal circulation system. The system for separating and collecting cells for angiogenesis therapy according to claim 16, wherein the blood extracorporeal circulation system is a hemodialysis system. 18. The system for separating and collecting angiogenic cells according to any one of claims 14 to 17, further comprising a cell concentrating device connected to the cell collecting means. The system for separating and collecting angiogenesis cells according to claim 18, wherein the cell concentrator is a hollow fiber membrane type cell concentrator.

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