JP6285649B2 - Method for producing cell concentrate - Google Patents

Description

 The present invention relates to a hollow fiber module filled with a hollow fiber separation membrane and a method for producing a cell concentrate using the hollow fiber module.

 In the field of cell medicine, a method of inoculating a patient with cells collected from a living body directly or after in vitro culture is used. The cells used for these treatments are suspended in a solution suitable for the treatment, or adjusted to an appropriate concentration and transplanted. However, when cells are collected from a living body, the cell concentration may not be sufficient for treatment, and when cells collected from a living body are further cultured outside the body, they may contain tissue-derived contaminants or culture media. Many. Therefore, in order to use cells collected or cultured from a living body for treatment, impurities and culture media are removed, suspended in a solution suitable for treatment (washing), and concentrated to a cell concentration suitable for treatment. It is necessary to

  In order to achieve the above object, concentration and washing operations using centrifugation are known. For example, a method using centrifugation to separate and concentrate regenerative cells from human tissue is disclosed (Patent Document). 1). However, the method using centrifugation in this way limits the facilities that can be used due to the increase in size and cost of the apparatus. Further, when washing the cells, the supernatant of the cells precipitated by centrifugation is removed, but there is a problem that the cells are released to the atmosphere and there is a risk of contamination and the like.

Therefore, in order to solve the problems listed in Patent Document 1, a compact and simple apparatus using a hollow fiber type separation membrane has been proposed (Patent Document 2).
When concentrating a cell suspension for cell medical applications using the hollow fiber type separation membrane as described above, the cell suspension speed is increased in order to recover the cells at a high recovery rate. It is necessary to prevent adhesion to the film. However, when the flow rate of the cell suspension is increased, a strong force (share stress) is applied to the cells, so that the cells are damaged and the cell survival rate is reduced. As a result, there is a possibility of causing a reduction in the recovery rate of surviving cells (Patent Document 3).

Special Table 2007-524396 Japanese Patent No. 2928913 JP-A-10-108673

 An object of the present invention is to solve problems in the production of a cell concentrate using the hollow fiber separation membrane. Specifically, a method for producing a cell concentrate that can maintain an excellent cell recovery rate without increasing the cell flow rate when concentrating a cell suspension is provided.

 As a result of intensive studies to solve the above problems, the present inventors have used a hollow fiber module having a specific number of hollow fibers, membrane area, and membrane inner diameter for cell concentration. The inventors have found that a cell concentrate can be produced while maintaining an excellent cell recovery rate without increasing the speed, and the present invention has been completed.

That is, the gist of the present invention is as follows.
(1) having a cell suspension inlet, a filtrate outlet, a cell suspension outlet, and a hollow fiber type separation membrane disposed between the cell suspension inlet and the cell suspension outlet, the hollow fiber type separation membrane inner diameter 540～600Myuemu, a thread number is present 200-350, and, using a hollow fiber module membrane area is 0.04m ² ~0.13m ^2,
(A) a step of concentrating the cell suspension while introducing the cell suspension from the cell suspension inlet and discharging the filtrate from the filtrate outlet;
(B) introducing the cell suspension from the cell suspension inlet and collecting the cell concentrate derived from the cell suspension outlet in a collection container;
A method for producing a cell concentrate, comprising:
(2) The method for producing a cell concentrate according to (1), wherein the volume of the hollow fiber module is 57 to 152 mL.
(3) The method for producing a cell concentrate according to (1) or (2), wherein the pore diameter of the hollow fiber type separation membrane is 0.1 μm to 1 μm.
(4) The method for producing a cell concentrate according to any one of (1) to (3), wherein the material of the hollow fiber type separation membrane is polyethersulfone.
(5) Before the step (b), the method includes the step of repeating dilution and concentration using a diluent and washing the cell suspension, according to any one of (1) to (4), A method for producing a cell concentrate.
(6) The method for producing a cell concentrate according to any one of (1) to (5), wherein the collection container has one or more ports through which liquid can flow in and out.
(7) The method for producing a cell concentrate according to (6), comprising a step of taking out the cell concentrate through a port after the step (b).
(8) The method for producing a cell concentrate according to (6) or (7), comprising a step of adding a drug through a port after the step (b).
(9) The method for producing a cell concentrate according to any one of (1) to (8), comprising a step of sealing and dividing the collection container after the step (b).
(10) A cell culture method comprising culturing again a cell concentrate obtained by the method for producing a cell concentrate according to any one of (1) to (9).
(11) A cryopreservation method comprising cryopreserving a cell concentrate obtained by the method for producing a cell concentrate according to any one of (1) to (9) using liquid nitrogen.

 According to the present invention, when concentrating a cell suspension, it is possible to produce a cell concentrate while maintaining a high cell recovery rate without increasing the cell flow rate. Furthermore, since the present invention can efficiently remove contaminants such as proteins present in the suspension and can be processed in a sterile closed system, the concentrated cells are provided for therapeutic use. it can.

Hollow fiber module used in examples of the present invention

The present invention will be described below.
Although the hollow fiber module of this invention is illustrated as shown in FIG. 1, it is not limited to this. The cylindrical container 1 is composed of a straight body portion 2, head portions 3 on both sides thereof, and header portions 9, and the head portion 3 is provided with a filtrate outlet 4. The filtrate outlet may be provided on one side or on both ends. In this example, the header portion 9 is provided with a cell suspension inlet 8a and a cell suspension outlet 8b. Inside the cylindrical container 1, a bundle 5 of hollow fiber type separation membranes loaded and a bundle 5 of hollow fiber type separation membranes provided inside the header portion 9 are fixed inside the container and hollow fiber type separation is performed. The resin layer portion 7 forming the opening end 6 of the film and the structure equivalent to this provided inside the head portion 3 are provided. The resin layer portion 7 and the open end 6 have a structure covered with a header portion 9 (or head portion 3), and the cell suspension inlet / outlet ports 8a and 8b and the filtrate outlet port 4 are hollow fiber type separation membranes. It is separated by the wall material which comprises, and has a non-continuous structure.

  In the example shown in FIG. 1, each part is distinguished as a barrel 2, a head 3, and a header 9 of a cylindrical container, but this is for convenience. Even when the header portion 9 is integrated with the head portion 3 of the cylindrical container, or when the trunk portion 2 and the head portion 3 of the cylindrical container are formed of separate parts, The cell suspension outlet is continuous without being separated by the wall material constituting the hollow fiber type separation membrane, and the cell suspension outlet and the filtrate outlet are further separated by the wall material constituting the hollow fiber type separation membrane. Various structures can be designed as long as the structure is provided.

  In the hollow fiber module of the present invention, the arrangement of the hollow fiber type separation membrane may be linear, bent, or spiral, and the cell suspension inlet and the cell suspension outlet may be arranged. The shape is not particularly limited as long as both ends of the hollow fiber type separation membrane are held therebetween.

  As a material for the cylindrical container of the hollow fiber module of the present invention, acrylonitrile polymer such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymer of tetrafluoroethylene and hexafluoropropylene, polyvinyl chloride, etc. Halogenated polymer: Polyamide, polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, acrylonitrile, butadiene styrene, polystyrene, polymethylpentene and the like can be used. In particular, a material having sterilization resistance, specifically, polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, polystyrene and the like are preferable.

  As a material of the resin layer portion for fixing the hollow fiber type separation membrane, a general adhesive material such as polyurethane resin, epoxy resin, and silicon resin can be used, and polyurethane resin is particularly preferable from the viewpoint of achievement in dialysis. preferable.

  The number of hollow fiber separation membranes in the present invention represents the number of hollow fiber separation membranes contained in the hollow fiber separation membrane bundle. The number of yarns of the hollow fiber type separation membrane in the present invention is preferably 200 to 350, more preferably 250 to 300. When the number of yarns is less than 200, the concentration of cells passing through the inside of the hollow fiber type separation membrane becomes too high when the cell suspension is processed, and there is a concern that the cells are clogged with the hollow fiber type separation membrane. Further, when the number of yarns is more than 350, the total cross-sectional area of the hollow fiber type separation membrane increases, and the flow rate when the cells pass through the inside of the hollow fiber type separation membrane decreases, so that the cells become hollow fiber type. There is a problem that the recovery rate decreases due to adhesion to the separation membrane, which is not preferable.

 The inner diameter of the hollow fiber type separation membrane in the present invention represents the diameter of the hollow portion in the hollow fiber. The inner diameter of the hollow fiber type separation membrane of the present invention is preferably 540 to 600 μm, and more preferably 560 to 580 μm. When the cell suspension is less than 540 μm, the concentration of cells passing through the inside of the hollow fiber type separation membrane becomes too high when the cell suspension is processed, and there is a concern that the cells are clogged with the hollow fiber type separation membrane. On the other hand, if it exceeds 600 μm, the total cross-sectional area of the hollow fiber type separation membrane increases, so that the speed when cells pass through the inside of the hollow fiber type separation membrane decreases. As a result, the cells adhere to the hollow fiber separation membrane and the recovery rate is lowered, which is not preferable.

The membrane area (m ² ) of the hollow fiber type separation membrane in the present invention is (the inner diameter of the hollow fiber type separation membrane (μm)) × π × (number of yarns) × (filtering sandwiched between hollow fiber adhesives) The area represented by the length (cm) of the side portion is represented. Membrane area is preferably 0.04~0.13m ^2, 0.09~0.11m ² is more preferable. If the membrane area is less than 0.04 m ² , a sufficient amount of the inner membrane area for treating the cell suspension cannot be secured, and there is a concern that the membrane may become clogged during the treatment. On the other hand, when the membrane area exceeds 0.13 m ² , the cross-sectional area of the hollow fiber type separation membrane increases, so that the speed when cells pass through the inside of the hollow fiber type separation membrane decreases. As a result, the cells adhere to the hollow fiber separation membrane and the recovery rate is lowered, which is not preferable.

The volume (mL) of the hollow fiber module in the present invention is expressed by (the length of the filtration side portion (cm) sandwiched between the hollow fiber adhesives) × (the cross-sectional area (cm ² ) of the casing). Represent. The volume is preferably 57 to 152 mL, more preferably 100 to 152 mL, and even more preferably 145 to 152 mL. When the volume of the hollow fiber module is smaller than 57 mL, a sufficient membrane area cannot be ensured, and there is a concern that the cells are clogged. If the volume of the hollow fiber module is larger than 152 mL, the amount of filtrate inside the hollow fiber module increases, and the cleaning efficiency is lowered, which is not preferable. This is not preferable because the protein concentration contained in the filtrate outside the hollow fiber is difficult to decrease.

 The pore diameter of the hollow fiber type separation membrane in the present invention is the diameter of the portion that communicates the inside and the outside of the hollow fiber. The lower limit of the pore diameter of the hollow fiber type separation membrane is preferably 0.1 μm or more, more preferably 0.2 μm or more, and further preferably 0.3 μm or more. The upper limit of the pore diameter of the hollow fiber separation membrane is preferably 1 μm or less, more preferably 0.8 μm or less, and even more preferably 0.6 μm or less. If it is less than 0.1 μm, it is not preferable because a large filtration flow rate cannot be obtained or unnecessary impurities such as proteins cannot be efficiently removed. Moreover, when larger than 1 micrometer, there exists a problem that a cell leaks from a film | membrane, and is not preferable. Here, the pore diameter inside the hollow fiber type separation membrane can be measured by a bubble point method known as a general method.

  As the material of the hollow fiber type separation membrane used in the present invention, a synthetic polymer material can be preferably used from the viewpoint of the safety and stability of the material. Among these, polysulfone-based, polyolefin-based, or cellulose-based polymer materials can be preferably used. Furthermore, polyethersulfone, polyethylene, and cellulose ester can be most suitably used because of the safety, stability, and availability of the material.

 Examples of cells that can be concentrated in the present invention include artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mesenchymal stem cells, adipose-derived mesenchymal cells, adipose-derived stromal stem cells, pluripotency Multipotent biological stem cells such as adult stem cells, bone marrow stromal cells, hematopoietic stem cells, lymphocytes such as T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT cells, regulatory T cells Cells, macrophages, monocytes, dendritic cells, granulocytes, erythrocytes, platelets, neurons, somatic cells such as myocytes, fibroblasts, hepatocytes, cardiomyocytes, or treatments such as gene transfer or differentiation The cell which performed is illustrated. Among them, it can be suitably used for immune cells such as granulocytes, T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT cells, regulatory T cells, macrophages and dendritic cells. .

  The cell suspension used in the present invention is not particularly limited as long as it is a suspension containing cells. For example, fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle , Suspensions, blood and bone marrow fluid after enzymatic treatment, crushing treatment, extraction treatment, degradation treatment, sonication, etc. of biological tissues such as prostate, intestine, amniotic membrane, placenta, umbilical cord and filtration Examples thereof include cell suspensions prepared by pretreatment such as treatment, enzyme treatment, decomposition treatment, and ultrasonic treatment. Alternatively, the cells suspended after culturing the cells exemplified above in vitro using a culture solution such as DMEM, α-MEM, MEM, IMEM, RPMI-1640, and stimulating factors such as cytokines, antibodies and peptides. A turbid liquid is illustrated.

 The cell concentrate produced according to the present invention may be further washed. Washing means replacing a body fluid or a medium in which cells are suspended with a diluted solution. Thereby, unnecessary impurities in the concentrate can be removed, and it can be suitably used for transplantation to humans and animals. Examples of the diluent include physiological saline, infusion, medium, distilled water, inorganic salt, saccharide, serum, protein-containing liquid, buffer, medium, plasma, and the like. Can be suitably used.

  In addition, the cells isolated according to the present invention can be used for leukemia treatment, myocardial regeneration and vascular regeneration, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation, heart disease, immunity Regenerative medicine such as failure, coulomb disease, breast augmentation, wrinkle removal, cosmetic molding, tissue enlargement such as tissue depression, immunotherapy such as T cell therapy, NKT cell therapy, dendritic cell transfer therapy, gene transfer It can be used for gene therapy using cells, but is not limited thereto. In addition, the separated cells can be seeded on a structural material such as a scaffold and used for treatment.

 The cell concentrate produced and washed according to the present invention may be further cultured. By replacing the liquid suspended by washing and further culturing, cell number increase, cell differentiation, transformation, gene introduction, and the like can be performed. Examples of the medium used for cell growth include DMEM, α-MEM, MEM, IMEM, and RPMI-1640, and culture may be performed using stimulating factors such as cytokines, antibodies, and peptides.

 A pharmaceutical composition can be produced according to the present invention. A pharmaceutical composition can be produced by concentrating cells according to the method for producing a cell concentrate and mixing the concentrated cells with a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include anticoagulants, nutrient sources such as vitamins, antibiotics, and the like.

 The cell concentrate prepared and washed according to the present invention may be further cryopreserved. From the point that damage to cells can be reduced, it is better to cryopreserve using liquid nitrogen. In addition, cryopreserved cells can be thawed and used for transplantation into humans and animals, research, or re-culture. Since the cells produced and washed according to the present invention are less damaged by the concentration operation, they can be suitably used for cryopreservation and use after thawing.

  The hollow fiber module of the present invention may be used after sterilization. The sterilization method is not particularly limited, and sterilization methods widely used for sterilization of medical devices such as γ-ray sterilization, electron beam sterilization, EOG sterilization, and high-pressure steam sterilization can be suitably used.

  Hereinafter, although the method of isolate | separating a cell using the hollow fiber module of this invention is illustrated, this invention is not limited to this, A various change can be made in the range which is not contrary to the main point of this invention.

  The step of introducing the cell suspension from the cell suspension inlet using the hollow fiber module of the present invention and concentrating the cell suspension while discharging the filtrate from the filtrate outlet is provided in the module. The cell suspension that has flowed into the hollow fiber type separation membrane is subjected to internal pressure filtration, and the filtrate that is substantially free of cells is filtered out of the hollow fiber type separation membrane. It flows out from the cell suspension outlet. Here, the filtrate substantially free of cells means that the number of cells does not exceed 0.1% of the number of cells in the cell suspension before inflow.

  First, a tube or the like is attached to the cell suspension inlet and outlet of the hollow fiber module (becomes a part of the circuit in which the cell suspension flows into and out of the hollow fiber in the processor), and the attached tube is attached to the cell suspension. The cell suspension is connected to a container such as a cell bag containing the suspension so that the cell suspension can be circulated between the container such as the bag and the hollow fiber module. It is conceivable that a machine such as a pump is interposed in this circuit in order to circulate the liquid. Moreover, it is preferable to connect the tube connected to the waste liquid tank etc. to the filtrate outlet. At this time, it is preferable that the entire circuit is attached in an aseptic environment. At this time, it is possible to apply pressure to the separation membrane by narrowing the flow path on the outlet side of the cell suspension, or to perform filtration while applying pressure to the tube on the filtration side using a pump or the like. Various filtration methods generally used in hollow fiber separation membranes may be used in combination.

  On the other hand, after the concentration has progressed, a washing solution such as a buffer is added, and the concentration can be repeated to wash the cells and exchange the medium. At this time, the cleaning liquid is supplied from an inlet provided in the tube of the circulation circuit. At this time, it is preferable to use an inlet capable of aseptically injecting the liquid.

  The step of introducing the cell suspension from the cell suspension inlet using the hollow fiber module in the present invention and collecting the cell concentrate derived from the cell suspension outlet in the collection container includes the concentration step and washing In this step, the cell suspension is discharged from the cell suspension outlet, and the concentrated cell suspension is collected in a collection container. The collection into the collection container is preferably performed aseptically by separately providing an outlet such as a three-way stopcock on the tube of the circulation circuit. After collecting the cell suspension, the collection container can be sealed from the circuit and divided. Preferably it is possible. It is also conceivable that a machine such as a pump is interposed in this circuit in order to send the cell suspension from the circulation circuit to the collection container. Examples of the method for dividing the collection container include a method in which a tube is closed using a clamp and the upstream is cut, and a circuit is cut after heat welding using a tube sealer. Further, it is preferable that the recovery container has one or more ports through which liquid can flow in and out. At this time, the cell concentrate can be taken out through the port or a drug can be added. Ports include mixed injection ports.

  Hereinafter, the present invention will be described using experimental results. Here, the cell recovery rate is the value obtained by dividing the number of cells in the cell suspension collected in the collection bag by the number of cells in the cell suspension before processing. The higher the value, the higher the recovery efficiency. It is excellent in. The number of cells is determined by measuring the cell suspension with a blood cell counter (Sysmex, K-4500), calculating the cell concentration of the leukocyte fraction as the cell concentration in this example, and calculating from the cell suspension amount and the cell concentration. did.

  In the method of cell concentration in each experimental example, a PVC tube was connected to the inlet and outlet of the hollow fiber module described in each example. The cell suspension was stored in a plastic container and both ends of a PVC tube extending from both ends of the hollow fiber module were suspended in the suspension so that the suspension could circulate through the module and the tube. A pump was installed in the PVC tube so that the suspension flow could be set to an appropriate flow rate. A tube was also attached to the filtrate outlet of the hollow fiber module, which was set up to be poured into a waste container. The flow rate was adjusted by a pump, and filtration was performed while circulating the cell suspension in the circuit. After concentrating to a certain amount (80-120 mL), the cell suspension in the tube or hollow fiber type separation membrane is pushed out with air, then the pump is stopped and the number of cells in the cell suspension in the plastic container is measured. The recovery rate was calculated. Examples are shown below.

Example 1
200 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.07 m ² and a volume of 152 mL to form a module. The culture activated lymphocytes were concentrated at a filtration rate of 200 mL / min while flowing in the module at a rate of 450 mL / min. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 87%.

(Example 2)
250 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.09 m ² and a volume of 152 mL to form a module. The culture activated lymphocytes were concentrated at a filtration rate of 200 mL / min while flowing in the module at a rate of 450 mL / min. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 87%.

(Example 3)
300 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.11 m ² and a volume of 152 mL to form a module. The culture activated lymphocytes were concentrated at a filtration rate of 200 mL / min while flowing in the module at a rate of 450 mL / min. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 92%.

Example 4
350 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.13 m ² and a volume of 152 mL to form a module. The culture activated lymphocytes were concentrated at a filtration rate of 200 mL / min while flowing in the module at a rate of 450 mL / min. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 92%.

(Example 5)
300 hollow fibers having an inner diameter of 540 to 600 μm were put in a case having a membrane area of 0.08 m ² and a volume of 106 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluted solution, and a cell suspension was recovered by carrying out a recovery operation. The cell recovery rate was 96%.

(Example 6)
300 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.04 m ² and a volume of 57 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluent, and the collection operation was performed to collect the cell suspension. The cell recovery rate was 88%.

(Comparative Example 1)
400 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.14 m ² and a volume of 152 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 59%.

(Comparative Example 2)
Sixty hollow fibers having an inner diameter of 800 μm were put in a case having a membrane area of 0.013 m ² and a volume of 32 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluent, and the collection operation was carried out to collect the cell suspension. The cell recovery rate was 78%.

(Comparative Example 3)
590 hollow fibers having an inner diameter of 630 μm were put in a case having a membrane area of 0.15 m ² and a volume of 150 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 59%.

(Comparative Example 4)
300 hollow fibers having an inner diameter of 540 to 600 μm were put into a case having a membrane area of 0.02 m ² and a volume of 28 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluent, and the collection operation was carried out to collect the cell suspension. The cell recovery rate was 78%.

(Comparative Example 5)
230 hollow fibers having an inner diameter of 1000 μm were put in a case having a membrane area of 0.15 m ² and a volume of 128 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using the diluted solution, and the collection operation was performed to collect the cell suspension. As a result, the cell recovery rate was 67%.

(Comparative Example 6)
3000 hollow fibers having an inner diameter of 350 μm were put into a case having a membrane area of 0.8 m ² and a volume of 185 mL to form a module. The culture-activated lymphocytes were concentrated at a filtration rate of 200 mL / min while the culture-activated lymphocytes flowed at a rate of 450 mL / min inside the module. Next, the cells were washed using a diluent, and the cell suspension was recovered by carrying out a recovery operation. The cell recovery rate was 75%.

From Table 1, a hollow fiber inner diameter of the separation membrane 540～600Myuemu, yarn number is present 200-350, and the membrane area of 0.04m ² ~0.13m ^2, the volume of the hollow fiber module 57~152mL It is apparent that a cell concentrate can be produced with a high cell recovery rate by treating a cell suspension using a hollow fiber type separation membrane having a pore diameter of 0.1 μm to 1 μm.

  From the above, by using the production method of the present invention, cells can be recovered with high efficiency while removing impurities such as proteins other than cells in the suspension, and the cell concentrate is less clogged with cells in the hollow fiber. Can be manufactured. Furthermore, since no washing method or the like that causes a load on the cells is used, damage to the cells is reduced.

  Therefore, if the cell concentrate produced by the method of the present invention is used, it is possible to efficiently concentrate only the target cells by aseptic operation, and it is possible to prevent leakage of materials because a plurality of hollow fibers are not used. Because of its high nature, cells for cell therapy can also be provided.

1. Tubular container Torso part 3. Head 4. 4. Filtrate outlet 5. bundle of hollow fiber type separation membranes Open end (dotted line)
7). Resin layer (shaded area)
8a. Cell suspension inlet 8b. Cell suspension outlet 9. Header part

Claims (11)

A cell suspension inlet, a filtrate outlet, a cell suspension outlet, and a hollow fiber type separation membrane disposed between the cell suspension inlet and the cell suspension outlet, the hollow fiber type separation membrane the inner diameter 540～600Myuemu, a thread number is present 200-350, and, using a hollow fiber module membrane area is 0.04m ² ~0.13m ^2,
(A) introducing the cell suspension from the cell suspension inlet and concentrating the cell suspension while discharging the filtrate from the filtrate outlet;
(B) introducing the cell suspension from the cell suspension inlet and collecting the cell concentrate derived from the cell suspension outlet in a collection container;
A method for producing a cell concentrate, comprising:   The method for producing a cell concentrate according to claim 1, wherein the volume of the hollow fiber module is 57 to 152 mL.   The method for producing a cell concentrate according to claim 1 or 2, wherein the hollow fiber separation membrane has a pore size of 0.1 µm to 1 µm.   The method for producing a cell concentrate according to any one of claims 1 to 3, wherein the material of the hollow fiber type separation membrane is polyethersulfone.   The process for producing a cell concentrate according to any one of claims 1 to 4, further comprising a step of washing the cell suspension by repeating dilution and concentration using a diluent before the step (b). Method.   The method for producing a cell concentrate according to any one of claims 1 to 5, wherein the collection container has one or more ports through which liquid can flow in and out.   The manufacturing method of the cell concentrate of Claim 6 including the process of taking out a cell concentrate through a port after the process of (b).   The method for producing a cell concentrate according to claim 6 or 7, further comprising a step of adding a drug through a port after the step (b).   The manufacturing method of the cell concentrate in any one of Claims 1-8 including the process of sealing and dividing a collection container after the process of (b). To produce a cell concentrate by way of any of claims 1 to 9, the cell concentrate, the cell culture method characterized by culturing again. Cryopreservation method to produce a cell concentrate by way of any of claims 1 to 9, the cell concentrate, characterized by cryopreservation with liquid nitrogen.

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