JP2003250820A - Method of blood vessel regeneration and method and device for cell separation and recovery - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a device for separating and recoverying endothelial progenitor cells of a blood vessel efficiently by an easy operation and in an imperfect open system, and to provide a method to regenerate blood vessels by utilizing the endothelial progenitor cells of the blood vessel. <P>SOLUTION: The method of blood vessel regeneration includes the following processes. A cell suspending liquid containing the endothelial progenitor cells of the blood vessel and admixed cells is filtered through a cell separation filter which enables the passage of at least the admixed cells and capture of the endothelial progenitor cells of the blood vessel. The endothelial progenitor cells of the blood vessel trapped in the cell separation filter are recovered by introducing a fluid into the cell separation filter. The recovered endothelial progenitor cells of the blood vessel are used for the blood vessel regeneration. The invention relates to the method and the device to separate and recover the endothelial progenitor cells of the blood vessel used for the method of the blood vessel regeneration. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and apparatus for separating and recovering vascular endothelial progenitor cells from a cell suspension containing vascular endothelial progenitor cells and foreign cells used for regenerating blood vessels and using them for vascular regeneration. Regarding More specifically, the present invention relates to the separation and recovery of vascular endothelial progenitor cells used to repair and regenerate lesions and / or defects of blood vessels, and the repair and regeneration of blood vessels using the separated and recovered cells.

[0002]

BACKGROUND ART Lifestyle-related diseases such as diabetes, hypertension, hyperlipidemia, and obesity are risk factors for vascular disorders, and eventually obstructive arteriosclerosis (ASO (Arteriosclerosis obliteran
s)), causing organ failure such as myocardial infarction and renal 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, in arteriosclerosis obliterans that causes gangrene of the lower extremities, amputation of the lower leg is often required,
The patient's QOL (Quality of life) decreases significantly (150,000 in the United States annually, 2,000 in Japan annually, Volume 56 of the latest medicine, Vol.
No. 8 1748-1754 (2001)). As methods for treating these ischemic tissues / organs, vasodilation using a catheter and surgically reconstructing blood circulation using a vein graft have been performed, but they are effective for severe patients. It cannot be a means.

In recent years, as a new therapeutic method, angiogenic therapy (therapeutic angiogenesis) is being performed. Angiogenesis therapy is broadly divided into gene therapy and cell transplantation. In gene therapy, VEGF (Vascular endothelial growth
factor) and HGF (Hepatocyte growth factor) plasmids have been reported to be injected into ischemic lesions to improve blood flow.
(Circulation, 98: 2800-2804, 1998 / Hypertension, 3
3: 1379-1384, 1999). On the other hand, in the cell transplantation method, bone marrow-derived mononuclear cells were injected into the lower leg of a patient with arteriosclerosis obliterans, and good results have been obtained (Latest Medicine Vol. 56, No. 1 1755-1764 (200).
1)). In addition, transplantation of vascular endothelial progenitor cells, which are named as attaching cells from umbilical cord blood and peripheral blood mononuclear cells, that attach to the culture dish, to blood flow was more effective than in the untreated control group. Have been reported to have been improved (The Journal of Cli
nical Investigation, 105: 1527-1536, 2000).

Generally, in the cell transplantation method, vascular endothelial precursor cells are efficiently vascularized at the ischemic site, and
In order to efficiently perform cell expansion in vitro, erythrocytes, platelets, granulocytes, etc. in blood are removed and mononuclear cells containing vascular endothelial progenitor cells are separated and concentrated. As the concentration method, the gravity centrifuge method such as Ficoll manufactured by Pharmacia is mainstream, but the work is complicated such as not shaking the interface, the processing time is long (about 2 hours), and the contamination of bacteria is open system. There are many problems such as concerns, and a cell treatment method suitable for clinical application is desired. In addition, the centrifugation method has a problem that the cell recovery rate is low, and it is necessary to collect a large amount of raw blood in order to obtain vascular endothelial progenitor cells necessary for transplantation, which imposes a heavy burden on blood donors.

On the other hand, in the field of hematology, hematopoietic stem cell transplantation, which is regeneration of hematopoietic tissue, that is, bone marrow, has already been established as a normal medical treatment, and isolation and concentration of hematopoietic stem cells used in the field are described in, for example, Japanese Patent Application Laid-Open No. The filter method, which is characterized by simple operation, is proposed in Japanese Patent Laid-Open No. 8-104643.

[0007]

The object of the present invention is to provide a method and a device for efficiently separating and collecting vascular endothelial progenitor cells in a simple and incompletely open system, and further utilizing the vascular endothelial progenitor cells. To provide a method for regenerating blood vessels.

[0008]

[Means for Solving the Problems] The inventors of the present invention have made earnest studies to solve the problems. As a result, we made a surprising discovery that it is possible to concentrate and separate vascular endothelial progenitor cells using a filter used for the concentrated separation of hematopoietic stem cells, and it is possible to revascularize by using the separated and concentrated vascular endothelial progenitor cells. I found that there is.

That is, according to the present invention, a cell suspension containing vascular endothelial progenitor cells and foreign cells is passed through a cell separation filter that passes at least foreign cells and captures vascular endothelial precursor cells, and the liquid is passed through the cell separation filter. The present invention relates to a method for regenerating blood vessels, which comprises recovering vascular endothelial progenitor cells once introduced into the cell separation filter and using the collected vascular endothelial progenitor cells for regenerating blood vessels.
In the present invention, it is preferable to include a step of passing the cell suspension through the cell separation filter and then introducing a liquid into the cell separation filter to substantially remove contaminating cells remaining in the cell separation filter. The present invention also relates to a method and device for separating and collecting vascular endothelial progenitor cells used in such a method for regenerating blood vessels.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The vascular endothelial progenitor cells referred to in the present invention are present in blood such as bone marrow, peripheral blood and cord blood, and have at least KDR on the cell surface.
A cell expressing (Kinase Domain-containing Receptor). The attaching cells referred to in the present invention are present in blood such as bone marrow, peripheral blood and cord blood, and include fibronectin, vitronectin,
Cells attached when cultured on a surface coated with an extracellular matrix such as collagen or gelatin. The majority of attaching cells have a spindle-shaped morphology. In addition, analysis of surface markers on attaching cells (Kinase Domain-c
About 30 to 70% of the cells express ontaining Receptor), and vascular endothelial progenitor cells are abundantly contained in the attaching cells.

The term "contaminant cells" as used in the present invention means cells that do not have the ability to differentiate into blood vessels, such as erythrocytes, platelets and granulocytes, that are often present in the site where vascular endothelial precursor cells are present. Granulocytes cause an inflammatory reaction even after autologous transplantation, and red blood cells significantly reduce the efficiency of inducing differentiation into blood vessels, and may cause immune side effects when mixed with non-self individuals in vivo. In the present invention, it is a cell group that is particularly desired to be removed.

In the present invention, the "cell suspension containing vascular endothelial progenitor cells and foreign cells" may be any cell suspension containing at least vascular endothelial progenitor cells, such as bone marrow fluid and umbilical cord blood (collected from umbilical cord blood vessels. (Including those collected from placental blood vessels), peripheral blood, lymph, and those that have been subjected to some treatment such as centrifugation, or cells extracted from various organs and tissues were resuspended in some liquid. Something is included. Among them, bone marrow fluid, umbilical cord blood, and G-CSF-mobilized peripheral blood are cell suspensions containing a large amount of vascular endothelial progenitor cells and are preferably used in the present invention.

"A cell separation filter that passes at least contaminating cells and captures vascular endothelial progenitor cells" in the present invention
Is a container filled with a filter medium having a liquid inlet and a liquid outlet. As the shape of the filter medium, a nonwoven fabric or a sponge-like structure has a large surface area per volume, is easy to handle, and has good flowability of a cell suspension, which is preferable in the present invention.

In the case of non-woven fabric, the fiber diameter is 1.0 μm or more 3
0 μm or less, preferably 1.0 μm or more and 20 μm
Or less, and even more preferably 1.5 μm or more and 10
μm or less. If it is less than 1.0 μm, the vascular endothelial progenitor cells may be captured tightly and may be difficult to collect. If it exceeds 30 μm, the vascular endothelial progenitor cells are more likely to pass through without being captured by the fibers. Either case is not preferable because it may lead to a reduction in the recovery rate.

When a sponge-like structure is used, the pore size is usually 2.0 μm or more and 25 μm or less, preferably 3.0 μm or more and 20 μm or less, and more preferably 4.0 μm or more and 15 μm or less. 2.0 μm
If it is less than 25 μm, the flowability is remarkably inferior, and it may be difficult to pass the liquid. If it exceeds 25 μm, the capture rate of vascular endothelial progenitor cells is lowered and the recovery rate is lowered.

As a material of the filter medium, preferable examples are those having low moldability, sterilization property and low cytotoxicity, and synthetic materials such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide and polyurethane can be used. Examples include molecules, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginates, inorganic materials such as hydroxyapatite, glass, alumina, and titania, and metals such as stainless steel, titanium, and aluminum. In order to make the cell suspension flow uniformly through the filter medium, the filter medium may be hydrophilized with, for example, a synthetic polymer containing hydroxyethyl methacrylate as a main component proposed in Japanese Patent Publication No. 6-51060.

As a material of a container having a liquid inlet and a liquid outlet, which is filled with this filter medium, preferred examples are polyethylene, polypropylene, polystyrene, which are excellent in moldability and sterilization and have low cytotoxicity. Acrylic resins, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, synthetic polymers such as vinyl chloride, hydroxyapatite, inorganic materials such as glass, alumina and titania, and metals such as stainless steel, titanium and aluminum. It is not limited.

Examples of the structure of the container include a rectangular parallelepiped, a cube, a cylinder, an elliptic cylinder, and the like, but any shape may be used. Further, the positions of the liquid inlet and the liquid outlet may be any positions as long as the liquid inlet can introduce the liquid to the uppermost layer of the filter medium, and the liquid outlet is a position where the liquid can be led out from the lowermost layer of the filter medium. Good.

The "liquid for substantially removing the contaminating cells remaining in the cell separation filter" in the present invention can be any liquid as long as it does not adversely affect the cells, but some examples will be given. Physiological saline, Dulbecco's phosphate buffer (D-PBS) and Hanks' solution (HBS)
Buffer solutions such as S) and culture media such as RPMI-1640. The direction of introduction of this liquid is considered to be the same direction as the direction of passage of the cell suspension containing vascular endothelial progenitor cells and foreign cells to the cell separation filter, or the opposite direction,
The same direction is more preferable because the trapped cells tend to leak less.

In the present invention, "substantially passing contaminated cells" means passing 60% or more of the contaminated cells in the cell suspension, "substantially capturing vascular endothelial progenitor cells". Means capturing 60% or more of vascular endothelial progenitor cells in the cell suspension, and “substantially removing contaminating cells” means that 6 of contaminating cells remaining in the cell separation filter are removed.
It means to remove 0% or more.

The "fluid for recovering vascular endothelial progenitor cells trapped by the cell separation filter" in the present invention can be any fluid as long as it does not adversely affect the cells. Saline, D-PBS
(Dulbeccolate buffer), buffer such as HBSS (Hank's solution), and medium such as RPMI-1640. These liquids may be supplemented with albumin, globulin, glucose, etc. as necessary for the purpose of cell protection, nutritional supplementation, anticoagulant protection, frost damage prevention during cryopreservation, viscosity improvement (may be effective in improving recovery rate), etc. Saccharose, trehalose, citric acid compounds, EDTA, dimethyl sulfoxide, dextran, polyvinylpyrrolidone, glycerin, chitin derivatives, hydroxyethyl starch, gelatin and the like may be added. Further, the fluid mentioned here includes not only a liquid simple substance but also a mixture of air, argon, nitrogen, and other gases that do not adversely affect cells. The fluid may be introduced in the same direction as the direction in which the cell suspension containing vascular endothelial progenitor cells and foreign cells is passed through the cell separation filter, or in the opposite direction, but the reverse direction tends to yield a higher recovery rate. It is more preferable because it is

The term "use the recovered vascular endothelial progenitor cells for the regeneration of blood vessels" in the present invention means not only that the vascular endothelial progenitor cells once captured by the cell separation filter and recovered can be transplanted into an individual, It is used to transplant to another individual or to regenerate blood vessels in vitro. Furthermore, the vascular endothelial progenitor cells obtained in the present invention may be used as they are, or if necessary, further separation and purification, culture, activation, differentiation induction,
After undergoing various treatments such as amplification, gene transfer, cryopreservation, complexation with artificial blood vessels proposed in the 34th Annual Meeting of the Japanese Society for Artificial Organs S1-1, lesions and / or defects of blood vessels of each part It is used in the treatment of and for research in basic science fields.

The red blood cells that have passed through the cell separation filter can be used as a red blood cell sample for basic science experiments and for the purpose of collecting hemoglobin, which is a raw material for artificial red blood cells. In addition, it can be transfused into a patient as blood for transfusion, and in particular, transplantation of vascular endothelial progenitor cells into an autologous individual is more preferable because it prevents anemia of the individual.

In the present invention, "collecting the attaching cells attached to the extracellular matrix" means that the cells collected by the cell separation filter are attached to the substrate coated with the extracellular matrix. It means to collect cells. More specifically, for example, the cells collected by the cell separation filter are suspended in a medium for cell culture, seeded on the surface of a substrate coated with an extracellular matrix, for example, a plastic dish for cell culture, and cultured for several days. , The erythrocytes, granulocytes, and other non-adherent cells are removed, and the attached cells are attached.
Peel with an enzyme treatment such as EDTA containing trypsin, or
This means adding PBS and incubating at 37 ° C, suspending and collecting. By collecting the attaching cells from the cell suspension containing the vascular endothelial progenitor cells thus separated by the filter, erythrocytes that inhibit vascular regeneration and granulocytes that cause an inflammatory reaction can be efficiently removed. Highly preferred for revascularization. The cell culture medium mentioned here is an attaching cell (attaching cell).
Cells) that adhere to the substrate and maintain the expression of the surface marker of vascular endothelial progenitor cells, but those containing serum components, heparin, endothelial cell growth factor, etc. are preferably used. The serum component is not particularly limited, but for example, inactivated serum, autologous serum, fetal bovine serum, etc. are used.

[0025] In the present invention, "attaching cells (attach
ing cells) on the extracellular matrix with human umbilical vein endothelial cells "means that the seeding cell concentration is 0.1-5 ×
10 ⁵ / ml, the cell ratio is attaching cells / human umbilical vein endothelial cells = 0.3 to 3.0, and the cell culture medium is co-cultured under the conditions of only 2
Since a blood vessel-like network can be confirmed in about 3 hours, it is also preferable as a method for confirming the function of vascular endothelial progenitor cells before transplantation.

The extracellular matrix referred to in the present invention refers to fibronectin, vitronectin, gelatin, collagen, laminin, etc., which are single or Ma.
A composite of collagen and laminin such as trigel (manufactured by Becton Dickinson) may be used. Also
A commercially available product in which an extracellular matrix such as BIO-COAT (manufactured by Becton Dickinson) is coated on a plastic dish may be used.

[0027]

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 A method of regenerating blood vessels in vitro by separating vascular endothelial progenitor cells from cord blood using a cell separation filter will be illustrated. 1. Cell separation / collection device Polyester nonwoven fabric with an average fiber diameter of 2.3 μm
0 g / m ² , bulkiness of about 0.3 mm) 18 sheets and average fiber diameter 1
2 μm polyester non-woven fabric (Basis weight: approx. 100 g / m ² ,
16 pieces having a bulkiness of about 0.47 mm) were piled up and cut into 35 mm square with a push-cutting cutter to obtain a filter medium. This filter medium is 41 x 41 x 18 mm in outer dimensions (length x width x thickness) of a polycarbonate container having a liquid outlet and a liquid inlet on a diagonal line, and a polyester non-woven fabric having an average fiber diameter of 2.3 μm is provided on the inlet side. The cell separation filter 8 was filled with a polyester nonwoven fabric having an average fiber diameter of 12 μm on the side. Further, for the purpose of hydrophilicizing the nonwoven fabric, a 1% ethanol solution of hydroxyethyl methacrylate-dimethylaminoethyl methacrylate copolymer (molar ratio 97: 3) was passed through the liquid outlet of the cell separation filter to obtain nitrogen gas. After purging the excess polymer solution with, the product was vacuum dried at 60 ° C. for 16 hours or more. As shown in FIG. 1, a tube 2 having a spike 1 at its tip and a T-shaped tube 4 branching to a cell recovery bag 7 was connected to the inlet side of the cell separation filter 8 as shown in FIG. Further, on the outlet side of the cell separation / collection filter 8, there is provided an adapter 10 which is usually covered with a cap and which can be connected to a syringe by removing the cap, and a tube 9 whose end is connected to a drain bag 12 is connected. The cell separation and collection device was used.

2. An injection needle connected to the tip of a tube connected to a blood bag containing 28 ml of raw blood and an anticoagulant (CPD) was punctured into the umbilical vein, and umbilical cord blood was collected by a drop. During collection, the blood bag was tilted horizontally to prevent it from coagulating, and was thoroughly mixed with the anticoagulant (CPD). Stored at room temperature until filtered and processed within 48 hours. The total blood volume including anticoagulant was 100 ml.

3. Cell separation and collection operation 1. 1. In spike 1 of the cell separation and collection device prepared in 2. A blood bag containing cord blood (hereinafter, referred to as a blood bag) was connected. The clamp 5 is closed so that only the blood bag and the cell separation filter 8 communicate with each other, the adapter 10 is capped, and only the cell separation filter 8 and the drain bag 12 communicate with each other so that the cord blood is separated from the cell separation filter 8. The cell separation filter 8 is filtered by passing through the head.
The filtrate flowing out of the drain bag 12 was collected. After injecting physiological saline into the empty blood bag, 40 ml of physiological saline is passed through the cell separation filter 8 to wash away the erythrocytes remaining in the cell separation filter 8, and this washing solution is also collected in the drain bag 12. did. Next adapter 10
30 ml of 2 ml human serum albumin (25% solution) and 19 ml air in 16 ml dextran 40
l Syringe (luer lock port) was connected, clamp 11 was closed so that only syringe and cell separation filter 8 were in communication, and clamp 3 was closed so that only cell separation filter 8 and cell collection bag 7 were in communication. . Next, the cells captured by the cell separation / collection filter 8 were collected in the collection bag 7 by manually pushing the plunger of the syringe.

4. Regeneration Method and Evaluation Results The mononuclear cells collected in the collection bag were 80% of the mononuclear cells in the cord blood. The recovered mononuclear cells 20% FBS, (manufactured by Gibco Co.) bovine pituitary extract, heparin (10 units / ml), Medium 199 in suspended at a concentration of 3 × 10 ⁷ / 10ml was added,
The seeds were seeded on a gelatin-coated 100 mm plastic dish and cultured in a 5% CO ₂ incubator at 37 ° C. The results are shown in Fig. 2. According to FIG. 2, a cell mass was generated within 48 hours of culture (FIG. 2-1), and spindle-shaped attaching cells appeared from the end of the cell mass attached to the plastic dish on the 3rd day of culture. Can be seen (FIGS. 2-2 and 2-3). 70% or more of these attaching cells had the lectin binding ability and the acetyl LDL uptake ability which are the characteristics of endothelial cells. Fig. 2-4 is an attaching cell before fluorescent staining, and Figs. 2-5 and 2-6 are fluorescent micrographs after fluorescent staining for showing lectin binding ability and acetyl LDL uptake ability. is there. Moreover, from these RT-PCR analysis results, these attaching cells (attaching cells) were characterized by KDR, CD31, ecNOS, and AC13, which are characteristic of endothelial cells.
3, endothelin-1, LOX-1, and GAPDH were expressed (Fig. 2
-7). FIG. 3 illustrates the micrographs shown in FIGS. 2-1 to 2-6 in an easy-to-understand manner. 3-1 to 3-6 correspond to FIGS. 2-1 to 2-6, respectively. Attaching cells obtained on the 7th day of culturing were 10% of the seeded mononuclear cells, which were KD by flow cytometry.
From the analysis of R-positive cells, vascular endothelial progenitor cells were 6% of the mononuclear cells of the raw blood. Attaching cells that had been cultivated for 7 days were incubated with 1 mM PBS containing EDTA at 37 ° C for 1
After incubating for 5 minutes to suspend and further fluorescently label with PKH2-GL, the total cell concentration is 1 × 10 ⁵ cells / ml with PKH26-labeled HUVEC (human umbilical vein endothelial cells) at a 1: 1 ratio. As described above, the cell culture medium was prepared. Matrigel (Becton Dickinson)
2 ml was plated on a 35 mm plastic dish coated with and co-cultured in a 5% CO ₂ incubator at 37 ° C. 2-3
When observed with a fluorescence microscope after a lapse of time, red HUVEC
A blood vessel-like network structure in which the green and green attaching cells mixed together appeared, and the differentiation into blood vessels was confirmed (Fig. 4). The analysis used in Example 1 is The Jo
urnal of Clinical Investigation, 105,1527-1536 (200
It was carried out by the method disclosed in 0).

[0031]

Example 2 A method for regenerating blood vessels in vivo by separating vascular endothelial progenitor cells from cord blood using a cell separation filter will be illustrated. 1. Cell separation / collection device Same as in Example 1. 2. Raw blood Same as in Example 1. 3. Cell separation and collection procedure Same as in Example 1. 4. Regeneration Method and Evaluation Results The left hindpaw of nude rats was placed in an ischemic state, and 3 × 10 ⁵ attaching cells (attaching cells) separated in the same manner as in Example 1 were transplanted to the ischemic region. As a result of examining the blood flow with a laser Doppler blood flow meter (LDI; made by Moor Instruments) that detects the flow velocity after transplantation, recovery of the blood flow was observed on the 7th day, and an ischemic state was established on the 21st day. Blood flow equivalent to that of the right hind paw, which was not present, could be confirmed (Fig. 5).

[0032]

Comparative Example 1 A method of regenerating blood vessels in vitro by separating vascular endothelial progenitor cells from cord blood by a gravity centrifuge method will be exemplified. 1. Raw blood Same as in Example 1. 2. Cell Separation and Collection Operation 5 ml of Histopaque (manufactured by Sigma) was injected into a 15 ml conical tube, and 3 ml of cord blood diluted with phosphate buffer to 6 ml was layered on the Histopaque without shaking the interface. After centrifugation at 475 xg for 45 minutes at room temperature, the mononuclear cell layer was collected. All 100 ml cord blood was processed this way. 3. Evaluation result 2. The mononuclear cells obtained in 1. were collected and washed twice with phosphate buffer. The recovery rate of mononuclear cells was 25% of the raw blood. Collected mononuclear cells were treated with 20% FBS, bovine pituitary extract (Gi
Medico with bco) and heparin (10 unit / ml) added
m199 in suspended at a concentration of 3 × 10 ⁷ / 10ml, seeded in 100mm plastic dish coated with gelatin, 5 of 37 ° C.
Cultured in a% CO ₂ incubator. Cell clusters were generated within 48 hours of culture, and spindle-shaped attaching cells appeared from the end of the cell clusters attached to the plastic dish on day 3 of culture. Attaching cells obtained on day 7 of culture were 1 of seeded mononuclear cells.
At 0%, analysis of KDR-positive cells by flow cytometry revealed that vascular endothelial progenitor cells were 1% of source blood mononuclear cells. The analysis used in Comparative Example 1 is based on The Journal of Cli.
nical Investigation, 105, 1527-1536 (2000).

[0033]

As described above, according to the present invention, vascular endothelial progenitor cells can be separated and collected by a simple and short-time operation and in an incompletely open system, and efficient blood vessel regeneration is possible. Therefore, the present invention is extremely important to contribute to the development of routine medical procedures by breaking away from conventional laboratory-level experimental medical care. In addition, since extremely rare vascular endothelial progenitor cells can be collected at a high rate, the amount of blood collected can be reduced, which greatly contributes to reducing the burden on blood donors. Furthermore, the function of vascular endothelial progenitor cells collected before transplantation can be confirmed in a short time, which greatly contributes to the improvement of transplantation results.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a cell separation / collection device used in Example 1.

FIG. 2 Attaching cells (attach) obtained in Example 1
ing cells) optical microscope photograph, fluorescence microscope photograph and RT-PC
The R analysis diagram is shown.

FIG. 3 Attaching cells (attach) obtained in Example 1
ing cell) and an optical microscope photograph and a fluorescence microscope photograph of the cells.

FIG. 4 is a fluorescence micrograph of blood vessels regenerated in vitro in Example 1.

FIG. 5 is a diagram showing the recovery of blood flow in the ischemic region of a nude rat over time measured by a laser Doppler analyzer in Example 2.

[Explanation of symbols]

1 spike 2 tubes 3 clamps 4 T-tube 5 clamps 6 tubes 7 collection bags 8 Cell separation filter 9 tubes 10 Adapter 11 clamps 12 drain bag

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shuji Terashima             2111-2 Ozai, Oita-shi, Oita Asahi Medica             Le Co., Ltd. (72) Inventor Mikitomo Yasutake             2111-2 Ozai, Oita-shi, Oita Asahi Medica             Le Co., Ltd. F-term (reference) 4B065 AA90 BD18 CA44                 4C081 AB13 CD29 CD34 DA03 EA02                 4C097 AA15 BB01 DD15 MM04 MM05

Claims (7)

[Claims] 1. A cell suspension containing vascular endothelial progenitor cells and foreign cells is passed through a cell separation filter that substantially passes at least foreign cells and substantially captures the vascular endothelial precursor cells. A method for regenerating blood vessels, which comprises introducing a fluid into a separation filter to recover vascular endothelial progenitor cells captured by the cell separation filter, and using the recovered vascular endothelial progenitor cells for regenerating blood vessels. 2. The method further comprising, after passing the cell suspension through a cell separation filter, further introducing a liquid into the cell separation filter to substantially remove contaminating cells remaining in the cell separation filter. The method for regenerating blood vessels according to. 3. A cell suspension containing vascular endothelial progenitor cells and foreign cells, which is cord blood, bone marrow, or G-CSF (Granulocyte-Colony Sti).
Mulator Factor) Mobilization Peripheral blood either.
Or the method for regenerating a blood vessel according to 2. 4. The method further comprising the step of further culturing the vascular endothelial progenitor cells recovered from the cell separation filter on an extracellular matrix to recover the attaching cells attached to the extracellular matrix. ~ 3
The method for regenerating a blood vessel according to any one of 1. 5. The method for regenerating blood vessels according to claim 4, further comprising co-culturing the attaching cells with human umbilical vein endothelial cells on an extracellular matrix. 6. A cell suspension containing vascular endothelial progenitor cells and foreign cells is passed through a cell separation filter that substantially passes at least foreign cells and substantially captures the vascular endothelial precursor cells. A method for separating and collecting vascular endothelial progenitor cells, which comprises introducing a fluid into a separation filter to recover the vascular endothelial progenitor cells captured by the cell separation filter. 7. A cell separation filter having at least an inlet and an outlet, a cell suspension injection means and a vascular endothelial progenitor cell recovery means upstream from the inlet of the cell separation filter,
A device for separating and recovering vascular endothelial progenitor cells, comprising a means for injecting a fluid for recovering vascular endothelial progenitor cells from a cell separation filter downstream of the outlet of the cell separation filter and a means for recovering contaminant cells.