JP2006050976A - Method for recovering cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently recovering cells which are effective for regenerating the diseased part of an ischemic disease patient and has a high neovascularization-assisting function, and additionally to provide a method for using the recovered cells to regenerate an ischemic disease part. <P>SOLUTION: This method for recovering the cells, sequentially including a process for making a cell-floated liquid to pass through a filter prepared by charging a polymer porous material for catching nucleated cells in a container, and a process for releasing and recovering the cells caught in the polymer porous material, is characterized in that the cell-catching time in the filter from a cell-floated liquid passage-starting time in the filter to a cell release-starting time is controlled to 2 to 12 hours. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for separating and recovering useful cells such as vascular endothelial progenitor cells and vascular endothelial growth factor-producing cells effective for regeneration treatment of ischemic disease sites from cell suspensions. Furthermore, the present invention relates to a cell-containing solution that can be collected by the above-described method and can be used to repair and regenerate lesion sites and / or defects of ischemic diseases such as obstructive arteriosclerosis and Buerger's disease.

  Lifestyle-related diseases such as diabetes, hypertension, hyperlipidemia, and obesity are risk factors for vascular disorders. Eventually, arteriosclerosis obliterans (ASO), myocardial infarction, renal failure, etc. Causes organ failure. The number of patients with these lifestyle-related diseases is extremely large, and it is no exaggeration to say that vascular treatment is the most important issue in modern medicine.

  For example, obstructive arteriosclerosis, which causes limb gangrene, is often forced to have lower leg amputations, which is said to be 150,000 per year in the United States and 2,000 per year in Japan, and the patient's quality of life (QOL) decreases significantly. (Non-Patent Document 1). As treatment methods for these ischemic tissues and organs, vascular dilatation using catheters and surgical reconstruction of blood circulation using vein grafts have been performed, but this is effective for severe patients. It cannot be a means.

  In recent years, therapeutic angiogenesis is being performed as a new treatment. There are two main types of angiogenesis: gene therapy and cell transplantation. In gene therapy, VEGF (Vascular endothelial growth factor) or HGF (Hepatocyte growth factor) plasmids are injected into ischemic lesions to improve blood flow (Non-patent Documents 2 and 3). On the other hand, in the cell transplantation method, bone marrow-derived mononuclear cells are injected into the lower leg of patients with obstructive arteriosclerosis, and good results have been obtained (Non-patent Document 4). In addition, transplantation of vascular endothelial progenitor cells, which are named attaching cells (attaching cells) attached to culture dishes from mononuclear cells of umbilical cord blood and peripheral blood, to blood flow more effectively than the untreated control group. There is a report that improvement of the above has been possible (Non-patent Document 5).

  In the mononuclear cells to be transplanted in these cell transplantation methods, focusing on monocytes, which are one of the subgroups, assisting in angiogenesis by inducing differentiation into vascular endothelium or by cell growth factor protein released from cells. Various studies have also been made on the ability (Non-Patent Documents 6 and 7).

  In general, in cell transplantation, vascular endothelial progenitor cells efficiently form blood vessels at ischemic sites, and in order to efficiently perform in vitro cell amplification, red blood cells, platelets and granules in blood. The mononuclear cells containing vascular endothelial progenitor cells are separated and concentrated by removing spheres and the like. As a concentration method, the specific gravity centrifugation method such as Ficoll made by Pharmacia is the mainstream, but the work is complicated such as not shaking the interface, the treatment time is long (about 2 hours), and the open system is free of germs. There are many problems such as concern about contamination, and a cell treatment method suitable for clinical application is desired. Further, the centrifugation method has a low cell recovery rate, and it is necessary to collect a large amount of raw material blood in order to obtain vascular endothelial progenitor cells necessary for transplantation, and there is a problem that the burden on the blood donor is large.

  On the other hand, in the field of hematology, hematopoietic stem cell transplantation, which is the regeneration of hematopoietic tissue, ie, bone marrow, has already been established as normal medicine. For example, Patent Document 1 proposes the isolation and concentration of hematopoietic stem cells used in this field. The filter method characterized by simple operation is used. Further, Patent Document 2 discloses a method for concentrating and separating vascular endothelial progenitor cells using this hematopoietic stem cell concentration and separation filter method, and that revascularization is possible using the separated and concentrated vascular endothelial progenitor cells. ing.

JP-A-8-104643 JP 2003-250820 A Latest Medicine Vol.56, No.8 1748-1754, 2001 Circulation, 98: 2800-2804, 1998 Hypertension, 33: 1379-1384, 1999 Latest Medicine Volume 56, No.8 1755-1764, 2001 The Journal of Clinical Investigation, 105: 1527-1536, 2000 PNAS, vol.100 No.5: 2426-2431, 2003 Cardiovasc Research, 49 (3): 671-680, 2001

  An object of the present invention is to provide a method for efficiently recovering cells that are effective for regeneration of affected areas of patients with ischemic disease and have high angiogenesis assisting function, and further using a cell-containing solution obtained by the above method The object is to provide a method of regenerating an ischemic disease site.

  The present inventors have intensively studied to solve the above problems. As a result, in the cell collection method for collecting the captured cells after passing the cell suspension through a filter filled with the polymer porous material to capture the cells in the polymer porous material, By setting the trapping time of the cells in the filter from the start of passage of the liquid to the filter to the start of cell detachment to be 2 hours or more, contrary to conventional common sense, nucleated cells, particularly mononuclear cells such as single cells. It was found that the recovery rate of spheres was significantly increased as compared with the conventional method, and that the amount of vascular endothelial growth factor (VEGF) obtained from the recovered mononuclear cells was significantly increased. The present invention has been completed based on these findings.

  That is, according to the present invention, the step of passing the cell suspension through a filter filled with a polymer porous body that captures nucleated cells in the container, and the cells captured by the polymer porous body are detached and collected. In the cell collection method including the steps in order, the cell capture time of the trapped cells from the start of passage of the cell suspension to the filter to the start of cell detachment is 2 hours or more and 12 hours or less, A method is provided.

Preferably, the cell passage time from the start of passage of the cell suspension to the filter to the end of passage of the cell suspension is from 30 minutes to 12 hours.
Preferably, the cell collection method of the present invention includes a step of washing the filter between the step of passing the cell suspension and the step of peeling and collecting the captured cells.

Preferably, the washing liquid replacement time from the filter cleaning start time to the cell peeling start time is 1 hour or more.
Preferably, the cells to be recovered are mononuclear cells, more preferably monocytes.

According to another aspect of the present invention, a cell-containing solution obtained by the above-described cell recovery method of the present invention is provided.
Preferably, the VEGF concentration of the cell-containing solution of the present invention is 100 pg / mL or more.
Preferably, the VEGF producing ability of the cell-containing solution of the present invention is 60 pg / 10 ⁶ cells or more.

Preferably, the cell-containing solution of the present invention can be used for the treatment of ischemic diseases.
Preferably, the ischemic disease is obstructive arteriosclerosis or Buerger's disease.

According to still another aspect of the present invention, there is provided a therapeutic agent for ischemic disease comprising the above-described cell-containing solution of the present invention.
According to still another aspect of the present invention, there is provided a method for treating ischemic disease in which the above-mentioned therapeutic agent for ischemic disease of the present invention is transplanted to an affected area of an ischemic disease patient.

  According to the present invention, in a filter method capable of recovering cells by a simple operation, a cell-containing solution having a higher angiogenesis ability, that is, a mononuclear cell, particularly a cell-containing solution with an increased ratio of monocytes is obtained in high yield It is possible to obtain in Moreover, under the specific conditions of the present invention, it is also possible to obtain a cell-containing solution having a high cell ratio that is particularly excellent in the ability to produce cell growth factors having a high ability to assist angiogenesis. Therefore, according to the present invention, the amount of collection of cell sources such as blood or bone marrow fluid necessary for obtaining angiogenesis necessary for cell transplantation treatment for ischemic diseases can be reduced, and the burden during blood supply is increased. Can be reduced. In addition, since a cell concentrate having higher angiogenic performance can be obtained from a limited amount of cell sources, highly effective treatment can be performed.

Hereinafter, the present invention will be described in detail.
The cell recovery method of the present invention includes a step of passing a cell suspension through a filter filled with a polymer porous body that captures nucleated cells, and detaching and collecting the cells captured by the polymer porous body. In the cell recovery method including the step of performing in order, the trapping time of trapped cells in the filter from the start of passage of the cell suspension to the filter to the start of cell detachment is 2 hours or more and 12 hours or less It is.

  The cell recovered in the present invention refers to a cell effective for repairing and regenerating an ischemic disease site and / or defect. Any cell can be used as long as it is effective for this purpose, but it is preferably a mononuclear cell, more preferably a monocyte.

  The cell suspension referred to in the present invention refers to blood such as bone marrow, peripheral blood or umbilical cord blood, and cells collected from these blood by known cell separation methods such as centrifugation and density gradient centrifugation (eg, Ficoll). It may be made.

  The “filter in which a container is filled with a polymer porous body that captures nucleated cells” as used in the present invention is preferably a filter medium made of a polymer porous body in a container having a liquid inlet and a liquid outlet. Filled. The shape of the polymer porous body that is a filter medium is preferable in the present invention because a nonwoven fabric and a sponge-like structure have a large surface area per volume, easy handling, and good flowability of the cell suspension.

  In the case of a nonwoven fabric, the average fiber diameter is suitably 1.0 μm or more and 30 μm or less, preferably 1.0 μm or more and 20 μm or less, and more preferably 1.5 μm or more and 10 μm or less. If it is less than 1.0 μm, the cells are firmly captured and may be difficult to recover. On the other hand, if it exceeds 30 μm, the possibility that the cells pass through without being captured by the fibers increases. In either case, the recovery rate may be reduced, which is not preferable.

  In the case of using a sponge-like structure, the pore diameter is usually 2.0 μm or more and 25 μm or less, preferably 3.0 μm or more and 20 μm or less, more preferably 4.0 μm or more and 15 μm or less. If it is less than 2.0 μm, the flowability is remarkably inferior, and there is a possibility that the liquid permeation itself may be difficult. If it exceeds 25 μm, the cell capture rate decreases and the recovery rate decreases, which is not preferable.

  As the material of the filter medium made of the polymer porous body, any of a synthetic polymer porous body, a semi-synthetic polymer porous body, a natural polymer porous body, and an inorganic porous body can be used. Examples that are preferable in terms of low moldability, sterility, and cytotoxicity include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane, agarose, cellulose, and acetic acid. Examples thereof include natural polymers such as cellulose, chitin, chitosan, and alginate. Further, the filter medium may be hydrophilized with a synthetic polymer mainly composed of hydroxyethyl methacrylate proposed in Japanese Patent Publication No. 6-50060 so that the cell suspension can flow uniformly through the filter medium.

  Depending on the use conditions and the purpose of use, it is possible to select a polymer porous body that captures nucleated cells with an appropriately modified pore inner surface. 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 method of surface modification may be by constructing hydrophilic surfaces in this way, by constructing an appropriate distribution of hydrophilicity and hydrophobicity, and by being charged to these surfaces. A known method such as imparting 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. 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 having a liquid inlet and a liquid outlet filled with the filter medium are excellent in moldability and sterilization property and low in cytotoxicity. For example, polyethylene, polypropylene, polystyrene, acrylic resin, Examples include, but are not limited to, synthetic polymers such as nylon, polyester, polycarbonate, polyacrylamide, polyurethane, and vinyl chloride, inorganic materials such as hydroxyapatite, glass, alumina, and titania, and metals such as stainless steel, titanium, and aluminum. It is not a thing.

  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 cell suspension inlet and the outlet of the cell suspension may be any position where the liquid can be introduced into the uppermost layer of the filter medium, and the cell suspension outlet is the lowermost layer of the filter medium. Any position can be used as long as the liquid can be derived from the liquid.

  The “passing the cell suspension through the filter” as used in the present invention may be any method as long as the cell suspension is introduced and led out from the filter. For example, a continuous extracorporeal circulation method for cell donors may be used, or a method of treating a cell suspension previously collected by gravity drop may be used, or collected in a syringe or other injector. A method of introducing the cell suspension liquid into the filter manually or using an apparatus (for example, a syringe pump, a blood pump, a perista pump, etc.) may be used.

  The cell passage time from the start of passage of the cell suspension to the filter to the end of passage of the cell suspension is preferably 12 hours or less, preferably 10 hours or less, more preferably 8 hours or less. Exceeding 12 hours is not preferable because the cell itself is left in an in vitro environment for too long, and the viability of the cells decreases. Although the minimum of cell passage time is not specifically limited, For example, it can be 30 minutes or more.

  In the present invention, “removing and collecting the cells trapped in the polymer porous body” means, for example, taking out the polymer porous body from a filter through which a cell suspension is passed, In order to obtain ease of implementation and a high cell recovery rate, a cell recovery fluid is introduced into the filter from the cell suspension introduction port in the container, and a filter medium is used. Use the method of separating and extruding the trapped cells by the fluid to be introduced and recovering from the cell suspension outlet in the container, or introducing the cell recovery fluid from the cell suspension outlet in the container to the filter It is preferable to use a method in which the cells trapped in the filter medium are peeled and extruded by a fluid to be introduced and recovered from the cell suspension introduction port in the container. The latter method is more preferable from the viewpoint of cell recovery rate.

  When the fluid for recovering cells is a liquid, any fluid can be used as long as it does not adversely affect the cells. For example, physiological saline, D-PBS (Dulbeccolate buffer), Examples thereof include a buffer solution such as HBSS (Hank's solution) and a medium such as RPMI-1640. To these liquids, albumin, globulin, glucose, as needed for purposes such as cell protection, nutritional supplementation, anticoagulation, prevention of freezing damage during cryopreservation, viscosity improvement (may be effective in improving recovery) Saccharose, trehalose, citrate compounds, EDTA, dimethyl sulfoxide, dextran, polyvinyl pyrrolidone, glycerin, chitin derivatives, hydroxyethyl starch, gelatin and the like may be added. The fluid referred to here includes not only a single liquid but also a gas that does not adversely affect cells, such as air, argon, nitrogen, or a mixture of these gases with the liquid. The direction of fluid introduction may be the same direction or the reverse direction of the cell suspension liquid passing through the cell separation filter, but the reverse direction is more preferable because a higher recovery rate tends to be obtained.

  According to the present invention, the “capture time of trapped cells in the filter from the start of passage of the cell suspension to the filter to the start of cell detachment” refers to filling the container with a polymer porous body that captures the cells. In the so-called “filtration” in which the cell suspension is passed through the filter, the cells are trapped in the porous body from the point when the trapping of the cells in the cell suspension is substantially started in the polymer porous body. Until the time when the detachment of the cells is substantially started. The “capture time of trapped cells from the start of passage of the cell suspension to the filter to the start of cell detachment” is 2 hours to 12 hours, preferably 2 hours to 10 hours, more preferably Is 3 hours or more and 10 hours or less, more preferably 3 hours or more and 8 hours or less. If it is less than 2 hours, the cell surface change due to the interaction between the filter medium surface and the captured cells cannot be sufficiently obtained, and a sufficient increase in monocyte recovery rate cannot be obtained, which is not preferable. On the other hand, if it exceeds 12 hours, the cell itself is left in an in vitro environment for too long, and the viability of the cells decreases, which is not preferable.

  As a method for supplying the cell suspension to the filter, that is, for supplying the cell suspension, (a) continuously supplying the polymer porous body over the capture time in the filter, (b) within the capture time in the filter To the polymer porous body intermittently, (c) to stop the supply completely after the supply, or a combination of (b) and (c) can be considered. Good. For example, when the cell suspension is an extracorporeal circulation method of peripheral blood, the method (a) can be continuously processed by incorporating this filter into the main circuit of extracorporeal circulation or by providing a bypass circuit. Yes, the methods (b) and (c) can be intermittently processed or completely stopped by providing and incorporating a bypass circuit. The same applies to batch processing that is not extracorporeal circulation, and it may be properly used depending on processing conditions such as processing amount and processing speed or the purpose of enforcement.

  In the above detachment and collection, if cells that are not necessary for treatment such as red blood cells are mixed in the cell suspension, the cell suspension is passed through the filter to remove this in advance, and then "collection" is performed. It is preferable to perform “cleaning” before performing. As the fluid used for “washing”, the same fluid as that used for the separation and recovery may be used, or any fluid may be used as long as it does not adversely affect the cells even if it is different from the fluid. Means for allowing the cleaning liquid to pass through the filter include devices using devices such as a syringe pump, a blood pump, a peristaltic pump, a method of pushing the syringe by hand as a simple method, and a method of inducing a liquid flow by pressing a bag storing liquid And drop processing.

  When “washing” is performed, the washing solution replacement time from the start of “washing” to the start of cell detachment is not particularly limited, and the trapping time of the trapped cells in the filter is not less than 2 hours and not more than 12 hours. The time can be set as appropriate, but is preferably 1 hour or longer, more preferably 2 hours or longer. As a result, substances that promote the aggregation of cells that are produced before or during filtration and cells that release such substances can be removed by washing, so that useful cells trapped on the surface of the filter medium can be detached. It is possible to increase the ability to produce cell growth factors by changing to an easy state or by activating trapped cells.

The cell-containing liquid obtained by the above-described cell recovery method of the present invention is also within the scope of the present invention. In the cell-containing solution of the present invention, cells that are effective for regenerative treatment of ischemic disease sites such as monocytes, particularly monocytes, are collected. The cell-containing solution of the present invention preferably has a VEGF concentration of 100 pg / mL or more, or preferably has a VEGF production ability of 60 pg / 10 ⁶ cells or more. By having such a high VEGF concentration or VEGF production ability, the cell-containing solution of the present invention can be used for the treatment of ischemic diseases.

  The ischemic disease referred to in the present invention refers to a disease in which sufficient blood supply is not maintained due to arteriosclerosis of blood vessels, and organs are damaged. Specific examples of ischemic diseases include obstructive arteriosclerosis, Buerger's disease (a disease that is not an arteriosclerotic disease but caused by insufficient blood flow), angina pectoris, myocardial infarction, and the like. The cell-containing solution of the present invention can be used particularly for the treatment of obstructive arteriosclerosis or Buerger's disease. Moreover, as obstructive arteriosclerosis, obstructive arteriosclerosis derived from diabetes is mentioned as an example, and examples thereof include coronary artery occlusion or leg occlusive arteriosclerosis.

When transplanting the cell-containing solution of the present invention to an affected area of an ischemic disease to treat the ischemic disease, the number of cells to be transplanted into the affected area is not particularly limited as long as the therapeutic effect can be exerted. The number of cells in the sphere is 1 × 10 ⁶ to 1 × 10 ¹² cells, preferably 1 × 10 ⁷ to 1 × 10 ¹² cells, more preferably about 1 × 10 ⁸ to 1 × 10 ¹¹ cells per time. The administration frequency of the cell-containing solution is not particularly limited, and an appropriate number of cells may be administered once every several months to several years, or may be regularly administered at a higher frequency.

  The cell-containing solution of the present invention can be transfused directly or after some treatment, to the above-mentioned disease site. 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, the patient who receives the treatment by infusion may be different from the person who provides cells effective for angiogenesis in the blood, or the same person. In the case of the same person, rejection associated with transplantation can be basically avoided.

  For example, when the cell suspension is peripheral blood, an extracorporeal circulation method in which the peripheral blood that has passed through the filter is returned to the blood donor can be employed. In this case, it is preferable to provide a dedicated flow path and a cleaning liquid recovery container so that the cleaning fluid does not mix with the peripheral blood to be returned. In addition, when extracorporeal circulation is performed on patients with ischemic disease themselves, autologous transplantation treatment using self cells can also be performed.

  The anticoagulant in the case of extracorporeal circulation can be 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.

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

Example 1
Three polypropylene non-woven fabrics with a mean fiber diameter of 1.9 μm cut into a circular shape with a diameter of 6.8 mm (approx. 66 g / m ² , bulk height of about 0.6 mm) were laminated to obtain a filter medium. The filter medium was packed in a Mobilol column (Mo Bi Tec, A5310) to obtain a cell trapping filter. Connect a blood circuit for cell suspension introduction to one of the entrance / exit nozzles of this cell capture filter, and connect a blood circuit to lead the filtered cell suspension to the other nozzle. A filter system was used.

  Peripheral blood was collected from a healthy donor, and 5000 units of heparin per liter of blood was added as an anticoagulant to obtain heparinized whole blood. As a result of measuring the number of cells in heparinized whole blood using a microcell counter (Sysmex Corporation), the leukocyte concentration was 3,890 cells / μL, the mononuclear cell concentration was 1,470 cells / μL, and the monocyte concentration was The number was 350 / μL.

  2 mL of this heparinized whole blood was collected in a plastic syringe and connected to the blood circuit for introduction of the cell capture filter system. The plastic syringe was set in a syringe pump.

  The heparinized whole blood was passed through the cell capture filter at a constant flow rate of 2.67 mL / hour, and the passed blood was collected in a plastic tube installed at the end of the circuit on the outlet side of the cell capture filter system. When the plastic syringe became empty, the pump was stopped and the introduction side circuit was closed with forceps. The time when heparinized whole blood flowed into the cell trapping filter was taken as the filtration start point, and the time when the pump was stopped was taken as the filtration end point, and the filtration time was measured and designated as “treatment time A”. The processing time A in this example was 45 minutes.

  After leaving still for 90 minutes at room temperature, a plastic syringe filled with 3 mL of a recovery solution in which dextran 40 was mixed with physiological saline was connected to the end of the cell suspension discharge side circuit of the filter system. At the end of the cell suspension introduction side circuit, a collection plastic tube is installed, a plastic syringe is pushed with a pressure of 0.2 MPa, the collection solution is injected from the system cell suspension introduction side, and the collection plastic The cells detached from the filter medium in the tube were collected together with the collected liquid. The amount of the collected liquid was 3.8 mL.

  The point in time when the pressurization of the plastic syringe for recovered liquid was started by the pressurizer was taken as the peeling start point, the time from the filtration start point was measured, and this was set as “treatment time B”. In this example, the processing time B was 135 minutes.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation), the leukocyte concentration was 1,200 cells / μL, the mononuclear cell concentration was 700 cells / μL, and monocytes The concentration was 110 / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, leukocytes were 59%, mononuclear cells were 90%, and monocytes were 60%.

After measuring the number of mononuclear cells in the collected liquid finally collected using a microcell counter (Sysmex Corporation, K-4500), the number of mononuclear cells is 1.6 × 10 ⁶ , What was added to 1.0 mL of FBS 5% culture medium (CLONETICS, EBM2) was seeded on a plastic dish (IWAKI, 35 mm Fibrinectin dish) and cultured at 37 ° C. (CO ₂ concentration 5%) for 72 hours. It was. The medium after the culture was collected, and the VEGF concentration was measured by ELISA (ENDOGEN, VEGF ELISA KIT). As a result, it was 124 pg / mL.

In order to express the vascular endothelial growth effect by the cell recovery method of this example, the VEGF production amount “VEGF production ability” per unit number of mononuclear cells filtered using the following (Formula 1) was determined.
Collected solution VEGF production amount = VEGF concentration × 1.0 × {(number of recovered mononuclear cells) / (number of seeded mononuclear cells)}
VEGF production ability = (recovered solution VEGF production amount) / (number of treated mononuclear cells)
... (Formula 1)
Number of seeded mononuclear cells: 1.6 × 10 ⁶ pieces Number of recovered mononuclear cells: Amount (μL) of the collected liquid finally collected to the measured mononuclear cell concentration (number / μL) in the collected liquid The number of processed mononuclear cells multiplied by: The measured mononuclear cell concentration (cells / μL) in the heparinized whole blood multiplied by the amount (μL) used for filtration. The ability to produce VEGF in the examples was 70 pg / 10 ⁶ pieces.

Comparative Example 1
The treatment was performed in the same manner as in Example 1 except that the standing time at room temperature from the end of filtration to the peeling start point was not allowed. That is, the same heparin-added whole blood was used, and this was processed using a cell capture filter system having the same configuration, and the collected liquid was collected. The operation was performed under conditions.

  The time from the start of filtration of heparinized whole blood to the cell capture filter until the end of filtration, ie, the treatment time A was 45 minutes. Further, the time from the start of filtration to the start of peeling, that is, the processing time B was 45 minutes. Moreover, the amount of the finally obtained recovered liquid was 3.8 mL.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation, K-4500), the leukocyte concentration was 1,110 cells / μL, the mononuclear cell concentration was 620 cells / μL, The monocyte concentration was 70 / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, the leukocytes were 54%, the mononuclear cells were 80%, and the monocytes were 38%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 87 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 44pg / 10 < ⁶ > pieces.

Example 2
Cells were collected in the same manner as in Example 1 except that the heparinized whole blood to be filtered was 6 ml, and that the standing time from the end of filtration to the start of detachment was not allowed.

  The time from the start of filtration of heparinized whole blood to the cell capture filter to the end of filtration, ie, the treatment time A was 135 minutes. Further, the time from the start of filtration to the start of peeling, that is, the processing time B was 135 minutes. Moreover, the amount of the finally obtained recovered liquid was 4.0 mL.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation, K-4500), the leukocyte concentration was 2,260 / μL, and the mononuclear cell concentration was 1,120 / μL and the monocyte concentration was 130 cells / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, leukocytes were 39%, monocytes were 51%, and monocytes were 25%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 210 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 67pg / 10 < ⁶ >.

Example 3
Cells were collected in the same manner as in Example 1 except that the heparinized whole blood to be filtered was 8 ml, and that the standing time from the end of filtration to the start of peeling was not allowed.

  The time from the start of filtration of heparinized whole blood to the cell trapping filter until the end of filtration, that is, the processing time A was 180 minutes. Further, the time from the start of filtration to the start of peeling, that is, the processing time B was 180 minutes. Moreover, the amount of the finally obtained recovered liquid was 4.0 mL.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation, K-4500), the leukocyte concentration was 2,020 cells / μL, and the mononuclear cell concentration was 1,200 cells / μL. μL and the monocyte concentration was 130 cells / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, the leukocytes were 26%, the mononuclear cells were 41%, and the monocytes were 19%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 292 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 74pg / 10 < ⁶ > pieces.

Example 4
Cells were collected by the same operation as in Example 1 except that washing was performed after filtration and a 90-minute static time was allowed from the start of washing to the beginning of peeling.

Washing was performed by the following method.
After the filtration is completed, the empty plastic syringe for cell suspension is immediately replaced with a plastic syringe filled with a washing solution in which dextran 40 is mixed with physiological saline, and the forceps that has closed the introduction side circuit is removed. I removed it. The syringe pump was started and the washing solution was passed through the cell capture filter for 20 minutes at a constant flow rate of 2.67 mL / hour for washing. After the pump was stopped with the cleaning liquid filled in the filter to finish the cleaning, peeling was started in the same manner as in Example 1. The time when the plastic syringe for recovered liquid was started to be pressurized by the pressurizer was defined as the peeling start point, and the time from the cleaning start point was measured to be “processing time C”. In this example, the processing time C was 90 minutes. The time from the start of filtration of heparinized whole blood to the cell trapping filter to the end of filtration, ie, the processing time A was 45 minutes. Further, the time from the start of filtration to the start of peeling, that is, the processing time B was 135 minutes. Moreover, the amount of the finally obtained recovered liquid was 3.8 mL.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation, K-4500), the leukocyte concentration was 1,190 cells / μL, the mononuclear cell concentration was 730 cells / μL, The monocyte concentration was 110 cells / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, the leukocytes were 58%, the mononuclear cells were 94%, and the monocytes were 60%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 140 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 83pg / 10 < ⁶ > pieces.

Example 5
Cells were collected by the same operation as in Example 1, except that washing was performed after filtration and a standing time of 150 minutes was allowed from the start of washing to the beginning of peeling.

  The time from the start of filtration of heparinized whole blood to the cell capture filter until the end of filtration, ie, the treatment time A was 45 minutes. The time from the start of filtration to the start of peeling, that is, the processing time B was 195 minutes. The time from the start of cleaning to the start of peeling, that is, the processing time C was 150 minutes. Moreover, the amount of the finally obtained recovered liquid was 3.8 mL.

  As a result of measuring the number of cells in the finally collected collected liquid using a microcell counter (Sysmex Corporation, K-4500), leukocyte concentration = 1,600 cells / μL, mononuclear cell concentration was 880 cells / μL, The monocyte concentration was 130 / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, the leukocytes were 78%, the mononuclear cells were 100%, and the monocytes were 72%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 150 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 107pg / 10 < ⁶ > pieces.

Comparative Example 2
Cells were collected in the same manner as in Example 1 except that washing was performed after filtration, and a 45-minute static time was allowed from the start of washing to the beginning of peeling.

  The time from the start of filtration of heparinized whole blood to the cell trapping filter to the end of filtration, ie, the processing time A was 45 minutes. The time from the start of filtration to the start of peeling, that is, the processing time B was 90 minutes. The time from the start of cleaning to the start of peeling, that is, the processing time C was 45 minutes. Moreover, the amount of the finally obtained recovered liquid was 3.8 mL.

  As a result of measuring the number of cells in the finally collected liquid using a microcell counter (Sysmex Corporation, K-4500), the leukocyte concentration was 1,340 cells / μL, the mononuclear cell concentration was 650 cells / μL, The monocyte concentration was 70 / μL.

  Calculate the number of cells by multiplying the measured concentration of each cell by the amount of heparinized whole blood or the finally collected recovery solution, and divide the latter number by the former number. As a result, leukocytes were 65%, monocytes were 84%, and monocytes were 38%.

As a result of measuring the VEGF concentration in the same manner as in Example 1, it was 95 pg / mL. Moreover, the VEGF production ability in this example calculated | required by (Formula 1) similarly to Example 1 was 50pg / 10 < ⁶ >.

  The results of Examples 1 to 5 and Comparative Examples 1 and 2 described above are shown in Table 1 below.

According to the present invention, in a filter method capable of recovering cells by a simple operation, a cell-containing solution having a higher angiogenic ability, i.e., a cell-containing solution with an increased ratio of monocytes, particularly monocytes, is obtained at a high yield. Can be obtained at a rate. The cell-containing solution obtained by the method of the present invention can be used for cell transplantation treatment for ischemic disease, and highly effective treatment can be performed.

Claims (11)

A cell recovery method comprising sequentially passing a cell suspension through a filter filled with a polymer porous body for capturing nucleated cells, and separating and recovering the cells captured by the polymer porous body The cell recovery method according to claim 1, wherein the trapping time of the trapped cells in the filter from the start of passage of the cell suspension to the filter to the start of cell detachment is 2 hours or more and 12 hours or less. The cell recovery method according to claim 1, wherein the cell passage time from the start of passage of the cell suspension to the filter to the end of passage of the cell suspension is from 30 minutes to 12 hours. The cell collection method according to claim 1 or 2, comprising a step of washing the filter between the step of passing the cell suspension and the step of peeling and collecting the trapped cells. The cell recovery method according to claim 3, wherein the washing liquid replacement time from the filter washing start time to the cell peeling start time is 1 hour or more. The cell collection method according to any one of claims 1 to 4, wherein the cells to be collected are mononuclear cells. The cell collection method according to any one of claims 1 to 5, wherein the cells to be collected are monocytes. A cell-containing solution obtained by the cell recovery method according to any one of claims 1 to 6. The cell-containing solution according to claim 7, wherein the VEGF concentration is 100 pg / mL or more. The cell-containing solution according to claim 6 or 7, wherein the VEGF-producing ability is 60 pg / 10 ⁶ cells or more. The cell-containing solution according to any one of claims 7 to 9, which is used for treatment of ischemic disease. The cell-containing solution according to claim 10, wherein the ischemic disease is obstructive arteriosclerosis or Buerger's disease.